Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2013 Article ID 879845 10 pageshttpdxdoiorg1011552013879845

Research ArticleBee Venom Mitigates Cisplatin-Induced Nephrotoxicity byRegulating CD4+CD25+Foxp3+ Regulatory T Cells in Mice

Hyunseong Kim1 Gihyun Lee1 Soojin Park1 Hwan-Suck Chung1 Hyojung Lee1

Jong-Yoon Kim2 Sangsoo Nam2 Sun Kwang Kim1 and Hyunsu Bae13

1 Department of Physiology College of Oriental Medicine Kyung Hee University 1 Hoeki-Dong Dongdaemun-guSeoul 130-701 Republic of Korea

2Department of Internal Medicine College of Oriental Medicine Kyung Hee University Seoul 130-701 Republic of Korea3 Institute of Oriental Medicine Kyung Hee University 1 Hoeki-Dong Dongdaemun-gu Seoul 130-701 Republic of Korea

Correspondence should be addressed to Hyunsu Bae hbaekhuackr

Received 27 August 2012 Revised 17 December 2012 Accepted 26 December 2012

Academic Editor Bashar Saad

Copyright copy 2013 Hyunseong Kim et alThis is an open access article distributed under theCreativeCommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Cisplatin is used as a potent anticancer drug but it often causes nephrotoxicity Bee venom (BV) has been used for the treatment ofvarious inflammatory diseases and its renoprotective action was shown in NZBW mice However little is known about whetherBV has beneficial effects on cisplatin-induced nephrotoxicity and how such effects might be mediated In the present study theBV-injected group showed a significant increase in the population of Tregs in spleen Although there was no significant differencein the numbers of Tregs 3 days after cisplatin injection between the BV- and PBS-injected groups more migration of Tregs intothe kidney was observed 6 hours after cisplatin administration in BV group than in PBS group In addition BV-injected miceshowed reduced levels of serum creatinine blood urea nitrogen renal tissue damage proinflammatory cytokines and macrophageinfiltration into the kidney 3 days after cisplatin administration These renoprotective effects were abolished by the depletion ofTregs The anticancer effect of repeated administrations of cisplatin was not affected by BV injection These results suggest that BVhas protective effects on cisplatin-induced nephrotoxicity in mice at least in part through the regulation of Tregs without a biginfluence on the antitumor effects of cisplatin

1 Introduction

cis-Diamminedichloroplatinum (cisplatin) is widely used asa chemotherapeutic agent to treat various cancers [1] It iseffective against cancer of the lung head and neck testisovary cervix endometrium bladder and oropharynx [2]However side effects in normal tissues and organs partic-ularly nephrotoxicity limit the use of cisplatin and relatedplatinum-based therapeutics [1] The nephrotoxic effects ofcisplatin are manifested as a decrease in creatinine clearanceand electrolyte imbalances particularly hypomagnesemiamainly due to the acute cytotoxic effects of cisplatin onproximal and distal tubules [3]

Foxp3 is an important regulator of the activation andfunction of CD4+CD25+ regulatory T cells (Tregs) [4] Tregsplay a pivotal role in the maintenance of tolerance in the

immune system [5ndash7] Recently we provided clear evidencethat Tregs mitigated cisplatin-induced nephrotoxicity Theadoptive transfer of Tregs into mice successfully protectedcisplatin-induced renal damage whereas the depletion ofTregs accelerated cisplatin toxicity [8] From these findingsit is expected that agents capable of enhancing Treg functionwould have beneficial effects on cisplatin-induced nephro-toxicity Thus we screened a natural product library usinga Foxp3 promoter reporter assay system and found thatbee venom ((BV) from Apis melifera) significantly increasedTregs compared to other candidates

BV has been used for the treatment of various inflam-matory diseases such as rheumatoid arthritis and possessesstrong immune modulatory effects [9 10] BV injectionproduced amarked suppression of leukocyte migration and asignificant reduction in the concentration of tumor necrosis

2 Evidence-Based Complementary and Alternative Medicine

Foxp

3

CD25

Cont104

104

103

103

102

102

101

101

100

100

3618

BV

104103102101100

104

103

102

101

100

5356

(a)

Cont BV

7

6

5

4

3

2

CD4+ CD

25+ Fo

xp3+

T ce

lls in

Tce

lls (

)in

viv

o

lowastlowastlowast

(b)

Cont BV

8

6

4

2

0

lowast

CD4+ CD

25+ Fo

xp3+

T ce

lls in

Tce

lls (

)in

viv

o

(c)

Figure 1 Increased CD4+CD25+Foxp3+ Tregs in vitro and in vivo BV (1 120583gmL) was treated in splenocytes separated from Foxp3EGFP micefor 3 days There was no significant difference in the proportion of dead cells between BV- (207) and PBS-treated (193) groups Thisin vitro BV treatment significantly increased the number of CD25+Foxp3+ T cells among the CD4+T cells (a b) And Foxp3EGFP mice wereinjected with BV (1mgkg) or same volume PBS once a day for 5 days This in vivo BV injection also significantly increased the number ofCD25+Foxp3+ T cells among the CD4+T cells (c) Tregs were analyzed by flow cytometry gated for CD4-positive cells (a) The values shownindicate the mean plusmn SEM lowastlowastlowast119875 lt 0001 versus control 119899 = 5 (b) lowast119875 lt 005 versus control 119899 = 3 in vivo (c)

factor (TNF) inmice [11] Our recent study demonstrated thatBV causes immune tolerance by increasing the populationof Tregs in an autoimmune disease model lupus-proneNZBW mice [12] However it remains unknown whetherBV mitigates cisplatin-induced nephrotoxicity in mice byregulating Tregs

In the present study we examined the protective effectsof BV pretreatment on renal tissuefunction and Tregs incisplatin-injected mice We found that BV inhibited thelevels of proinflammatory cytokines renal dysfunction andtissue damage in cisplatin-injected mice which were due

to the regulation of Tregs in the kidney Thus BV mighthave a therapeutic potential in preventing cisplatin-inducednephrotoxicity

2 Materials and Methods

21 Animals Male C57BL6 mice (5-6 weeks old KoreaBiolink Chungbuk Korea) weighing 18ndash20 g each wereused in most of the experiments To analyze CD4+CD25+

Foxp3+ Tregs in spleen and kidney male Foxp3EGFPC57BL6

Evidence-Based Complementary and Alternative Medicine 3

150

100

50

0

Cis

0 48 96 144 192 240

Perc

ent s

urvi

val

Cis + BV

lowast

Figure 2 Survival in cisplatin-treated mice Survival curves of micetreated with 25mgkg of cisplatin that were followed up for up to240 h BV-treated mice had a 4375 survival rate whereas all of thecontrol mice expired The values shown indicate the mean plusmn SEMlowast

119875 lt 005 versus control 119899 = 16

mice (C Cg-Foxp3tm2TchJ 5-6 weeks old) were purchasedfrom The Jackson Laboratory (Bar Harbor ME USA) Theywere kept under specific pathogen-free conditions with airconditioning and a 12 h lightdark cycle Mice had freeaccess to food and water during experiments This study wasapproved by the Animal Care and Use Committee of KyungHee University (KHUASP (SE)-09-009)

22 BV and Cisplatin Administration Mice received BV ata concentration of 1mgkg body weight once a day for5 days The control group received an equal volume ofPBS Cisplatin (cis-diammineplatinum II dichloride Sigma-Aldrich St Louis MO USA) was dissolved in 09 ofPBS at a concentration of 1mgmL Two days after the fifthadministration of BV or PBS all mice received a singleintraperitoneal (ip) injection of cisplatin (25mgkg bodyweight) Mice were sacrificed under ether anesthesia 72 hafter cisplatin administration Blood spleen and kidneysamples were extracted for analysis

23 The Levels of CD4+CD25+Foxp3+ Tregs in Spleno-cytes Splenocytes were isolated from Foxp3EGFP mice forimmunofluorescence and T cells were incubated with anti-mouse CD3 and anti-CD28 antibodiesThe cells were treatedwith PBS or BV (1120583gmL) and cultured in complete RPMI1640 media containing 25 120583gmL anti-mouse CD3 antibodyand 2120583gmL of anti-mouse CD28 antibody for 72 hr Single-cell suspensions were incubated with fluorescently taggedAbs directed against a panel of cell surface markers andstained with CD4 and CD25 (eBioscience San Diego CAUSA) Foxp3EGFP mice were also injected BV (1mgkg) orPBS once a day for 5 days and sacrificed to measure thelevel of Treg in splenocytes The staining was performed inaccordance with the manufacturerrsquos instructions All FACSdata were acquired on a FACSCalibur flow cytometer (BD

Biosciences San Jose CA USA) and analyzed using Cel-lQuest Pro (BD Biosciences San Jose CA USA)

24 Survival Test After cisplatin injection mice were mon-itored every 6 h for 10 days The results were statisticallyanalyzed

25 Assessment of Renal Dysfunctions Blood samples wereobtained at 24 48 and 72 h after cisplatin injection Sampleswere left at room temperature for 1 hour and then centrifugedfor 10min at 1000 g to obtain the serum Renal functionwas assessed by measuring blood urea nitrogen (BUN) andcreatinine using a FUJI DRI-CHEM 3500i instrument (FujiPhoto Film Ltd Tokyo Japan)

Kidney tissues were fixed in 4 paraformaldehyde (PFA)for 1 day and then embedded in paraffin sliced into 5120583msections and stained with hematoxylin and eosin (HampE)Three pathologists who were blinded to the experimentsscored the degree of tubular injury Renal tubular injurywas assessed using a semiquantitative score in which thepercentage of cortical tubules showing epithelial necrosis wasassigned a score of 0 none 1 lt10 2 10ndash25 3 25ndash75 or4 gt75 [13]

26 The Depletion of CD4+CD25+Foxp3+ Regulatory T Cellsin Mice Anti-mouse CD25 rat IgG1 (anti-CD25 clonePC61) antibodies were generated in-house from hybridomasobtained from ATCC (Manassas VA USA) A dose of 01mgof anti-CD25 antibody was injected each day before BV andcisplatin administration The efficacy of CD4+CD25+Foxp3+Treg depletion was confirmed by flow cytometry analysisusing PE-anti-mouse CD25 and FITC-anti-mouse CD4

27 Assessment of Proinflammatory Cytokines To examineproinflammatory cytokine levels after cisplatin administra-tion the TNF-120572 and IL-6 protein levels in the kidney weremeasured using an enzyme-linked immunosorbent assay(ELISA BD Biosciences San Jose CA USA) Frozen kid-ney tissue was homogenized in a buffer containing 10mMHEPES 10mM KCl 01mM EGTA 1mM dithiothreitoland 10mM phenylmethanesulfonyl fluoride [14] incubatedfor 20min on ice and then centrifuged at 13000 rpm (4∘C)for 15min The supernatant was evaluated using a kidneyinflammatory cytokine array The protein concentrations ineach supernatant were determined by a BCATH ProteinAssayKit (Thermo Scientific Rockford IL USA) The cytokineprotein levels were corrected for total amount of protein andthe results were expressed as pgmg or ngmg

28 Foxp3-Positive Cells in the Kidney We injected BV(1mgkg) or same volume PBS once a day for 5 days inFoxp3EGFP mice before cisplatin administration To visualizeand quantify the degree of Treg migration Zeiss LSM5confocal microscope (Zeiss Jena Germany) was used withkidney samples obtained from Foxp3EGFP mice that werekilled at 6 hours after cisplatin injection And then kidneywas

4 Evidence-Based Complementary and Alternative Medicine

Cont ContCis

2

1

0

4

3

Crea

tinin

e (m

gdL

)

Cis + BV Cis Cis + BV

48 h 72 h

lowastlowastlowast

(a)

Cont ContCis Cis + BV Cis Cis + BV

48 h 72 h

lowastlowastlowast

lowast

200

300

100

0

BUN

(mg

dL)

(b)

CisCisCont + BV

(c)

Cont Cis Cis + BV

lowastlowastlowast

2

1

0

3

Tubu

lar d

amag

e sco

re

(d)

Figure 3 The protective effects of BV on cisplatin-induced nephrotoxicity in mice Mice received BV (1mgkg) once a day for 5 days Thecontrol group received the same volume of PBS After the fifth administration of BV or PBS all mice received a single injection of cisplatin(25mgkg) Blood samples were obtained at 24 h and 48 h after cisplatin administration and mice were sacrificed under ether anesthesia at72 h after cisplatin administration Blood and kidney samples were extracted for analysis Blood was obtained at 24 48 and 72 h after cisplatininjection (119899 = 18) Renal dysfunction wasmeasured as creatinine (a) and BUN (b) Kidney sections were stained with HampE 72 h after cisplatininjection PBS treatment alone (cont) PBS and cisplatin treatments (Cis) BV and cisplatin (Cis + BV) (400x bar = 50 um) (c) Tubular injurydamage was scored from 0 to 4 (0 none 1 lt10 2 10ndash25 3 25ndash75 4 gt75) (d) The values shown indicate the mean plusmn SEM NS nosignificance (119875 gt 005) lowast119875 lt 005 lowastlowastlowast119875 lt 0001 versus Cis

sliced into 20120583m cryosection after blood flow The numbersof Foxp3-positive cell were counted in 10 fields per slide

29 Macrophage Infiltration into the Kidney To evalu-ate macrophage infiltration into the kidneys after cis-platin administration immunohistochemical staining formacrophages was performed on paraffin-embedded kidneytissue The slides were incubated overnight at 4∘C with ratanti-mouse F480 antibody (dilution 1 50 Serotec OxfordUK) The primary antibody was localized using the Vec-tastain ABC Elite Kit (Vector Laboratories) according tothe manufacturerrsquos instructions followed by reaction with a331015840-diaminobenzidine substrate-chromogen solution (Vec-tor Laboratories Burlingame CA USA) The slides werecounterstained with Harris hematoxylin The numbers ofF480-positive cells in each section were calculated by count-ing the number of positively stained cells in 5 fields per slideat a magnification of times200

210 Tumor-Bearing Mice with a Low Dose of CisplatinEL4 lymphoma cells were purchased from KCLB (SeoulKorea) Cells were cultured in minimum essential mediumsupplemented with DMEM (Welgene Daegu Korea) 10fetal bovine serum (FBS GIBCO BRL Grand Island USA)and 1 penicillinstreptomycin (Invitrogen Carlsbad CAUSA) Cells were grown at 37∘C in a humidified atmospherecontaining 5 CO

2 C57BL6 mice were divided randomly

into 3 groups (group I PBS group II PBS + cisplatin groupIII BV + cisplatin) At day 0 EL4 lymphoma cells (2 times 106cells in 01mL mediamouse) were injected with the samevolume of Matrigel (BD Biosciences San Jose CA USA)subcutaneously into the right flank of all mice BV (1mgkg)or PBS was given once a day for 5 days Thereafter cisplatinwas injected intraperitoneally 3 times (on days 11 14 and 17)at a concentration of 5mgkg each time to groups I and IIThe control group received the same volume of PBS All micewere sacrificed on day 20The size of the tumorwasmeasured

Evidence-Based Complementary and Alternative Medicine 5

0 2412 4836 7260 9684 120

NS

108

150

100

50

0

Perc

ent s

urvi

val

CisCis + BV

Figure 4 Survival test in CD25-depleted mice After CD25 deple-tion mice were treated with 25mg of cisplatinkg intraperitoneallyand followed up for up to 114 h to produce survival curves At 114 hall of the control mice and BV-treated mice had expired (NS 119875 gt005 versus cont 119899 = 8)

every three days (6 times in total) using calipers The volumeof the tumor was calculated as length times width22

211 Statistical Analysis All results are expressed as themeans plusmn SEMThe data were analyzed using two-tailed 119905-testor one-way ANOVA with Tukeyrsquos post hoc test Differenceswere considered significant at 119875 lt 005

3 Results

31 Population of Tregs in Spleen To confirm the immune-modulating effect of BV we isolated splenocytes from sac-rificed Foxp3EGFP mice We treated BV (1120583gmL) or PBS toincubated cells for 3 days The CD4+CD25+Foxp3+ cell pop-ulation was significantly increased in the BV-treated groupcompared to the PBS-treated group in vitro (cont 362 plusmn 017BV 536 plusmn 024 119899 = 5 resp) (Figures 1(a) and 1(b)) We alsoexamined the population of the CD4+CD25+Foxp3+ cells inspleen in vivo The similar increase in Tregs numbers wasobserved in BV-injected mice before cisplatin administration(cont 384 plusmn 031 BV 548 plusmn 025 119899 = 3 resp) (Figure 1(c))Three days after cisplatin administration however the pop-ulation of Tregs markedly increased in splenocytes fromboth of BV- and PBS-injected mice There was no significantdifference in Treg numbers between BV- and PBS-treatedanimals (cont 1369 plusmn 052 BV 1381 plusmn 038 119899 = 4 resp)

32 Survival after Administration of High-Dose CisplatinAfter cisplatin injection (25mgkg ip) the mice weremonitored for 10 days All controlmice had died by 134 h aftercisplatin administration whereas 7 of the 16 BV-treated micesurvived The BV-treated mice that died at early timepointsexpressed features similar to those of the saline-treated miceHowever all of the BV-treated mice that survived to 134 hsurvived until the end of the experiment (Figure 2)

33 Protective Effects of BV on Cisplatin-Induced Nephrotoxic-ity Mice were treated with intraperitoneal (ip) injections ofcisplatin (25mgkg) after BV treatment Blood urea nitrogen(BUN) and serum creatinine levels are common indicatorsof renal function Blood samples were collected at 24 48and 72 h after cisplatin injection and the levels of bloodurea nitrogen (BUN) and creatinine were measured in the48 and 72 h samples Creatinine and BUN were significantlyincreased in the control group compared with BV-treatedgroup (creatinine 48 h cont 016 plusmn 001mgdL Cis 083 plusmn005mgdL Cis + BV 046 plusmn 006mgdL creatinine 72 hcont 016 plusmn 001mgdL Cis 363 plusmn 014mgdL Cis + BV218 plusmn 034mgdL BUN 48 h Cont 2896 plusmn 106mgdL Cis1091plusmn1400mgdL Cis + BV 5538plusmn535mgdL BUN 72 hcont 2259 plusmn 081mgdL Cis 2675 plusmn 1073mgdL Cis + BV1938 plusmn 1812mgdL)

To investigate the effects of BV on cisplatin-induced renaldamage we stained kidney tissue sections with hematoxylinand eosin (HampE) Pretreatmentwith BV significantly reducedthe tubular injury score compared to the score in sectionsfrom control mice treated with cisplatin alone (renal damagescore Cis 2667 plusmn 1556 Cis + BV 1556 plusmn 02318) (Figure 3)

34 Survival in CD25-Depleted Mice After CD25 depletionall mice received an injection of cisplatin (25mgkg ip) andwere evaluated for 114 h All mice in both the control and BV-treated groups had died at 114 h (Figure 4)

35 The Effects of BV in CD25-Depleted Mice To confirmthat the effect of BV on cisplatin-induced nephrotoxicity wasmediated by Tregs we tested the combination treatment ina CD4+CD25+ Treg cell depletion model in vivo by treatingmice with an anti-CD25 antibody (01mgmouse ip) Wemeasured the amount of creatinine and BUN in the serumThese results demonstrated that Treg depletion abolished BV-mediated antinephrotoxic effects (creatinine 48 h Cis 066 plusmn006mgdL Cis + BV 066 plusmn 008mgdL creatinine 72 h Cis257 plusmn 027mgdL Cis + BV 242 plusmn 028mgdL BUN 48 hCis 6113 plusmn 358mgdL Cis + BV 6147 plusmn 473mgdL BUN72 h Cis 2252 plusmn 1116mgdL Cis + BV 2197 plusmn 749mgdL)In addition histological examination revealed that BV hadno protective effect on kidney tissues in CD25-depleted mice(renal damage score Cis 283 plusmn 023 Cis + BV 287 plusmn 019)(Figure 5)

36 Proinflammatory Cytokines in Kidney To evaluate theproinflammatory molecules generated by cisplatin-inducedrenal injury the levels of TNF-120572 and IL-6 proteins in thekidney were measured at 72 h after cisplatin administration

Only cisplatin-treated mice exhibited increased levels ofTNF-120572 and IL-6 at 72 h after cisplatin injection HoweverBV-treated mice showed significantly lower levels of theseproinflammatory cytokines than did control mice while Tregdepletion caused increases in the levels of these cytokines inboth the BV-treated and nontreated groups (Figure 6)

37 Migration of Tregs into Kidney Confocal microscopy wasused with kidney samples obtained from Foxp3EGFP mice

6 Evidence-Based Complementary and Alternative Medicine

2

1

0

3

Cis

NS

NSCrea

tinin

e (m

gdL

)

Cis + BV Cis Cis + BV

48 h 72 h

(a)

250

200

150

100

50

0

NS

NSBUN

(mg

dL)

Cis Cis + BV Cis Cis + BV

48 h 72 h

(b)

Cis Cis + BV

(c)

2

1

0

4

3Tu

bula

r dam

age s

core

NS

Cis Cis + BV

(d)

Figure 5 The inefficacy of bee venom in CD25-depleted mice All mice were treated with anti-CD25 antibody (01mgmouse ip) twicebefore and after BV injection Nephrotoxicity was induced by cisplatin (25mgkg ip) Blood was obtained at 24 48 and 72 h after cisplatininjection (119899 = 12) Renal dysfunction was reflected by the levels of creatinine at 48 h 72 h (a) and BUN at 48 h 72 h (b) Kidney sections fromCD25-depleted mice were stained with HampE at 72 h after cisplatin injection PBS treatment alone (cont) PBS and cisplatin treatments (Cis)BV and cisplatin (Cis + BV) (400x bar = 50 120583m) (c) Tubular injury damage was scored from 0ndash4 (0 none 1 lt10 2 10ndash25 3 25ndash75 4gt75) (d) The values shown indicate the mean plusmn SEM NS 119875 gt 005 versus Cis

that were killed at 6 hours after cisplatin injection MoreFoxp3-positive cells were observed in the kidney in BV-pretreated group compared to the PBS-treated group 6 hoursafter cisplatin injection (Figure 7) suggesting the increasedmigration of Tregs into the kidney in the early phase ofnephrotoxic process caused by cisplatin

38 Effects of BV on Cisplatin-Induced Macrophage Infil-tration Macrophages were counted in the kidney sec-tions at 72 h after cisplatin administration F480-positivemacrophages were rarely detected in BV-treated micecompared with control mice whereas more infiltrationof macrophages was measured in CD25-depleted mice(Figure 8)

39 The Influence of BV on Cisplatin-Induced AnticancerEffects To determine whether the preadministration of BVinfluenced the anticancer effects of cisplatin the experimentwas repeated in a tumor-bearing (EL4 lymphoma) mouse

model with a low dose of cisplatin (5mgkg) Cisplatin wasinjected 3 times at a concentration of 5mgkgThemice wereweighed and the size of the tumor was measured over thewhole experimental period All mice were sacrificed on day21 and the separated tumors were collected

Tumor size in all groups of mice was gradually increasedbefore cisplatin administration (until day 11 in Figure 9(b))There was no significant difference in tumor size beforecisplatin administration between BV and PBS groups Inaddition irrespective of BV pretreatment repeated admin-istrations of cisplatin exerted very potent anticancer effects(a remarkable reduction in tumor size) in EL4 tumor modelresulting in no difference in tumor size between PBS- andBV-preinjected groups (Figures 9(a)ndash9(c)) In contrast tumorsize in controlmice (tumormodel without cisplatin) continu-ously increased after cisplatin administrations (Figure 9(b))The amounts of creatinine and BUN were within the normalrange in all groups (data not shown) These results suggestthat BV has not a big influence on the anticancer effects ofcisplatin

Evidence-Based Complementary and Alternative Medicine 7

5

4

3

2

1

0

TNF

(ng

mg)

lowast NS

Cis CisCis + BV Cis + BVCD25-depleted mice

(a)

120

100

80

60

40

20

0

lowast

NS

Cis CisCis + BV Cis + BV

CD25-depleted mice

IL-6

(pg

mg)

(b)

Figure 6 Proinflammatory cytokines in the kidney Proinflammatory cytokines were measured in kidney tissue from both of normal miceand CD25-depleted mice by ELISA Renal IL-6 and TNF120572were decreased in the BV-treated group (Cis + BV) compared with the PBS-treatedgroup (Cis) However there was no difference between the BV-treated group and PBS-treated group in CD25-depleted mice The valuesshown indicate the mean plusmn SEM lowast119875 lt 005 versus Cis

Cont BV

Cis Cis + BV

(a)

CisCis + BVBV

NS

5

4

3

2

1

0Cont

lowastlowast

Foxp

3-po

sitiv

e cel

ls10

HPF

(b)

Figure 7The number of Foxp3-positive cells in the kidney Tregs in the kidney were detected by EGFP signal under the confocal microscopeimaging Foxp3-positive cells were indicated by arrows (a)There was no difference in the number of Foxp3 positive cells between PBS-treatedgroup and BV-treated group before cisplatin administration However Foxp3-positive cells in the kidney increased 6 hours after cisplatinadministration In BV-pretreated group after cisplatin administration the number of Foxp3-positive cells in the kidney was significantlyhigher than in PBS-pretreated group (b) All data are presented as the mean plusmn SEM NS 119875 gt 005 lowast119875 lt 005 versus cont and

119875 lt 005

versus Cis as determined by one-way ANOVA followed by Tukeyrsquos multiple comparison test

4 Discussion

The development of side effects after cisplatin treatment iscommon and acute renal failure may develop after expo-sure to a single dose [2] Acute renal failure as inducedby cisplatin leads to the generation of danger signals bytubular cells resulting in inflammation mediated by T lym-phocytes and NK cells [15] Tregs are lymphocytes with

immunosuppressive properties and play a pivotal role inthe maintenance of immune tolerance [5] Previous studieshave reported that the anti-inflammatory actions of Tregscan protect against renal injury [16ndash18] Our recent studydemonstrated that CD4+CD25+ Tregs attenuate cisplatin-induced nephrotoxicity in vivo [8] Thus we evaluated theeffects of many candidate agents on CD4+CD25+Foxp3+Tregs in vitro and found that BV potently increases the

8 Evidence-Based Complementary and Alternative Medicine

Cont

(a)

Cis

(b)

Cis + BV

(c)

Cis Cis + BVCis Cis + BV

NS40

30

20

10

0Cont

lowastlowastlowast

F48

0-po

sitiv

e cel

lsfie

ld

CD25-depleted mice

(d)

Cis

CD25-depleted

(e)

Cis + BV

CD25-depleted

(f)

Figure 8 Macrophage infiltration into kidney Macrophage accumulation in the kidney was detected by immunostaining for F480-positivecells at 72 h after cisplatin administration The number of F480-positive cells in each section was counted in 10 fields per slide at an originalmagnification of 200xThe infiltration in BV-treatedmice was compared with those of PBS-treatedmice and CD25-depletedmiceThe valuesshown indicate the mean plusmn SEM lowastlowastlowast119875 lt 0001 versus Cis

Cis

Cis

Cont

+ BV

CisCis

Cis

Cis

Cont

Cont

Cont

(c)

(b)

(a)

Tum

or w

eigh

t (m

g)

+ BV

6000500040003000

3000

2000

2000

10000

50

0

85 11 14 17 20Day

NS

NS

Cis + BV

Cis + BV

Tum

or si

ze (m

m3 )

Figure 9 Influence of BV on the antitumor effect of cisplatin All mice received injections of EL4 lymphoma cells (2 times 10601mL) sc intothe right flank BV (1mgkg) was injected once per day for 5 days from day 5 of tumor inoculation (119899 = 8) Cisplatin was injected 3 times (ondays 11 14 and 17) at a concentration of 5mgkg All mice were sacrificed at day 20 and the tumors were photographed (a) The tumor sizewas measured every three days after BV injection for 16 days After sacrifice the tumors were fully separated from the mice and the size (b)and weight (c) of the tumors were measured All data are presented as the mean plusmn SEM lowastlowastlowast119875 lt 0001 versus cont NS 119875 gt 005 versus Cisas determined by one-way ANOVA followed by Tukeyrsquos test

Evidence-Based Complementary and Alternative Medicine 9

population of CD4+CD25+Foxp3+ Tregs Moreover it wasshown that BV has remarkable renoprotective effects inNZBW lupus mice [12] Based on these lines of evidencewe hypothesized that BV may have therapeutic effects oncisplatin-induced nephrotoxicity through the upregulation ofTregs

In this study we first showed that BV indeed increases thepopulation of CD4+CD25+Foxp3+ Tregs in isolated mousesplenocytes in vitro and in vivo before cisplatin adminis-tration (Figure 1) corroborating the results of our previousstudy [12]Three days after cisplatin administration howeverthe population of Tregs markedly increased in splenocytesfrom both of BV- and PBS-injected mice with no significantdifference in Treg numbers between the two groups Wefurther examined how many Tregs are migrated into thekidney after cisplatin administration We found that moreTregs were migrated into the kidney 6 hours after cisplatininjection in BV-pretreated mice than in PBS-pretreated mice(Figure 7) Next we found that BV has protective effectson cisplatin-induced renal dysfunction and injury SerumBUN and creatinine are typical indicators of renal functionand an increase in these values could indicate renal failure[19] Our data showed that cisplatin treatment significantlyincreased the BUN and serum creatinine levels in the saline-treated control group and that this increase was preventedby BV treatment The histological analysis of the degree ofrenal tubular injury (epithelial necrosis) also suggests thatBV can effectively prevent the structural damage of renaltissuesThe degree of macrophage infiltration into the kidneyis a major parameter used to predict the progression ofrenal disease and activated macrophages express severalinflammatory mediators [20] including TNF-120572 and IL-6which play important roles in the pathogenesis of cisplatin-induced renal injury [21 22] Our data showed that BV treat-ment in cisplatin-injected mice decreased the expression ofproinflammatory cytokines as well asmacrophage infiltrationin the kidney Because renal toxicity is themain cause of deathin cisplatin-injected mice [23] we evaluated the survival ofBV- or saline-treated mice after cisplatin administrationTheBV-treated mice showed a higher survival rate than did thesaline-treated control mice Collectively these results mightsuggest that Tregs in spleen increase in number after BVtreatment before cisplatin administration which cause moremigration of Tregs into the kidney in the early phase ofcisplatin-induced nephrotoxicity Such action might lead tothe protective effects of BV on cisplatin-induced nephrotoxicevents as shown in the present study This is supported byour previous study [8] showing that adoptive transfer of Tregsto mice (iv) caused increased migration of Tregs into thekidney 6 h after cisplatin administration and then attenuatedthe renal injuryThe renoprotective effects of BV in cisplatin-injectedmice in the present data are comparable to the resultsshown in Treg-transfused mice [8]

Since BV is known to have direct anti-inflammatory andantitumor effects [24 25] we cannot rule out the possibleinvolvement of Tregs-independent mechanisms of BV in thepresent results However we demonstrated that depletionof Tregs using anti-CD25 antibody injection completelyinhibited the BV effects on cisplatin-induced nephrotoxic

eventsTherefore our findings strongly suggest that BV couldprevent cisplatin-induced severe side effects in the kidney atleast partly by modulating the Tregs

Acknowledgment

This work was supported by the National Research Founda-tion of Korea (NRF)Grant funded by the Korean government(MEST) (no 2011-006220)

References

[1] K B Meyer and N E Madias ldquoCisplatin nephrotoxicityrdquoMineral and Electrolyte Metabolism vol 20 no 4 pp 201ndash2131994

[2] A H Lau ldquoApoptosis induced by cisplatin nephrotoxic injuryrdquoKidney International vol 56 no 4 pp 1295ndash1298 1999

[3] R Safirstein J Winston M Goldstein et al ldquoCisplatin nephro-toxicityrdquo American Journal of Kidney Diseases vol 8 no 5 pp356ndash367 1986

[4] J D Fontenot M A Gavin and A Y Rudensky ldquoFoxp3 pro-grams the development and function ofCD4+CD25+ regulatoryT cellsrdquo Nature Immunology vol 4 no 4 pp 330ndash336 2003

[5] G Raimondi M S Turner A W Thomson and P A MorelldquoNaturally occurring regulatory T cells recent insights in healthand diseaserdquo Critical Reviews in Immunology vol 27 no 1 pp61ndash95 2007

[6] S Sakaguchi M Ono R Setoguchi et al ldquoFoxp3+CD25+CD4+natural regulatory T cells in dominant self-tolerance andautoimmune diseaserdquo Immunological Reviews vol 212 no 1 pp8ndash27 2006

[7] S Sakaguchi N Sakaguchi M Asano M Itoh and M TodaldquoImmunologic self-tolerance maintained by activated T cellsexpressing IL- 2 receptor 120572-chains (CD25) Breakdown of asingle mechanism of self- tolerance causes various autoimmunediseasesrdquo Journal of Immunology vol 155 no 3 pp 1151ndash11641995

[8] H Lee D Nho H S Chung et al ldquoCD4+CD25+ regulatoryT cells attenuate cisplatin-induced nephrotoxicity in micerdquoKidney International vol 78 no 11 pp 1100ndash1109 2010

[9] M H Park M S Choi D H Kwak et al ldquoAnti-cancer effect ofbee venomin prostate cancer cells through activation of caspasepathway via inactivation of NF-120581Brdquo Prostate vol 71 no 8 pp801ndash812 2011

[10] S Y Yoon Y B Kwon H W Kim et al ldquoBee venominjection produces a peripheral anti-inflammatory effect byactivation of a nitric oxide-dependent spinocoeruleus pathwayrdquoNeuroscience Letters vol 430 no 2 pp 163ndash168 2008

[11] Y B KwonHWKim TWHamet al ldquoThe anti-inflammatoryeffect of bee venom stimulation in a mouse air pouch modelis mediated by adrenal medullary activityrdquo Journal of Neuroen-docrinology vol 15 no 1 pp 93ndash96 2003

[12] H Lee E J Lee H Kim et al ldquoBee venom-associated Th1Th2immunoglobulin class switching results in immune toleranceof NZBW F1 murine lupus nephritisrdquo American Journal ofNephrology vol 34 no 2 pp 163ndash172 2011

[13] J Megyesi R L Safirstein and P M Price ldquoInduction ofp21WAF1CIP1SDI1 in kidney tubule cells affects the courseof cisplatin-induced acute renal failurerdquoThe Journal of ClinicalInvestigation vol 101 no 4 pp 777ndash782 1998

10 Evidence-Based Complementary and Alternative Medicine

[14] K K Meldrum P Metcalfe J A Leslie R Misseri K L Hileand D R Meldrum ldquoTNF-120572 neutralization decreases nuclearfactor-120581B activation and apoptosis during renal obstructionrdquoJournal of Surgical Research vol 131 no 2 pp 182ndash188 2006

[15] A Linkermann N Himmerkus L Rolver et al ldquoRenal tubularFas ligandmediates fratricide in cisplatin-induced acute kidneyfailurerdquo Kidney International vol 79 no 2 pp 169ndash178 2011

[16] T Muthukumar D Dadhania R Ding et al ldquoMessenger RNAfor FOXP3 in the urine of renal-allograft recipientsrdquo The NewEngland Journal of Medicine vol 353 no 22 pp 2342ndash23512005

[17] G R Kinsey R Sharma L Huang et al ldquoRegulatory Tcells suppress innate immunity in kidney ischemia-reperfusioninjuryrdquo Journal of the American Society of Nephrology vol 20no 8 pp 1744ndash1753 2009

[18] S Bunnag K Allanach G S Jhangri et al ldquoFOXP3 expressionin human kidney transplant biopsies is associatedwith rejectionand time post transplant but not with favorable outcomesrdquoAmerican Journal of Transplantation vol 8 no 7 pp 1423ndash14332008

[19] H Kim G Lee H Lee M Shin M Hong and H Bae ldquoEffectsof Scutellaria barbata on cisplatin induced nephrotoxicity inmicerdquoMolecular and Cellular Toxicology vol 6 no 3 pp 255ndash259 2010

[20] Y Wang Q Cai G Zheng et al ldquoBy homing to the kidneyactivated macrophages potently exacerbate renal injuryrdquo TheAmerican Journal of Pathology vol 172 no 6 pp 1491ndash14992008

[21] S Faubel E C Lewis L Reznikov et al ldquoCisplatin-inducedacute renal failure is associatedwith an increase in the cytokinesinterleukin (IL)-1120573 IL-18 IL-6 and neutrophil infiltrationin the kidneyrdquo Journal of Pharmacology and ExperimentalTherapeutics vol 322 no 1 pp 8ndash15 2007

[22] G Ramesh and W Brian Reeves ldquoTNF-120572mediates chemokineand cytokine expression and renal injury in cisplatin nephro-toxicityrdquoThe Journal of Clinical Investigation vol 110 no 6 pp835ndash842 2002

[23] F Ries and J Klastersky ldquoNephrotoxicity induced by cancerchemotherapy with special emphasis on cisplatin toxicityrdquoAmerican Journal of Kidney Diseases vol 8 no 5 pp 368ndash3791986

[24] N Orsol ldquoBee venom in cancer therapyrdquo Cancer andMetastasisReviews vol 31 no 1-2 pp 173ndash194 2012

[25] N Orsolic L Sver S Verstovsek S Terzic and I BasicldquoInhibition of mammary carcinoma cell proliferation in vitroand tumor growth in vivo by bee venomrdquo Toxicon vol 41 no 7pp 861ndash870 2003

2 Evidence-Based Complementary and Alternative Medicine

Foxp

3

CD25

Cont104

104

103

103

102

102

101

101

100

100

3618

BV

104103102101100

104

103

102

101

100

5356

(a)

Cont BV

7

6

5

4

3

2

CD4+ CD

25+ Fo

xp3+

T ce

lls in

Tce

lls (

)in

viv

o

lowastlowastlowast

(b)

Cont BV

8

6

4

2

0

lowast

CD4+ CD

25+ Fo

xp3+

T ce

lls in

Tce

lls (

)in

viv

o

(c)

Figure 1 Increased CD4+CD25+Foxp3+ Tregs in vitro and in vivo BV (1 120583gmL) was treated in splenocytes separated from Foxp3EGFP micefor 3 days There was no significant difference in the proportion of dead cells between BV- (207) and PBS-treated (193) groups Thisin vitro BV treatment significantly increased the number of CD25+Foxp3+ T cells among the CD4+T cells (a b) And Foxp3EGFP mice wereinjected with BV (1mgkg) or same volume PBS once a day for 5 days This in vivo BV injection also significantly increased the number ofCD25+Foxp3+ T cells among the CD4+T cells (c) Tregs were analyzed by flow cytometry gated for CD4-positive cells (a) The values shownindicate the mean plusmn SEM lowastlowastlowast119875 lt 0001 versus control 119899 = 5 (b) lowast119875 lt 005 versus control 119899 = 3 in vivo (c)

factor (TNF) inmice [11] Our recent study demonstrated thatBV causes immune tolerance by increasing the populationof Tregs in an autoimmune disease model lupus-proneNZBW mice [12] However it remains unknown whetherBV mitigates cisplatin-induced nephrotoxicity in mice byregulating Tregs

In the present study we examined the protective effectsof BV pretreatment on renal tissuefunction and Tregs incisplatin-injected mice We found that BV inhibited thelevels of proinflammatory cytokines renal dysfunction andtissue damage in cisplatin-injected mice which were due

to the regulation of Tregs in the kidney Thus BV mighthave a therapeutic potential in preventing cisplatin-inducednephrotoxicity

2 Materials and Methods

21 Animals Male C57BL6 mice (5-6 weeks old KoreaBiolink Chungbuk Korea) weighing 18ndash20 g each wereused in most of the experiments To analyze CD4+CD25+

Foxp3+ Tregs in spleen and kidney male Foxp3EGFPC57BL6

Evidence-Based Complementary and Alternative Medicine 3

150

100

50

0

Cis

0 48 96 144 192 240

Perc

ent s

urvi

val

Cis + BV

lowast

Figure 2 Survival in cisplatin-treated mice Survival curves of micetreated with 25mgkg of cisplatin that were followed up for up to240 h BV-treated mice had a 4375 survival rate whereas all of thecontrol mice expired The values shown indicate the mean plusmn SEMlowast

119875 lt 005 versus control 119899 = 16

mice (C Cg-Foxp3tm2TchJ 5-6 weeks old) were purchasedfrom The Jackson Laboratory (Bar Harbor ME USA) Theywere kept under specific pathogen-free conditions with airconditioning and a 12 h lightdark cycle Mice had freeaccess to food and water during experiments This study wasapproved by the Animal Care and Use Committee of KyungHee University (KHUASP (SE)-09-009)

22 BV and Cisplatin Administration Mice received BV ata concentration of 1mgkg body weight once a day for5 days The control group received an equal volume ofPBS Cisplatin (cis-diammineplatinum II dichloride Sigma-Aldrich St Louis MO USA) was dissolved in 09 ofPBS at a concentration of 1mgmL Two days after the fifthadministration of BV or PBS all mice received a singleintraperitoneal (ip) injection of cisplatin (25mgkg bodyweight) Mice were sacrificed under ether anesthesia 72 hafter cisplatin administration Blood spleen and kidneysamples were extracted for analysis

23 The Levels of CD4+CD25+Foxp3+ Tregs in Spleno-cytes Splenocytes were isolated from Foxp3EGFP mice forimmunofluorescence and T cells were incubated with anti-mouse CD3 and anti-CD28 antibodiesThe cells were treatedwith PBS or BV (1120583gmL) and cultured in complete RPMI1640 media containing 25 120583gmL anti-mouse CD3 antibodyand 2120583gmL of anti-mouse CD28 antibody for 72 hr Single-cell suspensions were incubated with fluorescently taggedAbs directed against a panel of cell surface markers andstained with CD4 and CD25 (eBioscience San Diego CAUSA) Foxp3EGFP mice were also injected BV (1mgkg) orPBS once a day for 5 days and sacrificed to measure thelevel of Treg in splenocytes The staining was performed inaccordance with the manufacturerrsquos instructions All FACSdata were acquired on a FACSCalibur flow cytometer (BD

Biosciences San Jose CA USA) and analyzed using Cel-lQuest Pro (BD Biosciences San Jose CA USA)

24 Survival Test After cisplatin injection mice were mon-itored every 6 h for 10 days The results were statisticallyanalyzed

25 Assessment of Renal Dysfunctions Blood samples wereobtained at 24 48 and 72 h after cisplatin injection Sampleswere left at room temperature for 1 hour and then centrifugedfor 10min at 1000 g to obtain the serum Renal functionwas assessed by measuring blood urea nitrogen (BUN) andcreatinine using a FUJI DRI-CHEM 3500i instrument (FujiPhoto Film Ltd Tokyo Japan)

Kidney tissues were fixed in 4 paraformaldehyde (PFA)for 1 day and then embedded in paraffin sliced into 5120583msections and stained with hematoxylin and eosin (HampE)Three pathologists who were blinded to the experimentsscored the degree of tubular injury Renal tubular injurywas assessed using a semiquantitative score in which thepercentage of cortical tubules showing epithelial necrosis wasassigned a score of 0 none 1 lt10 2 10ndash25 3 25ndash75 or4 gt75 [13]

26 The Depletion of CD4+CD25+Foxp3+ Regulatory T Cellsin Mice Anti-mouse CD25 rat IgG1 (anti-CD25 clonePC61) antibodies were generated in-house from hybridomasobtained from ATCC (Manassas VA USA) A dose of 01mgof anti-CD25 antibody was injected each day before BV andcisplatin administration The efficacy of CD4+CD25+Foxp3+Treg depletion was confirmed by flow cytometry analysisusing PE-anti-mouse CD25 and FITC-anti-mouse CD4

27 Assessment of Proinflammatory Cytokines To examineproinflammatory cytokine levels after cisplatin administra-tion the TNF-120572 and IL-6 protein levels in the kidney weremeasured using an enzyme-linked immunosorbent assay(ELISA BD Biosciences San Jose CA USA) Frozen kid-ney tissue was homogenized in a buffer containing 10mMHEPES 10mM KCl 01mM EGTA 1mM dithiothreitoland 10mM phenylmethanesulfonyl fluoride [14] incubatedfor 20min on ice and then centrifuged at 13000 rpm (4∘C)for 15min The supernatant was evaluated using a kidneyinflammatory cytokine array The protein concentrations ineach supernatant were determined by a BCATH ProteinAssayKit (Thermo Scientific Rockford IL USA) The cytokineprotein levels were corrected for total amount of protein andthe results were expressed as pgmg or ngmg

28 Foxp3-Positive Cells in the Kidney We injected BV(1mgkg) or same volume PBS once a day for 5 days inFoxp3EGFP mice before cisplatin administration To visualizeand quantify the degree of Treg migration Zeiss LSM5confocal microscope (Zeiss Jena Germany) was used withkidney samples obtained from Foxp3EGFP mice that werekilled at 6 hours after cisplatin injection And then kidneywas

4 Evidence-Based Complementary and Alternative Medicine

Cont ContCis

2

1

0

4

3

Crea

tinin

e (m

gdL

)

Cis + BV Cis Cis + BV

48 h 72 h

lowastlowastlowast

(a)

Cont ContCis Cis + BV Cis Cis + BV

48 h 72 h

lowastlowastlowast

lowast

200

300

100

0

BUN

(mg

dL)

(b)

CisCisCont + BV

(c)

Cont Cis Cis + BV

lowastlowastlowast

2

1

0

3

Tubu

lar d

amag

e sco

re

(d)

Figure 3 The protective effects of BV on cisplatin-induced nephrotoxicity in mice Mice received BV (1mgkg) once a day for 5 days Thecontrol group received the same volume of PBS After the fifth administration of BV or PBS all mice received a single injection of cisplatin(25mgkg) Blood samples were obtained at 24 h and 48 h after cisplatin administration and mice were sacrificed under ether anesthesia at72 h after cisplatin administration Blood and kidney samples were extracted for analysis Blood was obtained at 24 48 and 72 h after cisplatininjection (119899 = 18) Renal dysfunction wasmeasured as creatinine (a) and BUN (b) Kidney sections were stained with HampE 72 h after cisplatininjection PBS treatment alone (cont) PBS and cisplatin treatments (Cis) BV and cisplatin (Cis + BV) (400x bar = 50 um) (c) Tubular injurydamage was scored from 0 to 4 (0 none 1 lt10 2 10ndash25 3 25ndash75 4 gt75) (d) The values shown indicate the mean plusmn SEM NS nosignificance (119875 gt 005) lowast119875 lt 005 lowastlowastlowast119875 lt 0001 versus Cis

sliced into 20120583m cryosection after blood flow The numbersof Foxp3-positive cell were counted in 10 fields per slide

29 Macrophage Infiltration into the Kidney To evalu-ate macrophage infiltration into the kidneys after cis-platin administration immunohistochemical staining formacrophages was performed on paraffin-embedded kidneytissue The slides were incubated overnight at 4∘C with ratanti-mouse F480 antibody (dilution 1 50 Serotec OxfordUK) The primary antibody was localized using the Vec-tastain ABC Elite Kit (Vector Laboratories) according tothe manufacturerrsquos instructions followed by reaction with a331015840-diaminobenzidine substrate-chromogen solution (Vec-tor Laboratories Burlingame CA USA) The slides werecounterstained with Harris hematoxylin The numbers ofF480-positive cells in each section were calculated by count-ing the number of positively stained cells in 5 fields per slideat a magnification of times200

210 Tumor-Bearing Mice with a Low Dose of CisplatinEL4 lymphoma cells were purchased from KCLB (SeoulKorea) Cells were cultured in minimum essential mediumsupplemented with DMEM (Welgene Daegu Korea) 10fetal bovine serum (FBS GIBCO BRL Grand Island USA)and 1 penicillinstreptomycin (Invitrogen Carlsbad CAUSA) Cells were grown at 37∘C in a humidified atmospherecontaining 5 CO

2 C57BL6 mice were divided randomly

into 3 groups (group I PBS group II PBS + cisplatin groupIII BV + cisplatin) At day 0 EL4 lymphoma cells (2 times 106cells in 01mL mediamouse) were injected with the samevolume of Matrigel (BD Biosciences San Jose CA USA)subcutaneously into the right flank of all mice BV (1mgkg)or PBS was given once a day for 5 days Thereafter cisplatinwas injected intraperitoneally 3 times (on days 11 14 and 17)at a concentration of 5mgkg each time to groups I and IIThe control group received the same volume of PBS All micewere sacrificed on day 20The size of the tumorwasmeasured

Evidence-Based Complementary and Alternative Medicine 5

0 2412 4836 7260 9684 120

NS

108

150

100

50

0

Perc

ent s

urvi

val

CisCis + BV

Figure 4 Survival test in CD25-depleted mice After CD25 deple-tion mice were treated with 25mg of cisplatinkg intraperitoneallyand followed up for up to 114 h to produce survival curves At 114 hall of the control mice and BV-treated mice had expired (NS 119875 gt005 versus cont 119899 = 8)

every three days (6 times in total) using calipers The volumeof the tumor was calculated as length times width22

211 Statistical Analysis All results are expressed as themeans plusmn SEMThe data were analyzed using two-tailed 119905-testor one-way ANOVA with Tukeyrsquos post hoc test Differenceswere considered significant at 119875 lt 005

3 Results

31 Population of Tregs in Spleen To confirm the immune-modulating effect of BV we isolated splenocytes from sac-rificed Foxp3EGFP mice We treated BV (1120583gmL) or PBS toincubated cells for 3 days The CD4+CD25+Foxp3+ cell pop-ulation was significantly increased in the BV-treated groupcompared to the PBS-treated group in vitro (cont 362 plusmn 017BV 536 plusmn 024 119899 = 5 resp) (Figures 1(a) and 1(b)) We alsoexamined the population of the CD4+CD25+Foxp3+ cells inspleen in vivo The similar increase in Tregs numbers wasobserved in BV-injected mice before cisplatin administration(cont 384 plusmn 031 BV 548 plusmn 025 119899 = 3 resp) (Figure 1(c))Three days after cisplatin administration however the pop-ulation of Tregs markedly increased in splenocytes fromboth of BV- and PBS-injected mice There was no significantdifference in Treg numbers between BV- and PBS-treatedanimals (cont 1369 plusmn 052 BV 1381 plusmn 038 119899 = 4 resp)

32 Survival after Administration of High-Dose CisplatinAfter cisplatin injection (25mgkg ip) the mice weremonitored for 10 days All controlmice had died by 134 h aftercisplatin administration whereas 7 of the 16 BV-treated micesurvived The BV-treated mice that died at early timepointsexpressed features similar to those of the saline-treated miceHowever all of the BV-treated mice that survived to 134 hsurvived until the end of the experiment (Figure 2)

33 Protective Effects of BV on Cisplatin-Induced Nephrotoxic-ity Mice were treated with intraperitoneal (ip) injections ofcisplatin (25mgkg) after BV treatment Blood urea nitrogen(BUN) and serum creatinine levels are common indicatorsof renal function Blood samples were collected at 24 48and 72 h after cisplatin injection and the levels of bloodurea nitrogen (BUN) and creatinine were measured in the48 and 72 h samples Creatinine and BUN were significantlyincreased in the control group compared with BV-treatedgroup (creatinine 48 h cont 016 plusmn 001mgdL Cis 083 plusmn005mgdL Cis + BV 046 plusmn 006mgdL creatinine 72 hcont 016 plusmn 001mgdL Cis 363 plusmn 014mgdL Cis + BV218 plusmn 034mgdL BUN 48 h Cont 2896 plusmn 106mgdL Cis1091plusmn1400mgdL Cis + BV 5538plusmn535mgdL BUN 72 hcont 2259 plusmn 081mgdL Cis 2675 plusmn 1073mgdL Cis + BV1938 plusmn 1812mgdL)

To investigate the effects of BV on cisplatin-induced renaldamage we stained kidney tissue sections with hematoxylinand eosin (HampE) Pretreatmentwith BV significantly reducedthe tubular injury score compared to the score in sectionsfrom control mice treated with cisplatin alone (renal damagescore Cis 2667 plusmn 1556 Cis + BV 1556 plusmn 02318) (Figure 3)

34 Survival in CD25-Depleted Mice After CD25 depletionall mice received an injection of cisplatin (25mgkg ip) andwere evaluated for 114 h All mice in both the control and BV-treated groups had died at 114 h (Figure 4)

35 The Effects of BV in CD25-Depleted Mice To confirmthat the effect of BV on cisplatin-induced nephrotoxicity wasmediated by Tregs we tested the combination treatment ina CD4+CD25+ Treg cell depletion model in vivo by treatingmice with an anti-CD25 antibody (01mgmouse ip) Wemeasured the amount of creatinine and BUN in the serumThese results demonstrated that Treg depletion abolished BV-mediated antinephrotoxic effects (creatinine 48 h Cis 066 plusmn006mgdL Cis + BV 066 plusmn 008mgdL creatinine 72 h Cis257 plusmn 027mgdL Cis + BV 242 plusmn 028mgdL BUN 48 hCis 6113 plusmn 358mgdL Cis + BV 6147 plusmn 473mgdL BUN72 h Cis 2252 plusmn 1116mgdL Cis + BV 2197 plusmn 749mgdL)In addition histological examination revealed that BV hadno protective effect on kidney tissues in CD25-depleted mice(renal damage score Cis 283 plusmn 023 Cis + BV 287 plusmn 019)(Figure 5)

36 Proinflammatory Cytokines in Kidney To evaluate theproinflammatory molecules generated by cisplatin-inducedrenal injury the levels of TNF-120572 and IL-6 proteins in thekidney were measured at 72 h after cisplatin administration

Only cisplatin-treated mice exhibited increased levels ofTNF-120572 and IL-6 at 72 h after cisplatin injection HoweverBV-treated mice showed significantly lower levels of theseproinflammatory cytokines than did control mice while Tregdepletion caused increases in the levels of these cytokines inboth the BV-treated and nontreated groups (Figure 6)

37 Migration of Tregs into Kidney Confocal microscopy wasused with kidney samples obtained from Foxp3EGFP mice

6 Evidence-Based Complementary and Alternative Medicine

2

1

0

3

Cis

NS

NSCrea

tinin

e (m

gdL

)

Cis + BV Cis Cis + BV

48 h 72 h

(a)

250

200

150

100

50

0

NS

NSBUN

(mg

dL)

Cis Cis + BV Cis Cis + BV

48 h 72 h

(b)

Cis Cis + BV

(c)

2

1

0

4

3Tu

bula

r dam

age s

core

NS

Cis Cis + BV

(d)

Figure 5 The inefficacy of bee venom in CD25-depleted mice All mice were treated with anti-CD25 antibody (01mgmouse ip) twicebefore and after BV injection Nephrotoxicity was induced by cisplatin (25mgkg ip) Blood was obtained at 24 48 and 72 h after cisplatininjection (119899 = 12) Renal dysfunction was reflected by the levels of creatinine at 48 h 72 h (a) and BUN at 48 h 72 h (b) Kidney sections fromCD25-depleted mice were stained with HampE at 72 h after cisplatin injection PBS treatment alone (cont) PBS and cisplatin treatments (Cis)BV and cisplatin (Cis + BV) (400x bar = 50 120583m) (c) Tubular injury damage was scored from 0ndash4 (0 none 1 lt10 2 10ndash25 3 25ndash75 4gt75) (d) The values shown indicate the mean plusmn SEM NS 119875 gt 005 versus Cis

that were killed at 6 hours after cisplatin injection MoreFoxp3-positive cells were observed in the kidney in BV-pretreated group compared to the PBS-treated group 6 hoursafter cisplatin injection (Figure 7) suggesting the increasedmigration of Tregs into the kidney in the early phase ofnephrotoxic process caused by cisplatin

38 Effects of BV on Cisplatin-Induced Macrophage Infil-tration Macrophages were counted in the kidney sec-tions at 72 h after cisplatin administration F480-positivemacrophages were rarely detected in BV-treated micecompared with control mice whereas more infiltrationof macrophages was measured in CD25-depleted mice(Figure 8)

39 The Influence of BV on Cisplatin-Induced AnticancerEffects To determine whether the preadministration of BVinfluenced the anticancer effects of cisplatin the experimentwas repeated in a tumor-bearing (EL4 lymphoma) mouse

model with a low dose of cisplatin (5mgkg) Cisplatin wasinjected 3 times at a concentration of 5mgkgThemice wereweighed and the size of the tumor was measured over thewhole experimental period All mice were sacrificed on day21 and the separated tumors were collected

Tumor size in all groups of mice was gradually increasedbefore cisplatin administration (until day 11 in Figure 9(b))There was no significant difference in tumor size beforecisplatin administration between BV and PBS groups Inaddition irrespective of BV pretreatment repeated admin-istrations of cisplatin exerted very potent anticancer effects(a remarkable reduction in tumor size) in EL4 tumor modelresulting in no difference in tumor size between PBS- andBV-preinjected groups (Figures 9(a)ndash9(c)) In contrast tumorsize in controlmice (tumormodel without cisplatin) continu-ously increased after cisplatin administrations (Figure 9(b))The amounts of creatinine and BUN were within the normalrange in all groups (data not shown) These results suggestthat BV has not a big influence on the anticancer effects ofcisplatin

Evidence-Based Complementary and Alternative Medicine 7

5

4

3

2

1

0

TNF

(ng

mg)

lowast NS

Cis CisCis + BV Cis + BVCD25-depleted mice

(a)

120

100

80

60

40

20

0

lowast

NS

Cis CisCis + BV Cis + BV

CD25-depleted mice

IL-6

(pg

mg)

(b)

Figure 6 Proinflammatory cytokines in the kidney Proinflammatory cytokines were measured in kidney tissue from both of normal miceand CD25-depleted mice by ELISA Renal IL-6 and TNF120572were decreased in the BV-treated group (Cis + BV) compared with the PBS-treatedgroup (Cis) However there was no difference between the BV-treated group and PBS-treated group in CD25-depleted mice The valuesshown indicate the mean plusmn SEM lowast119875 lt 005 versus Cis

Cont BV

Cis Cis + BV

(a)

CisCis + BVBV

NS

5

4

3

2

1

0Cont

lowastlowast

Foxp

3-po

sitiv

e cel

ls10

HPF

(b)

Figure 7The number of Foxp3-positive cells in the kidney Tregs in the kidney were detected by EGFP signal under the confocal microscopeimaging Foxp3-positive cells were indicated by arrows (a)There was no difference in the number of Foxp3 positive cells between PBS-treatedgroup and BV-treated group before cisplatin administration However Foxp3-positive cells in the kidney increased 6 hours after cisplatinadministration In BV-pretreated group after cisplatin administration the number of Foxp3-positive cells in the kidney was significantlyhigher than in PBS-pretreated group (b) All data are presented as the mean plusmn SEM NS 119875 gt 005 lowast119875 lt 005 versus cont and

119875 lt 005

versus Cis as determined by one-way ANOVA followed by Tukeyrsquos multiple comparison test

4 Discussion

The development of side effects after cisplatin treatment iscommon and acute renal failure may develop after expo-sure to a single dose [2] Acute renal failure as inducedby cisplatin leads to the generation of danger signals bytubular cells resulting in inflammation mediated by T lym-phocytes and NK cells [15] Tregs are lymphocytes with

immunosuppressive properties and play a pivotal role inthe maintenance of immune tolerance [5] Previous studieshave reported that the anti-inflammatory actions of Tregscan protect against renal injury [16ndash18] Our recent studydemonstrated that CD4+CD25+ Tregs attenuate cisplatin-induced nephrotoxicity in vivo [8] Thus we evaluated theeffects of many candidate agents on CD4+CD25+Foxp3+Tregs in vitro and found that BV potently increases the

8 Evidence-Based Complementary and Alternative Medicine

Cont

(a)

Cis

(b)

Cis + BV

(c)

Cis Cis + BVCis Cis + BV

NS40

30

20

10

0Cont

lowastlowastlowast

F48

0-po

sitiv

e cel

lsfie

ld

CD25-depleted mice

(d)

Cis

CD25-depleted

(e)

Cis + BV

CD25-depleted

(f)

Figure 8 Macrophage infiltration into kidney Macrophage accumulation in the kidney was detected by immunostaining for F480-positivecells at 72 h after cisplatin administration The number of F480-positive cells in each section was counted in 10 fields per slide at an originalmagnification of 200xThe infiltration in BV-treatedmice was compared with those of PBS-treatedmice and CD25-depletedmiceThe valuesshown indicate the mean plusmn SEM lowastlowastlowast119875 lt 0001 versus Cis

Cis

Cis

Cont

+ BV

CisCis

Cis

Cis

Cont

Cont

Cont

(c)

(b)

(a)

Tum

or w

eigh

t (m

g)

+ BV

6000500040003000

3000

2000

2000

10000

50

0

85 11 14 17 20Day

NS

NS

Cis + BV

Cis + BV

Tum

or si

ze (m

m3 )

Figure 9 Influence of BV on the antitumor effect of cisplatin All mice received injections of EL4 lymphoma cells (2 times 10601mL) sc intothe right flank BV (1mgkg) was injected once per day for 5 days from day 5 of tumor inoculation (119899 = 8) Cisplatin was injected 3 times (ondays 11 14 and 17) at a concentration of 5mgkg All mice were sacrificed at day 20 and the tumors were photographed (a) The tumor sizewas measured every three days after BV injection for 16 days After sacrifice the tumors were fully separated from the mice and the size (b)and weight (c) of the tumors were measured All data are presented as the mean plusmn SEM lowastlowastlowast119875 lt 0001 versus cont NS 119875 gt 005 versus Cisas determined by one-way ANOVA followed by Tukeyrsquos test

Evidence-Based Complementary and Alternative Medicine 9

population of CD4+CD25+Foxp3+ Tregs Moreover it wasshown that BV has remarkable renoprotective effects inNZBW lupus mice [12] Based on these lines of evidencewe hypothesized that BV may have therapeutic effects oncisplatin-induced nephrotoxicity through the upregulation ofTregs

In this study we first showed that BV indeed increases thepopulation of CD4+CD25+Foxp3+ Tregs in isolated mousesplenocytes in vitro and in vivo before cisplatin adminis-tration (Figure 1) corroborating the results of our previousstudy [12]Three days after cisplatin administration howeverthe population of Tregs markedly increased in splenocytesfrom both of BV- and PBS-injected mice with no significantdifference in Treg numbers between the two groups Wefurther examined how many Tregs are migrated into thekidney after cisplatin administration We found that moreTregs were migrated into the kidney 6 hours after cisplatininjection in BV-pretreated mice than in PBS-pretreated mice(Figure 7) Next we found that BV has protective effectson cisplatin-induced renal dysfunction and injury SerumBUN and creatinine are typical indicators of renal functionand an increase in these values could indicate renal failure[19] Our data showed that cisplatin treatment significantlyincreased the BUN and serum creatinine levels in the saline-treated control group and that this increase was preventedby BV treatment The histological analysis of the degree ofrenal tubular injury (epithelial necrosis) also suggests thatBV can effectively prevent the structural damage of renaltissuesThe degree of macrophage infiltration into the kidneyis a major parameter used to predict the progression ofrenal disease and activated macrophages express severalinflammatory mediators [20] including TNF-120572 and IL-6which play important roles in the pathogenesis of cisplatin-induced renal injury [21 22] Our data showed that BV treat-ment in cisplatin-injected mice decreased the expression ofproinflammatory cytokines as well asmacrophage infiltrationin the kidney Because renal toxicity is themain cause of deathin cisplatin-injected mice [23] we evaluated the survival ofBV- or saline-treated mice after cisplatin administrationTheBV-treated mice showed a higher survival rate than did thesaline-treated control mice Collectively these results mightsuggest that Tregs in spleen increase in number after BVtreatment before cisplatin administration which cause moremigration of Tregs into the kidney in the early phase ofcisplatin-induced nephrotoxicity Such action might lead tothe protective effects of BV on cisplatin-induced nephrotoxicevents as shown in the present study This is supported byour previous study [8] showing that adoptive transfer of Tregsto mice (iv) caused increased migration of Tregs into thekidney 6 h after cisplatin administration and then attenuatedthe renal injuryThe renoprotective effects of BV in cisplatin-injectedmice in the present data are comparable to the resultsshown in Treg-transfused mice [8]

Since BV is known to have direct anti-inflammatory andantitumor effects [24 25] we cannot rule out the possibleinvolvement of Tregs-independent mechanisms of BV in thepresent results However we demonstrated that depletionof Tregs using anti-CD25 antibody injection completelyinhibited the BV effects on cisplatin-induced nephrotoxic

eventsTherefore our findings strongly suggest that BV couldprevent cisplatin-induced severe side effects in the kidney atleast partly by modulating the Tregs

Acknowledgment

This work was supported by the National Research Founda-tion of Korea (NRF)Grant funded by the Korean government(MEST) (no 2011-006220)

References

[1] K B Meyer and N E Madias ldquoCisplatin nephrotoxicityrdquoMineral and Electrolyte Metabolism vol 20 no 4 pp 201ndash2131994

[2] A H Lau ldquoApoptosis induced by cisplatin nephrotoxic injuryrdquoKidney International vol 56 no 4 pp 1295ndash1298 1999

[3] R Safirstein J Winston M Goldstein et al ldquoCisplatin nephro-toxicityrdquo American Journal of Kidney Diseases vol 8 no 5 pp356ndash367 1986

[4] J D Fontenot M A Gavin and A Y Rudensky ldquoFoxp3 pro-grams the development and function ofCD4+CD25+ regulatoryT cellsrdquo Nature Immunology vol 4 no 4 pp 330ndash336 2003

[5] G Raimondi M S Turner A W Thomson and P A MorelldquoNaturally occurring regulatory T cells recent insights in healthand diseaserdquo Critical Reviews in Immunology vol 27 no 1 pp61ndash95 2007

[6] S Sakaguchi M Ono R Setoguchi et al ldquoFoxp3+CD25+CD4+natural regulatory T cells in dominant self-tolerance andautoimmune diseaserdquo Immunological Reviews vol 212 no 1 pp8ndash27 2006

[7] S Sakaguchi N Sakaguchi M Asano M Itoh and M TodaldquoImmunologic self-tolerance maintained by activated T cellsexpressing IL- 2 receptor 120572-chains (CD25) Breakdown of asingle mechanism of self- tolerance causes various autoimmunediseasesrdquo Journal of Immunology vol 155 no 3 pp 1151ndash11641995

[8] H Lee D Nho H S Chung et al ldquoCD4+CD25+ regulatoryT cells attenuate cisplatin-induced nephrotoxicity in micerdquoKidney International vol 78 no 11 pp 1100ndash1109 2010

[9] M H Park M S Choi D H Kwak et al ldquoAnti-cancer effect ofbee venomin prostate cancer cells through activation of caspasepathway via inactivation of NF-120581Brdquo Prostate vol 71 no 8 pp801ndash812 2011

[10] S Y Yoon Y B Kwon H W Kim et al ldquoBee venominjection produces a peripheral anti-inflammatory effect byactivation of a nitric oxide-dependent spinocoeruleus pathwayrdquoNeuroscience Letters vol 430 no 2 pp 163ndash168 2008

[11] Y B KwonHWKim TWHamet al ldquoThe anti-inflammatoryeffect of bee venom stimulation in a mouse air pouch modelis mediated by adrenal medullary activityrdquo Journal of Neuroen-docrinology vol 15 no 1 pp 93ndash96 2003

[12] H Lee E J Lee H Kim et al ldquoBee venom-associated Th1Th2immunoglobulin class switching results in immune toleranceof NZBW F1 murine lupus nephritisrdquo American Journal ofNephrology vol 34 no 2 pp 163ndash172 2011

[13] J Megyesi R L Safirstein and P M Price ldquoInduction ofp21WAF1CIP1SDI1 in kidney tubule cells affects the courseof cisplatin-induced acute renal failurerdquoThe Journal of ClinicalInvestigation vol 101 no 4 pp 777ndash782 1998

10 Evidence-Based Complementary and Alternative Medicine

[14] K K Meldrum P Metcalfe J A Leslie R Misseri K L Hileand D R Meldrum ldquoTNF-120572 neutralization decreases nuclearfactor-120581B activation and apoptosis during renal obstructionrdquoJournal of Surgical Research vol 131 no 2 pp 182ndash188 2006

[15] A Linkermann N Himmerkus L Rolver et al ldquoRenal tubularFas ligandmediates fratricide in cisplatin-induced acute kidneyfailurerdquo Kidney International vol 79 no 2 pp 169ndash178 2011

[16] T Muthukumar D Dadhania R Ding et al ldquoMessenger RNAfor FOXP3 in the urine of renal-allograft recipientsrdquo The NewEngland Journal of Medicine vol 353 no 22 pp 2342ndash23512005

[17] G R Kinsey R Sharma L Huang et al ldquoRegulatory Tcells suppress innate immunity in kidney ischemia-reperfusioninjuryrdquo Journal of the American Society of Nephrology vol 20no 8 pp 1744ndash1753 2009

[18] S Bunnag K Allanach G S Jhangri et al ldquoFOXP3 expressionin human kidney transplant biopsies is associatedwith rejectionand time post transplant but not with favorable outcomesrdquoAmerican Journal of Transplantation vol 8 no 7 pp 1423ndash14332008

[19] H Kim G Lee H Lee M Shin M Hong and H Bae ldquoEffectsof Scutellaria barbata on cisplatin induced nephrotoxicity inmicerdquoMolecular and Cellular Toxicology vol 6 no 3 pp 255ndash259 2010

[20] Y Wang Q Cai G Zheng et al ldquoBy homing to the kidneyactivated macrophages potently exacerbate renal injuryrdquo TheAmerican Journal of Pathology vol 172 no 6 pp 1491ndash14992008

[21] S Faubel E C Lewis L Reznikov et al ldquoCisplatin-inducedacute renal failure is associatedwith an increase in the cytokinesinterleukin (IL)-1120573 IL-18 IL-6 and neutrophil infiltrationin the kidneyrdquo Journal of Pharmacology and ExperimentalTherapeutics vol 322 no 1 pp 8ndash15 2007

[22] G Ramesh and W Brian Reeves ldquoTNF-120572mediates chemokineand cytokine expression and renal injury in cisplatin nephro-toxicityrdquoThe Journal of Clinical Investigation vol 110 no 6 pp835ndash842 2002

[23] F Ries and J Klastersky ldquoNephrotoxicity induced by cancerchemotherapy with special emphasis on cisplatin toxicityrdquoAmerican Journal of Kidney Diseases vol 8 no 5 pp 368ndash3791986

[24] N Orsol ldquoBee venom in cancer therapyrdquo Cancer andMetastasisReviews vol 31 no 1-2 pp 173ndash194 2012

[25] N Orsolic L Sver S Verstovsek S Terzic and I BasicldquoInhibition of mammary carcinoma cell proliferation in vitroand tumor growth in vivo by bee venomrdquo Toxicon vol 41 no 7pp 861ndash870 2003

Evidence-Based Complementary and Alternative Medicine 3

150

100

50

0

Cis

0 48 96 144 192 240

Perc

ent s

urvi

val

Cis + BV

lowast

Figure 2 Survival in cisplatin-treated mice Survival curves of micetreated with 25mgkg of cisplatin that were followed up for up to240 h BV-treated mice had a 4375 survival rate whereas all of thecontrol mice expired The values shown indicate the mean plusmn SEMlowast

119875 lt 005 versus control 119899 = 16

mice (C Cg-Foxp3tm2TchJ 5-6 weeks old) were purchasedfrom The Jackson Laboratory (Bar Harbor ME USA) Theywere kept under specific pathogen-free conditions with airconditioning and a 12 h lightdark cycle Mice had freeaccess to food and water during experiments This study wasapproved by the Animal Care and Use Committee of KyungHee University (KHUASP (SE)-09-009)

22 BV and Cisplatin Administration Mice received BV ata concentration of 1mgkg body weight once a day for5 days The control group received an equal volume ofPBS Cisplatin (cis-diammineplatinum II dichloride Sigma-Aldrich St Louis MO USA) was dissolved in 09 ofPBS at a concentration of 1mgmL Two days after the fifthadministration of BV or PBS all mice received a singleintraperitoneal (ip) injection of cisplatin (25mgkg bodyweight) Mice were sacrificed under ether anesthesia 72 hafter cisplatin administration Blood spleen and kidneysamples were extracted for analysis

23 The Levels of CD4+CD25+Foxp3+ Tregs in Spleno-cytes Splenocytes were isolated from Foxp3EGFP mice forimmunofluorescence and T cells were incubated with anti-mouse CD3 and anti-CD28 antibodiesThe cells were treatedwith PBS or BV (1120583gmL) and cultured in complete RPMI1640 media containing 25 120583gmL anti-mouse CD3 antibodyand 2120583gmL of anti-mouse CD28 antibody for 72 hr Single-cell suspensions were incubated with fluorescently taggedAbs directed against a panel of cell surface markers andstained with CD4 and CD25 (eBioscience San Diego CAUSA) Foxp3EGFP mice were also injected BV (1mgkg) orPBS once a day for 5 days and sacrificed to measure thelevel of Treg in splenocytes The staining was performed inaccordance with the manufacturerrsquos instructions All FACSdata were acquired on a FACSCalibur flow cytometer (BD

Biosciences San Jose CA USA) and analyzed using Cel-lQuest Pro (BD Biosciences San Jose CA USA)

24 Survival Test After cisplatin injection mice were mon-itored every 6 h for 10 days The results were statisticallyanalyzed

25 Assessment of Renal Dysfunctions Blood samples wereobtained at 24 48 and 72 h after cisplatin injection Sampleswere left at room temperature for 1 hour and then centrifugedfor 10min at 1000 g to obtain the serum Renal functionwas assessed by measuring blood urea nitrogen (BUN) andcreatinine using a FUJI DRI-CHEM 3500i instrument (FujiPhoto Film Ltd Tokyo Japan)

Kidney tissues were fixed in 4 paraformaldehyde (PFA)for 1 day and then embedded in paraffin sliced into 5120583msections and stained with hematoxylin and eosin (HampE)Three pathologists who were blinded to the experimentsscored the degree of tubular injury Renal tubular injurywas assessed using a semiquantitative score in which thepercentage of cortical tubules showing epithelial necrosis wasassigned a score of 0 none 1 lt10 2 10ndash25 3 25ndash75 or4 gt75 [13]

26 The Depletion of CD4+CD25+Foxp3+ Regulatory T Cellsin Mice Anti-mouse CD25 rat IgG1 (anti-CD25 clonePC61) antibodies were generated in-house from hybridomasobtained from ATCC (Manassas VA USA) A dose of 01mgof anti-CD25 antibody was injected each day before BV andcisplatin administration The efficacy of CD4+CD25+Foxp3+Treg depletion was confirmed by flow cytometry analysisusing PE-anti-mouse CD25 and FITC-anti-mouse CD4

27 Assessment of Proinflammatory Cytokines To examineproinflammatory cytokine levels after cisplatin administra-tion the TNF-120572 and IL-6 protein levels in the kidney weremeasured using an enzyme-linked immunosorbent assay(ELISA BD Biosciences San Jose CA USA) Frozen kid-ney tissue was homogenized in a buffer containing 10mMHEPES 10mM KCl 01mM EGTA 1mM dithiothreitoland 10mM phenylmethanesulfonyl fluoride [14] incubatedfor 20min on ice and then centrifuged at 13000 rpm (4∘C)for 15min The supernatant was evaluated using a kidneyinflammatory cytokine array The protein concentrations ineach supernatant were determined by a BCATH ProteinAssayKit (Thermo Scientific Rockford IL USA) The cytokineprotein levels were corrected for total amount of protein andthe results were expressed as pgmg or ngmg

28 Foxp3-Positive Cells in the Kidney We injected BV(1mgkg) or same volume PBS once a day for 5 days inFoxp3EGFP mice before cisplatin administration To visualizeand quantify the degree of Treg migration Zeiss LSM5confocal microscope (Zeiss Jena Germany) was used withkidney samples obtained from Foxp3EGFP mice that werekilled at 6 hours after cisplatin injection And then kidneywas

4 Evidence-Based Complementary and Alternative Medicine

Cont ContCis

2

1

0

4

3

Crea

tinin

e (m

gdL

)

Cis + BV Cis Cis + BV

48 h 72 h

lowastlowastlowast

(a)

Cont ContCis Cis + BV Cis Cis + BV

48 h 72 h

lowastlowastlowast

lowast

200

300

100

0

BUN

(mg

dL)

(b)

CisCisCont + BV

(c)

Cont Cis Cis + BV

lowastlowastlowast

2

1

0

3

Tubu

lar d

amag

e sco

re

(d)

Figure 3 The protective effects of BV on cisplatin-induced nephrotoxicity in mice Mice received BV (1mgkg) once a day for 5 days Thecontrol group received the same volume of PBS After the fifth administration of BV or PBS all mice received a single injection of cisplatin(25mgkg) Blood samples were obtained at 24 h and 48 h after cisplatin administration and mice were sacrificed under ether anesthesia at72 h after cisplatin administration Blood and kidney samples were extracted for analysis Blood was obtained at 24 48 and 72 h after cisplatininjection (119899 = 18) Renal dysfunction wasmeasured as creatinine (a) and BUN (b) Kidney sections were stained with HampE 72 h after cisplatininjection PBS treatment alone (cont) PBS and cisplatin treatments (Cis) BV and cisplatin (Cis + BV) (400x bar = 50 um) (c) Tubular injurydamage was scored from 0 to 4 (0 none 1 lt10 2 10ndash25 3 25ndash75 4 gt75) (d) The values shown indicate the mean plusmn SEM NS nosignificance (119875 gt 005) lowast119875 lt 005 lowastlowastlowast119875 lt 0001 versus Cis

sliced into 20120583m cryosection after blood flow The numbersof Foxp3-positive cell were counted in 10 fields per slide

29 Macrophage Infiltration into the Kidney To evalu-ate macrophage infiltration into the kidneys after cis-platin administration immunohistochemical staining formacrophages was performed on paraffin-embedded kidneytissue The slides were incubated overnight at 4∘C with ratanti-mouse F480 antibody (dilution 1 50 Serotec OxfordUK) The primary antibody was localized using the Vec-tastain ABC Elite Kit (Vector Laboratories) according tothe manufacturerrsquos instructions followed by reaction with a331015840-diaminobenzidine substrate-chromogen solution (Vec-tor Laboratories Burlingame CA USA) The slides werecounterstained with Harris hematoxylin The numbers ofF480-positive cells in each section were calculated by count-ing the number of positively stained cells in 5 fields per slideat a magnification of times200

210 Tumor-Bearing Mice with a Low Dose of CisplatinEL4 lymphoma cells were purchased from KCLB (SeoulKorea) Cells were cultured in minimum essential mediumsupplemented with DMEM (Welgene Daegu Korea) 10fetal bovine serum (FBS GIBCO BRL Grand Island USA)and 1 penicillinstreptomycin (Invitrogen Carlsbad CAUSA) Cells were grown at 37∘C in a humidified atmospherecontaining 5 CO

2 C57BL6 mice were divided randomly

into 3 groups (group I PBS group II PBS + cisplatin groupIII BV + cisplatin) At day 0 EL4 lymphoma cells (2 times 106cells in 01mL mediamouse) were injected with the samevolume of Matrigel (BD Biosciences San Jose CA USA)subcutaneously into the right flank of all mice BV (1mgkg)or PBS was given once a day for 5 days Thereafter cisplatinwas injected intraperitoneally 3 times (on days 11 14 and 17)at a concentration of 5mgkg each time to groups I and IIThe control group received the same volume of PBS All micewere sacrificed on day 20The size of the tumorwasmeasured

Evidence-Based Complementary and Alternative Medicine 5

0 2412 4836 7260 9684 120

NS

108

150

100

50

0

Perc

ent s

urvi

val

CisCis + BV

Figure 4 Survival test in CD25-depleted mice After CD25 deple-tion mice were treated with 25mg of cisplatinkg intraperitoneallyand followed up for up to 114 h to produce survival curves At 114 hall of the control mice and BV-treated mice had expired (NS 119875 gt005 versus cont 119899 = 8)

every three days (6 times in total) using calipers The volumeof the tumor was calculated as length times width22

211 Statistical Analysis All results are expressed as themeans plusmn SEMThe data were analyzed using two-tailed 119905-testor one-way ANOVA with Tukeyrsquos post hoc test Differenceswere considered significant at 119875 lt 005

3 Results

31 Population of Tregs in Spleen To confirm the immune-modulating effect of BV we isolated splenocytes from sac-rificed Foxp3EGFP mice We treated BV (1120583gmL) or PBS toincubated cells for 3 days The CD4+CD25+Foxp3+ cell pop-ulation was significantly increased in the BV-treated groupcompared to the PBS-treated group in vitro (cont 362 plusmn 017BV 536 plusmn 024 119899 = 5 resp) (Figures 1(a) and 1(b)) We alsoexamined the population of the CD4+CD25+Foxp3+ cells inspleen in vivo The similar increase in Tregs numbers wasobserved in BV-injected mice before cisplatin administration(cont 384 plusmn 031 BV 548 plusmn 025 119899 = 3 resp) (Figure 1(c))Three days after cisplatin administration however the pop-ulation of Tregs markedly increased in splenocytes fromboth of BV- and PBS-injected mice There was no significantdifference in Treg numbers between BV- and PBS-treatedanimals (cont 1369 plusmn 052 BV 1381 plusmn 038 119899 = 4 resp)

32 Survival after Administration of High-Dose CisplatinAfter cisplatin injection (25mgkg ip) the mice weremonitored for 10 days All controlmice had died by 134 h aftercisplatin administration whereas 7 of the 16 BV-treated micesurvived The BV-treated mice that died at early timepointsexpressed features similar to those of the saline-treated miceHowever all of the BV-treated mice that survived to 134 hsurvived until the end of the experiment (Figure 2)

33 Protective Effects of BV on Cisplatin-Induced Nephrotoxic-ity Mice were treated with intraperitoneal (ip) injections ofcisplatin (25mgkg) after BV treatment Blood urea nitrogen(BUN) and serum creatinine levels are common indicatorsof renal function Blood samples were collected at 24 48and 72 h after cisplatin injection and the levels of bloodurea nitrogen (BUN) and creatinine were measured in the48 and 72 h samples Creatinine and BUN were significantlyincreased in the control group compared with BV-treatedgroup (creatinine 48 h cont 016 plusmn 001mgdL Cis 083 plusmn005mgdL Cis + BV 046 plusmn 006mgdL creatinine 72 hcont 016 plusmn 001mgdL Cis 363 plusmn 014mgdL Cis + BV218 plusmn 034mgdL BUN 48 h Cont 2896 plusmn 106mgdL Cis1091plusmn1400mgdL Cis + BV 5538plusmn535mgdL BUN 72 hcont 2259 plusmn 081mgdL Cis 2675 plusmn 1073mgdL Cis + BV1938 plusmn 1812mgdL)

To investigate the effects of BV on cisplatin-induced renaldamage we stained kidney tissue sections with hematoxylinand eosin (HampE) Pretreatmentwith BV significantly reducedthe tubular injury score compared to the score in sectionsfrom control mice treated with cisplatin alone (renal damagescore Cis 2667 plusmn 1556 Cis + BV 1556 plusmn 02318) (Figure 3)

34 Survival in CD25-Depleted Mice After CD25 depletionall mice received an injection of cisplatin (25mgkg ip) andwere evaluated for 114 h All mice in both the control and BV-treated groups had died at 114 h (Figure 4)

35 The Effects of BV in CD25-Depleted Mice To confirmthat the effect of BV on cisplatin-induced nephrotoxicity wasmediated by Tregs we tested the combination treatment ina CD4+CD25+ Treg cell depletion model in vivo by treatingmice with an anti-CD25 antibody (01mgmouse ip) Wemeasured the amount of creatinine and BUN in the serumThese results demonstrated that Treg depletion abolished BV-mediated antinephrotoxic effects (creatinine 48 h Cis 066 plusmn006mgdL Cis + BV 066 plusmn 008mgdL creatinine 72 h Cis257 plusmn 027mgdL Cis + BV 242 plusmn 028mgdL BUN 48 hCis 6113 plusmn 358mgdL Cis + BV 6147 plusmn 473mgdL BUN72 h Cis 2252 plusmn 1116mgdL Cis + BV 2197 plusmn 749mgdL)In addition histological examination revealed that BV hadno protective effect on kidney tissues in CD25-depleted mice(renal damage score Cis 283 plusmn 023 Cis + BV 287 plusmn 019)(Figure 5)

36 Proinflammatory Cytokines in Kidney To evaluate theproinflammatory molecules generated by cisplatin-inducedrenal injury the levels of TNF-120572 and IL-6 proteins in thekidney were measured at 72 h after cisplatin administration

Only cisplatin-treated mice exhibited increased levels ofTNF-120572 and IL-6 at 72 h after cisplatin injection HoweverBV-treated mice showed significantly lower levels of theseproinflammatory cytokines than did control mice while Tregdepletion caused increases in the levels of these cytokines inboth the BV-treated and nontreated groups (Figure 6)

37 Migration of Tregs into Kidney Confocal microscopy wasused with kidney samples obtained from Foxp3EGFP mice

6 Evidence-Based Complementary and Alternative Medicine

2

1

0

3

Cis

NS

NSCrea

tinin

e (m

gdL

)

Cis + BV Cis Cis + BV

48 h 72 h

(a)

250

200

150

100

50

0

NS

NSBUN

(mg

dL)

Cis Cis + BV Cis Cis + BV

48 h 72 h

(b)

Cis Cis + BV

(c)

2

1

0

4

3Tu

bula

r dam

age s

core

NS

Cis Cis + BV

(d)

Figure 5 The inefficacy of bee venom in CD25-depleted mice All mice were treated with anti-CD25 antibody (01mgmouse ip) twicebefore and after BV injection Nephrotoxicity was induced by cisplatin (25mgkg ip) Blood was obtained at 24 48 and 72 h after cisplatininjection (119899 = 12) Renal dysfunction was reflected by the levels of creatinine at 48 h 72 h (a) and BUN at 48 h 72 h (b) Kidney sections fromCD25-depleted mice were stained with HampE at 72 h after cisplatin injection PBS treatment alone (cont) PBS and cisplatin treatments (Cis)BV and cisplatin (Cis + BV) (400x bar = 50 120583m) (c) Tubular injury damage was scored from 0ndash4 (0 none 1 lt10 2 10ndash25 3 25ndash75 4gt75) (d) The values shown indicate the mean plusmn SEM NS 119875 gt 005 versus Cis

that were killed at 6 hours after cisplatin injection MoreFoxp3-positive cells were observed in the kidney in BV-pretreated group compared to the PBS-treated group 6 hoursafter cisplatin injection (Figure 7) suggesting the increasedmigration of Tregs into the kidney in the early phase ofnephrotoxic process caused by cisplatin

38 Effects of BV on Cisplatin-Induced Macrophage Infil-tration Macrophages were counted in the kidney sec-tions at 72 h after cisplatin administration F480-positivemacrophages were rarely detected in BV-treated micecompared with control mice whereas more infiltrationof macrophages was measured in CD25-depleted mice(Figure 8)

39 The Influence of BV on Cisplatin-Induced AnticancerEffects To determine whether the preadministration of BVinfluenced the anticancer effects of cisplatin the experimentwas repeated in a tumor-bearing (EL4 lymphoma) mouse

model with a low dose of cisplatin (5mgkg) Cisplatin wasinjected 3 times at a concentration of 5mgkgThemice wereweighed and the size of the tumor was measured over thewhole experimental period All mice were sacrificed on day21 and the separated tumors were collected

Tumor size in all groups of mice was gradually increasedbefore cisplatin administration (until day 11 in Figure 9(b))There was no significant difference in tumor size beforecisplatin administration between BV and PBS groups Inaddition irrespective of BV pretreatment repeated admin-istrations of cisplatin exerted very potent anticancer effects(a remarkable reduction in tumor size) in EL4 tumor modelresulting in no difference in tumor size between PBS- andBV-preinjected groups (Figures 9(a)ndash9(c)) In contrast tumorsize in controlmice (tumormodel without cisplatin) continu-ously increased after cisplatin administrations (Figure 9(b))The amounts of creatinine and BUN were within the normalrange in all groups (data not shown) These results suggestthat BV has not a big influence on the anticancer effects ofcisplatin

Evidence-Based Complementary and Alternative Medicine 7

5

4

3

2

1

0

TNF

(ng

mg)

lowast NS

Cis CisCis + BV Cis + BVCD25-depleted mice

(a)

120

100

80

60

40

20

0

lowast

NS

Cis CisCis + BV Cis + BV

CD25-depleted mice

IL-6

(pg

mg)

(b)

Figure 6 Proinflammatory cytokines in the kidney Proinflammatory cytokines were measured in kidney tissue from both of normal miceand CD25-depleted mice by ELISA Renal IL-6 and TNF120572were decreased in the BV-treated group (Cis + BV) compared with the PBS-treatedgroup (Cis) However there was no difference between the BV-treated group and PBS-treated group in CD25-depleted mice The valuesshown indicate the mean plusmn SEM lowast119875 lt 005 versus Cis

Cont BV

Cis Cis + BV

(a)

CisCis + BVBV

NS

5

4

3

2

1

0Cont

lowastlowast

Foxp

3-po

sitiv

e cel

ls10

HPF

(b)

Figure 7The number of Foxp3-positive cells in the kidney Tregs in the kidney were detected by EGFP signal under the confocal microscopeimaging Foxp3-positive cells were indicated by arrows (a)There was no difference in the number of Foxp3 positive cells between PBS-treatedgroup and BV-treated group before cisplatin administration However Foxp3-positive cells in the kidney increased 6 hours after cisplatinadministration In BV-pretreated group after cisplatin administration the number of Foxp3-positive cells in the kidney was significantlyhigher than in PBS-pretreated group (b) All data are presented as the mean plusmn SEM NS 119875 gt 005 lowast119875 lt 005 versus cont and

119875 lt 005

versus Cis as determined by one-way ANOVA followed by Tukeyrsquos multiple comparison test

4 Discussion

The development of side effects after cisplatin treatment iscommon and acute renal failure may develop after expo-sure to a single dose [2] Acute renal failure as inducedby cisplatin leads to the generation of danger signals bytubular cells resulting in inflammation mediated by T lym-phocytes and NK cells [15] Tregs are lymphocytes with

immunosuppressive properties and play a pivotal role inthe maintenance of immune tolerance [5] Previous studieshave reported that the anti-inflammatory actions of Tregscan protect against renal injury [16ndash18] Our recent studydemonstrated that CD4+CD25+ Tregs attenuate cisplatin-induced nephrotoxicity in vivo [8] Thus we evaluated theeffects of many candidate agents on CD4+CD25+Foxp3+Tregs in vitro and found that BV potently increases the

8 Evidence-Based Complementary and Alternative Medicine

Cont

(a)

Cis

(b)

Cis + BV

(c)

Cis Cis + BVCis Cis + BV

NS40

30

20

10

0Cont

lowastlowastlowast

F48

0-po

sitiv

e cel

lsfie

ld

CD25-depleted mice

(d)

Cis

CD25-depleted

(e)

Cis + BV

CD25-depleted

(f)

Figure 8 Macrophage infiltration into kidney Macrophage accumulation in the kidney was detected by immunostaining for F480-positivecells at 72 h after cisplatin administration The number of F480-positive cells in each section was counted in 10 fields per slide at an originalmagnification of 200xThe infiltration in BV-treatedmice was compared with those of PBS-treatedmice and CD25-depletedmiceThe valuesshown indicate the mean plusmn SEM lowastlowastlowast119875 lt 0001 versus Cis

Cis

Cis

Cont

+ BV

CisCis

Cis

Cis

Cont

Cont

Cont

(c)

(b)

(a)

Tum

or w

eigh

t (m

g)

+ BV

6000500040003000

3000

2000

2000

10000

50

0

85 11 14 17 20Day

NS

NS

Cis + BV

Cis + BV

Tum

or si

ze (m

m3 )

Figure 9 Influence of BV on the antitumor effect of cisplatin All mice received injections of EL4 lymphoma cells (2 times 10601mL) sc intothe right flank BV (1mgkg) was injected once per day for 5 days from day 5 of tumor inoculation (119899 = 8) Cisplatin was injected 3 times (ondays 11 14 and 17) at a concentration of 5mgkg All mice were sacrificed at day 20 and the tumors were photographed (a) The tumor sizewas measured every three days after BV injection for 16 days After sacrifice the tumors were fully separated from the mice and the size (b)and weight (c) of the tumors were measured All data are presented as the mean plusmn SEM lowastlowastlowast119875 lt 0001 versus cont NS 119875 gt 005 versus Cisas determined by one-way ANOVA followed by Tukeyrsquos test

Evidence-Based Complementary and Alternative Medicine 9

population of CD4+CD25+Foxp3+ Tregs Moreover it wasshown that BV has remarkable renoprotective effects inNZBW lupus mice [12] Based on these lines of evidencewe hypothesized that BV may have therapeutic effects oncisplatin-induced nephrotoxicity through the upregulation ofTregs

In this study we first showed that BV indeed increases thepopulation of CD4+CD25+Foxp3+ Tregs in isolated mousesplenocytes in vitro and in vivo before cisplatin adminis-tration (Figure 1) corroborating the results of our previousstudy [12]Three days after cisplatin administration howeverthe population of Tregs markedly increased in splenocytesfrom both of BV- and PBS-injected mice with no significantdifference in Treg numbers between the two groups Wefurther examined how many Tregs are migrated into thekidney after cisplatin administration We found that moreTregs were migrated into the kidney 6 hours after cisplatininjection in BV-pretreated mice than in PBS-pretreated mice(Figure 7) Next we found that BV has protective effectson cisplatin-induced renal dysfunction and injury SerumBUN and creatinine are typical indicators of renal functionand an increase in these values could indicate renal failure[19] Our data showed that cisplatin treatment significantlyincreased the BUN and serum creatinine levels in the saline-treated control group and that this increase was preventedby BV treatment The histological analysis of the degree ofrenal tubular injury (epithelial necrosis) also suggests thatBV can effectively prevent the structural damage of renaltissuesThe degree of macrophage infiltration into the kidneyis a major parameter used to predict the progression ofrenal disease and activated macrophages express severalinflammatory mediators [20] including TNF-120572 and IL-6which play important roles in the pathogenesis of cisplatin-induced renal injury [21 22] Our data showed that BV treat-ment in cisplatin-injected mice decreased the expression ofproinflammatory cytokines as well asmacrophage infiltrationin the kidney Because renal toxicity is themain cause of deathin cisplatin-injected mice [23] we evaluated the survival ofBV- or saline-treated mice after cisplatin administrationTheBV-treated mice showed a higher survival rate than did thesaline-treated control mice Collectively these results mightsuggest that Tregs in spleen increase in number after BVtreatment before cisplatin administration which cause moremigration of Tregs into the kidney in the early phase ofcisplatin-induced nephrotoxicity Such action might lead tothe protective effects of BV on cisplatin-induced nephrotoxicevents as shown in the present study This is supported byour previous study [8] showing that adoptive transfer of Tregsto mice (iv) caused increased migration of Tregs into thekidney 6 h after cisplatin administration and then attenuatedthe renal injuryThe renoprotective effects of BV in cisplatin-injectedmice in the present data are comparable to the resultsshown in Treg-transfused mice [8]

Since BV is known to have direct anti-inflammatory andantitumor effects [24 25] we cannot rule out the possibleinvolvement of Tregs-independent mechanisms of BV in thepresent results However we demonstrated that depletionof Tregs using anti-CD25 antibody injection completelyinhibited the BV effects on cisplatin-induced nephrotoxic

eventsTherefore our findings strongly suggest that BV couldprevent cisplatin-induced severe side effects in the kidney atleast partly by modulating the Tregs

Acknowledgment

This work was supported by the National Research Founda-tion of Korea (NRF)Grant funded by the Korean government(MEST) (no 2011-006220)

References

[1] K B Meyer and N E Madias ldquoCisplatin nephrotoxicityrdquoMineral and Electrolyte Metabolism vol 20 no 4 pp 201ndash2131994

[2] A H Lau ldquoApoptosis induced by cisplatin nephrotoxic injuryrdquoKidney International vol 56 no 4 pp 1295ndash1298 1999

[3] R Safirstein J Winston M Goldstein et al ldquoCisplatin nephro-toxicityrdquo American Journal of Kidney Diseases vol 8 no 5 pp356ndash367 1986

[4] J D Fontenot M A Gavin and A Y Rudensky ldquoFoxp3 pro-grams the development and function ofCD4+CD25+ regulatoryT cellsrdquo Nature Immunology vol 4 no 4 pp 330ndash336 2003

[5] G Raimondi M S Turner A W Thomson and P A MorelldquoNaturally occurring regulatory T cells recent insights in healthand diseaserdquo Critical Reviews in Immunology vol 27 no 1 pp61ndash95 2007

[6] S Sakaguchi M Ono R Setoguchi et al ldquoFoxp3+CD25+CD4+natural regulatory T cells in dominant self-tolerance andautoimmune diseaserdquo Immunological Reviews vol 212 no 1 pp8ndash27 2006

[7] S Sakaguchi N Sakaguchi M Asano M Itoh and M TodaldquoImmunologic self-tolerance maintained by activated T cellsexpressing IL- 2 receptor 120572-chains (CD25) Breakdown of asingle mechanism of self- tolerance causes various autoimmunediseasesrdquo Journal of Immunology vol 155 no 3 pp 1151ndash11641995

[8] H Lee D Nho H S Chung et al ldquoCD4+CD25+ regulatoryT cells attenuate cisplatin-induced nephrotoxicity in micerdquoKidney International vol 78 no 11 pp 1100ndash1109 2010

[9] M H Park M S Choi D H Kwak et al ldquoAnti-cancer effect ofbee venomin prostate cancer cells through activation of caspasepathway via inactivation of NF-120581Brdquo Prostate vol 71 no 8 pp801ndash812 2011

[10] S Y Yoon Y B Kwon H W Kim et al ldquoBee venominjection produces a peripheral anti-inflammatory effect byactivation of a nitric oxide-dependent spinocoeruleus pathwayrdquoNeuroscience Letters vol 430 no 2 pp 163ndash168 2008

[11] Y B KwonHWKim TWHamet al ldquoThe anti-inflammatoryeffect of bee venom stimulation in a mouse air pouch modelis mediated by adrenal medullary activityrdquo Journal of Neuroen-docrinology vol 15 no 1 pp 93ndash96 2003

[12] H Lee E J Lee H Kim et al ldquoBee venom-associated Th1Th2immunoglobulin class switching results in immune toleranceof NZBW F1 murine lupus nephritisrdquo American Journal ofNephrology vol 34 no 2 pp 163ndash172 2011

[13] J Megyesi R L Safirstein and P M Price ldquoInduction ofp21WAF1CIP1SDI1 in kidney tubule cells affects the courseof cisplatin-induced acute renal failurerdquoThe Journal of ClinicalInvestigation vol 101 no 4 pp 777ndash782 1998

10 Evidence-Based Complementary and Alternative Medicine

[14] K K Meldrum P Metcalfe J A Leslie R Misseri K L Hileand D R Meldrum ldquoTNF-120572 neutralization decreases nuclearfactor-120581B activation and apoptosis during renal obstructionrdquoJournal of Surgical Research vol 131 no 2 pp 182ndash188 2006

[15] A Linkermann N Himmerkus L Rolver et al ldquoRenal tubularFas ligandmediates fratricide in cisplatin-induced acute kidneyfailurerdquo Kidney International vol 79 no 2 pp 169ndash178 2011

[16] T Muthukumar D Dadhania R Ding et al ldquoMessenger RNAfor FOXP3 in the urine of renal-allograft recipientsrdquo The NewEngland Journal of Medicine vol 353 no 22 pp 2342ndash23512005

[17] G R Kinsey R Sharma L Huang et al ldquoRegulatory Tcells suppress innate immunity in kidney ischemia-reperfusioninjuryrdquo Journal of the American Society of Nephrology vol 20no 8 pp 1744ndash1753 2009

[18] S Bunnag K Allanach G S Jhangri et al ldquoFOXP3 expressionin human kidney transplant biopsies is associatedwith rejectionand time post transplant but not with favorable outcomesrdquoAmerican Journal of Transplantation vol 8 no 7 pp 1423ndash14332008

[19] H Kim G Lee H Lee M Shin M Hong and H Bae ldquoEffectsof Scutellaria barbata on cisplatin induced nephrotoxicity inmicerdquoMolecular and Cellular Toxicology vol 6 no 3 pp 255ndash259 2010

[20] Y Wang Q Cai G Zheng et al ldquoBy homing to the kidneyactivated macrophages potently exacerbate renal injuryrdquo TheAmerican Journal of Pathology vol 172 no 6 pp 1491ndash14992008

[21] S Faubel E C Lewis L Reznikov et al ldquoCisplatin-inducedacute renal failure is associatedwith an increase in the cytokinesinterleukin (IL)-1120573 IL-18 IL-6 and neutrophil infiltrationin the kidneyrdquo Journal of Pharmacology and ExperimentalTherapeutics vol 322 no 1 pp 8ndash15 2007

[22] G Ramesh and W Brian Reeves ldquoTNF-120572mediates chemokineand cytokine expression and renal injury in cisplatin nephro-toxicityrdquoThe Journal of Clinical Investigation vol 110 no 6 pp835ndash842 2002

[23] F Ries and J Klastersky ldquoNephrotoxicity induced by cancerchemotherapy with special emphasis on cisplatin toxicityrdquoAmerican Journal of Kidney Diseases vol 8 no 5 pp 368ndash3791986

[24] N Orsol ldquoBee venom in cancer therapyrdquo Cancer andMetastasisReviews vol 31 no 1-2 pp 173ndash194 2012

[25] N Orsolic L Sver S Verstovsek S Terzic and I BasicldquoInhibition of mammary carcinoma cell proliferation in vitroand tumor growth in vivo by bee venomrdquo Toxicon vol 41 no 7pp 861ndash870 2003

4 Evidence-Based Complementary and Alternative Medicine

Cont ContCis

2

1

0

4

3

Crea

tinin

e (m

gdL

)

Cis + BV Cis Cis + BV

48 h 72 h

lowastlowastlowast

(a)

Cont ContCis Cis + BV Cis Cis + BV

48 h 72 h

lowastlowastlowast

lowast

200

300

100

0

BUN

(mg

dL)

(b)

CisCisCont + BV

(c)

Cont Cis Cis + BV

lowastlowastlowast

2

1

0

3

Tubu

lar d

amag

e sco

re

(d)

Figure 3 The protective effects of BV on cisplatin-induced nephrotoxicity in mice Mice received BV (1mgkg) once a day for 5 days Thecontrol group received the same volume of PBS After the fifth administration of BV or PBS all mice received a single injection of cisplatin(25mgkg) Blood samples were obtained at 24 h and 48 h after cisplatin administration and mice were sacrificed under ether anesthesia at72 h after cisplatin administration Blood and kidney samples were extracted for analysis Blood was obtained at 24 48 and 72 h after cisplatininjection (119899 = 18) Renal dysfunction wasmeasured as creatinine (a) and BUN (b) Kidney sections were stained with HampE 72 h after cisplatininjection PBS treatment alone (cont) PBS and cisplatin treatments (Cis) BV and cisplatin (Cis + BV) (400x bar = 50 um) (c) Tubular injurydamage was scored from 0 to 4 (0 none 1 lt10 2 10ndash25 3 25ndash75 4 gt75) (d) The values shown indicate the mean plusmn SEM NS nosignificance (119875 gt 005) lowast119875 lt 005 lowastlowastlowast119875 lt 0001 versus Cis

sliced into 20120583m cryosection after blood flow The numbersof Foxp3-positive cell were counted in 10 fields per slide

29 Macrophage Infiltration into the Kidney To evalu-ate macrophage infiltration into the kidneys after cis-platin administration immunohistochemical staining formacrophages was performed on paraffin-embedded kidneytissue The slides were incubated overnight at 4∘C with ratanti-mouse F480 antibody (dilution 1 50 Serotec OxfordUK) The primary antibody was localized using the Vec-tastain ABC Elite Kit (Vector Laboratories) according tothe manufacturerrsquos instructions followed by reaction with a331015840-diaminobenzidine substrate-chromogen solution (Vec-tor Laboratories Burlingame CA USA) The slides werecounterstained with Harris hematoxylin The numbers ofF480-positive cells in each section were calculated by count-ing the number of positively stained cells in 5 fields per slideat a magnification of times200

210 Tumor-Bearing Mice with a Low Dose of CisplatinEL4 lymphoma cells were purchased from KCLB (SeoulKorea) Cells were cultured in minimum essential mediumsupplemented with DMEM (Welgene Daegu Korea) 10fetal bovine serum (FBS GIBCO BRL Grand Island USA)and 1 penicillinstreptomycin (Invitrogen Carlsbad CAUSA) Cells were grown at 37∘C in a humidified atmospherecontaining 5 CO

2 C57BL6 mice were divided randomly

into 3 groups (group I PBS group II PBS + cisplatin groupIII BV + cisplatin) At day 0 EL4 lymphoma cells (2 times 106cells in 01mL mediamouse) were injected with the samevolume of Matrigel (BD Biosciences San Jose CA USA)subcutaneously into the right flank of all mice BV (1mgkg)or PBS was given once a day for 5 days Thereafter cisplatinwas injected intraperitoneally 3 times (on days 11 14 and 17)at a concentration of 5mgkg each time to groups I and IIThe control group received the same volume of PBS All micewere sacrificed on day 20The size of the tumorwasmeasured

Evidence-Based Complementary and Alternative Medicine 5

0 2412 4836 7260 9684 120

NS

108

150

100

50

0

Perc

ent s

urvi

val

CisCis + BV

Figure 4 Survival test in CD25-depleted mice After CD25 deple-tion mice were treated with 25mg of cisplatinkg intraperitoneallyand followed up for up to 114 h to produce survival curves At 114 hall of the control mice and BV-treated mice had expired (NS 119875 gt005 versus cont 119899 = 8)

every three days (6 times in total) using calipers The volumeof the tumor was calculated as length times width22

211 Statistical Analysis All results are expressed as themeans plusmn SEMThe data were analyzed using two-tailed 119905-testor one-way ANOVA with Tukeyrsquos post hoc test Differenceswere considered significant at 119875 lt 005

3 Results

31 Population of Tregs in Spleen To confirm the immune-modulating effect of BV we isolated splenocytes from sac-rificed Foxp3EGFP mice We treated BV (1120583gmL) or PBS toincubated cells for 3 days The CD4+CD25+Foxp3+ cell pop-ulation was significantly increased in the BV-treated groupcompared to the PBS-treated group in vitro (cont 362 plusmn 017BV 536 plusmn 024 119899 = 5 resp) (Figures 1(a) and 1(b)) We alsoexamined the population of the CD4+CD25+Foxp3+ cells inspleen in vivo The similar increase in Tregs numbers wasobserved in BV-injected mice before cisplatin administration(cont 384 plusmn 031 BV 548 plusmn 025 119899 = 3 resp) (Figure 1(c))Three days after cisplatin administration however the pop-ulation of Tregs markedly increased in splenocytes fromboth of BV- and PBS-injected mice There was no significantdifference in Treg numbers between BV- and PBS-treatedanimals (cont 1369 plusmn 052 BV 1381 plusmn 038 119899 = 4 resp)

32 Survival after Administration of High-Dose CisplatinAfter cisplatin injection (25mgkg ip) the mice weremonitored for 10 days All controlmice had died by 134 h aftercisplatin administration whereas 7 of the 16 BV-treated micesurvived The BV-treated mice that died at early timepointsexpressed features similar to those of the saline-treated miceHowever all of the BV-treated mice that survived to 134 hsurvived until the end of the experiment (Figure 2)

33 Protective Effects of BV on Cisplatin-Induced Nephrotoxic-ity Mice were treated with intraperitoneal (ip) injections ofcisplatin (25mgkg) after BV treatment Blood urea nitrogen(BUN) and serum creatinine levels are common indicatorsof renal function Blood samples were collected at 24 48and 72 h after cisplatin injection and the levels of bloodurea nitrogen (BUN) and creatinine were measured in the48 and 72 h samples Creatinine and BUN were significantlyincreased in the control group compared with BV-treatedgroup (creatinine 48 h cont 016 plusmn 001mgdL Cis 083 plusmn005mgdL Cis + BV 046 plusmn 006mgdL creatinine 72 hcont 016 plusmn 001mgdL Cis 363 plusmn 014mgdL Cis + BV218 plusmn 034mgdL BUN 48 h Cont 2896 plusmn 106mgdL Cis1091plusmn1400mgdL Cis + BV 5538plusmn535mgdL BUN 72 hcont 2259 plusmn 081mgdL Cis 2675 plusmn 1073mgdL Cis + BV1938 plusmn 1812mgdL)

To investigate the effects of BV on cisplatin-induced renaldamage we stained kidney tissue sections with hematoxylinand eosin (HampE) Pretreatmentwith BV significantly reducedthe tubular injury score compared to the score in sectionsfrom control mice treated with cisplatin alone (renal damagescore Cis 2667 plusmn 1556 Cis + BV 1556 plusmn 02318) (Figure 3)

34 Survival in CD25-Depleted Mice After CD25 depletionall mice received an injection of cisplatin (25mgkg ip) andwere evaluated for 114 h All mice in both the control and BV-treated groups had died at 114 h (Figure 4)

35 The Effects of BV in CD25-Depleted Mice To confirmthat the effect of BV on cisplatin-induced nephrotoxicity wasmediated by Tregs we tested the combination treatment ina CD4+CD25+ Treg cell depletion model in vivo by treatingmice with an anti-CD25 antibody (01mgmouse ip) Wemeasured the amount of creatinine and BUN in the serumThese results demonstrated that Treg depletion abolished BV-mediated antinephrotoxic effects (creatinine 48 h Cis 066 plusmn006mgdL Cis + BV 066 plusmn 008mgdL creatinine 72 h Cis257 plusmn 027mgdL Cis + BV 242 plusmn 028mgdL BUN 48 hCis 6113 plusmn 358mgdL Cis + BV 6147 plusmn 473mgdL BUN72 h Cis 2252 plusmn 1116mgdL Cis + BV 2197 plusmn 749mgdL)In addition histological examination revealed that BV hadno protective effect on kidney tissues in CD25-depleted mice(renal damage score Cis 283 plusmn 023 Cis + BV 287 plusmn 019)(Figure 5)

36 Proinflammatory Cytokines in Kidney To evaluate theproinflammatory molecules generated by cisplatin-inducedrenal injury the levels of TNF-120572 and IL-6 proteins in thekidney were measured at 72 h after cisplatin administration

Only cisplatin-treated mice exhibited increased levels ofTNF-120572 and IL-6 at 72 h after cisplatin injection HoweverBV-treated mice showed significantly lower levels of theseproinflammatory cytokines than did control mice while Tregdepletion caused increases in the levels of these cytokines inboth the BV-treated and nontreated groups (Figure 6)

37 Migration of Tregs into Kidney Confocal microscopy wasused with kidney samples obtained from Foxp3EGFP mice

6 Evidence-Based Complementary and Alternative Medicine

2

1

0

3

Cis

NS

NSCrea

tinin

e (m

gdL

)

Cis + BV Cis Cis + BV

48 h 72 h

(a)

250

200

150

100

50

0

NS

NSBUN

(mg

dL)

Cis Cis + BV Cis Cis + BV

48 h 72 h

(b)

Cis Cis + BV

(c)

2

1

0

4

3Tu

bula

r dam

age s

core

NS

Cis Cis + BV

(d)

Figure 5 The inefficacy of bee venom in CD25-depleted mice All mice were treated with anti-CD25 antibody (01mgmouse ip) twicebefore and after BV injection Nephrotoxicity was induced by cisplatin (25mgkg ip) Blood was obtained at 24 48 and 72 h after cisplatininjection (119899 = 12) Renal dysfunction was reflected by the levels of creatinine at 48 h 72 h (a) and BUN at 48 h 72 h (b) Kidney sections fromCD25-depleted mice were stained with HampE at 72 h after cisplatin injection PBS treatment alone (cont) PBS and cisplatin treatments (Cis)BV and cisplatin (Cis + BV) (400x bar = 50 120583m) (c) Tubular injury damage was scored from 0ndash4 (0 none 1 lt10 2 10ndash25 3 25ndash75 4gt75) (d) The values shown indicate the mean plusmn SEM NS 119875 gt 005 versus Cis

that were killed at 6 hours after cisplatin injection MoreFoxp3-positive cells were observed in the kidney in BV-pretreated group compared to the PBS-treated group 6 hoursafter cisplatin injection (Figure 7) suggesting the increasedmigration of Tregs into the kidney in the early phase ofnephrotoxic process caused by cisplatin

38 Effects of BV on Cisplatin-Induced Macrophage Infil-tration Macrophages were counted in the kidney sec-tions at 72 h after cisplatin administration F480-positivemacrophages were rarely detected in BV-treated micecompared with control mice whereas more infiltrationof macrophages was measured in CD25-depleted mice(Figure 8)

39 The Influence of BV on Cisplatin-Induced AnticancerEffects To determine whether the preadministration of BVinfluenced the anticancer effects of cisplatin the experimentwas repeated in a tumor-bearing (EL4 lymphoma) mouse

model with a low dose of cisplatin (5mgkg) Cisplatin wasinjected 3 times at a concentration of 5mgkgThemice wereweighed and the size of the tumor was measured over thewhole experimental period All mice were sacrificed on day21 and the separated tumors were collected

Tumor size in all groups of mice was gradually increasedbefore cisplatin administration (until day 11 in Figure 9(b))There was no significant difference in tumor size beforecisplatin administration between BV and PBS groups Inaddition irrespective of BV pretreatment repeated admin-istrations of cisplatin exerted very potent anticancer effects(a remarkable reduction in tumor size) in EL4 tumor modelresulting in no difference in tumor size between PBS- andBV-preinjected groups (Figures 9(a)ndash9(c)) In contrast tumorsize in controlmice (tumormodel without cisplatin) continu-ously increased after cisplatin administrations (Figure 9(b))The amounts of creatinine and BUN were within the normalrange in all groups (data not shown) These results suggestthat BV has not a big influence on the anticancer effects ofcisplatin

Evidence-Based Complementary and Alternative Medicine 7

5

4

3

2

1

0

TNF

(ng

mg)

lowast NS

Cis CisCis + BV Cis + BVCD25-depleted mice

(a)

120

100

80

60

40

20

0

lowast

NS

Cis CisCis + BV Cis + BV

CD25-depleted mice

IL-6

(pg

mg)

(b)

Figure 6 Proinflammatory cytokines in the kidney Proinflammatory cytokines were measured in kidney tissue from both of normal miceand CD25-depleted mice by ELISA Renal IL-6 and TNF120572were decreased in the BV-treated group (Cis + BV) compared with the PBS-treatedgroup (Cis) However there was no difference between the BV-treated group and PBS-treated group in CD25-depleted mice The valuesshown indicate the mean plusmn SEM lowast119875 lt 005 versus Cis

Cont BV

Cis Cis + BV

(a)

CisCis + BVBV

NS

5

4

3

2

1

0Cont

lowastlowast

Foxp

3-po

sitiv

e cel

ls10

HPF

(b)

Figure 7The number of Foxp3-positive cells in the kidney Tregs in the kidney were detected by EGFP signal under the confocal microscopeimaging Foxp3-positive cells were indicated by arrows (a)There was no difference in the number of Foxp3 positive cells between PBS-treatedgroup and BV-treated group before cisplatin administration However Foxp3-positive cells in the kidney increased 6 hours after cisplatinadministration In BV-pretreated group after cisplatin administration the number of Foxp3-positive cells in the kidney was significantlyhigher than in PBS-pretreated group (b) All data are presented as the mean plusmn SEM NS 119875 gt 005 lowast119875 lt 005 versus cont and

119875 lt 005

versus Cis as determined by one-way ANOVA followed by Tukeyrsquos multiple comparison test

4 Discussion

The development of side effects after cisplatin treatment iscommon and acute renal failure may develop after expo-sure to a single dose [2] Acute renal failure as inducedby cisplatin leads to the generation of danger signals bytubular cells resulting in inflammation mediated by T lym-phocytes and NK cells [15] Tregs are lymphocytes with

immunosuppressive properties and play a pivotal role inthe maintenance of immune tolerance [5] Previous studieshave reported that the anti-inflammatory actions of Tregscan protect against renal injury [16ndash18] Our recent studydemonstrated that CD4+CD25+ Tregs attenuate cisplatin-induced nephrotoxicity in vivo [8] Thus we evaluated theeffects of many candidate agents on CD4+CD25+Foxp3+Tregs in vitro and found that BV potently increases the

8 Evidence-Based Complementary and Alternative Medicine

Cont

(a)

Cis

(b)

Cis + BV

(c)

Cis Cis + BVCis Cis + BV

NS40

30

20

10

0Cont

lowastlowastlowast

F48

0-po

sitiv

e cel

lsfie

ld

CD25-depleted mice

(d)

Cis

CD25-depleted

(e)

Cis + BV

CD25-depleted

(f)

Figure 8 Macrophage infiltration into kidney Macrophage accumulation in the kidney was detected by immunostaining for F480-positivecells at 72 h after cisplatin administration The number of F480-positive cells in each section was counted in 10 fields per slide at an originalmagnification of 200xThe infiltration in BV-treatedmice was compared with those of PBS-treatedmice and CD25-depletedmiceThe valuesshown indicate the mean plusmn SEM lowastlowastlowast119875 lt 0001 versus Cis

Cis

Cis

Cont

+ BV

CisCis

Cis

Cis

Cont

Cont

Cont

(c)

(b)

(a)

Tum

or w

eigh

t (m

g)

+ BV

6000500040003000

3000

2000

2000

10000

50

0

85 11 14 17 20Day

NS

NS

Cis + BV

Cis + BV

Tum

or si

ze (m

m3 )

Figure 9 Influence of BV on the antitumor effect of cisplatin All mice received injections of EL4 lymphoma cells (2 times 10601mL) sc intothe right flank BV (1mgkg) was injected once per day for 5 days from day 5 of tumor inoculation (119899 = 8) Cisplatin was injected 3 times (ondays 11 14 and 17) at a concentration of 5mgkg All mice were sacrificed at day 20 and the tumors were photographed (a) The tumor sizewas measured every three days after BV injection for 16 days After sacrifice the tumors were fully separated from the mice and the size (b)and weight (c) of the tumors were measured All data are presented as the mean plusmn SEM lowastlowastlowast119875 lt 0001 versus cont NS 119875 gt 005 versus Cisas determined by one-way ANOVA followed by Tukeyrsquos test

Evidence-Based Complementary and Alternative Medicine 9

population of CD4+CD25+Foxp3+ Tregs Moreover it wasshown that BV has remarkable renoprotective effects inNZBW lupus mice [12] Based on these lines of evidencewe hypothesized that BV may have therapeutic effects oncisplatin-induced nephrotoxicity through the upregulation ofTregs

In this study we first showed that BV indeed increases thepopulation of CD4+CD25+Foxp3+ Tregs in isolated mousesplenocytes in vitro and in vivo before cisplatin adminis-tration (Figure 1) corroborating the results of our previousstudy [12]Three days after cisplatin administration howeverthe population of Tregs markedly increased in splenocytesfrom both of BV- and PBS-injected mice with no significantdifference in Treg numbers between the two groups Wefurther examined how many Tregs are migrated into thekidney after cisplatin administration We found that moreTregs were migrated into the kidney 6 hours after cisplatininjection in BV-pretreated mice than in PBS-pretreated mice(Figure 7) Next we found that BV has protective effectson cisplatin-induced renal dysfunction and injury SerumBUN and creatinine are typical indicators of renal functionand an increase in these values could indicate renal failure[19] Our data showed that cisplatin treatment significantlyincreased the BUN and serum creatinine levels in the saline-treated control group and that this increase was preventedby BV treatment The histological analysis of the degree ofrenal tubular injury (epithelial necrosis) also suggests thatBV can effectively prevent the structural damage of renaltissuesThe degree of macrophage infiltration into the kidneyis a major parameter used to predict the progression ofrenal disease and activated macrophages express severalinflammatory mediators [20] including TNF-120572 and IL-6which play important roles in the pathogenesis of cisplatin-induced renal injury [21 22] Our data showed that BV treat-ment in cisplatin-injected mice decreased the expression ofproinflammatory cytokines as well asmacrophage infiltrationin the kidney Because renal toxicity is themain cause of deathin cisplatin-injected mice [23] we evaluated the survival ofBV- or saline-treated mice after cisplatin administrationTheBV-treated mice showed a higher survival rate than did thesaline-treated control mice Collectively these results mightsuggest that Tregs in spleen increase in number after BVtreatment before cisplatin administration which cause moremigration of Tregs into the kidney in the early phase ofcisplatin-induced nephrotoxicity Such action might lead tothe protective effects of BV on cisplatin-induced nephrotoxicevents as shown in the present study This is supported byour previous study [8] showing that adoptive transfer of Tregsto mice (iv) caused increased migration of Tregs into thekidney 6 h after cisplatin administration and then attenuatedthe renal injuryThe renoprotective effects of BV in cisplatin-injectedmice in the present data are comparable to the resultsshown in Treg-transfused mice [8]

Since BV is known to have direct anti-inflammatory andantitumor effects [24 25] we cannot rule out the possibleinvolvement of Tregs-independent mechanisms of BV in thepresent results However we demonstrated that depletionof Tregs using anti-CD25 antibody injection completelyinhibited the BV effects on cisplatin-induced nephrotoxic

eventsTherefore our findings strongly suggest that BV couldprevent cisplatin-induced severe side effects in the kidney atleast partly by modulating the Tregs

Acknowledgment

This work was supported by the National Research Founda-tion of Korea (NRF)Grant funded by the Korean government(MEST) (no 2011-006220)

References

[1] K B Meyer and N E Madias ldquoCisplatin nephrotoxicityrdquoMineral and Electrolyte Metabolism vol 20 no 4 pp 201ndash2131994

[2] A H Lau ldquoApoptosis induced by cisplatin nephrotoxic injuryrdquoKidney International vol 56 no 4 pp 1295ndash1298 1999

[3] R Safirstein J Winston M Goldstein et al ldquoCisplatin nephro-toxicityrdquo American Journal of Kidney Diseases vol 8 no 5 pp356ndash367 1986

[4] J D Fontenot M A Gavin and A Y Rudensky ldquoFoxp3 pro-grams the development and function ofCD4+CD25+ regulatoryT cellsrdquo Nature Immunology vol 4 no 4 pp 330ndash336 2003

[5] G Raimondi M S Turner A W Thomson and P A MorelldquoNaturally occurring regulatory T cells recent insights in healthand diseaserdquo Critical Reviews in Immunology vol 27 no 1 pp61ndash95 2007

[6] S Sakaguchi M Ono R Setoguchi et al ldquoFoxp3+CD25+CD4+natural regulatory T cells in dominant self-tolerance andautoimmune diseaserdquo Immunological Reviews vol 212 no 1 pp8ndash27 2006

[7] S Sakaguchi N Sakaguchi M Asano M Itoh and M TodaldquoImmunologic self-tolerance maintained by activated T cellsexpressing IL- 2 receptor 120572-chains (CD25) Breakdown of asingle mechanism of self- tolerance causes various autoimmunediseasesrdquo Journal of Immunology vol 155 no 3 pp 1151ndash11641995

[8] H Lee D Nho H S Chung et al ldquoCD4+CD25+ regulatoryT cells attenuate cisplatin-induced nephrotoxicity in micerdquoKidney International vol 78 no 11 pp 1100ndash1109 2010

[9] M H Park M S Choi D H Kwak et al ldquoAnti-cancer effect ofbee venomin prostate cancer cells through activation of caspasepathway via inactivation of NF-120581Brdquo Prostate vol 71 no 8 pp801ndash812 2011

[10] S Y Yoon Y B Kwon H W Kim et al ldquoBee venominjection produces a peripheral anti-inflammatory effect byactivation of a nitric oxide-dependent spinocoeruleus pathwayrdquoNeuroscience Letters vol 430 no 2 pp 163ndash168 2008

[11] Y B KwonHWKim TWHamet al ldquoThe anti-inflammatoryeffect of bee venom stimulation in a mouse air pouch modelis mediated by adrenal medullary activityrdquo Journal of Neuroen-docrinology vol 15 no 1 pp 93ndash96 2003

[12] H Lee E J Lee H Kim et al ldquoBee venom-associated Th1Th2immunoglobulin class switching results in immune toleranceof NZBW F1 murine lupus nephritisrdquo American Journal ofNephrology vol 34 no 2 pp 163ndash172 2011

[13] J Megyesi R L Safirstein and P M Price ldquoInduction ofp21WAF1CIP1SDI1 in kidney tubule cells affects the courseof cisplatin-induced acute renal failurerdquoThe Journal of ClinicalInvestigation vol 101 no 4 pp 777ndash782 1998

10 Evidence-Based Complementary and Alternative Medicine

[14] K K Meldrum P Metcalfe J A Leslie R Misseri K L Hileand D R Meldrum ldquoTNF-120572 neutralization decreases nuclearfactor-120581B activation and apoptosis during renal obstructionrdquoJournal of Surgical Research vol 131 no 2 pp 182ndash188 2006

[15] A Linkermann N Himmerkus L Rolver et al ldquoRenal tubularFas ligandmediates fratricide in cisplatin-induced acute kidneyfailurerdquo Kidney International vol 79 no 2 pp 169ndash178 2011

[16] T Muthukumar D Dadhania R Ding et al ldquoMessenger RNAfor FOXP3 in the urine of renal-allograft recipientsrdquo The NewEngland Journal of Medicine vol 353 no 22 pp 2342ndash23512005

[17] G R Kinsey R Sharma L Huang et al ldquoRegulatory Tcells suppress innate immunity in kidney ischemia-reperfusioninjuryrdquo Journal of the American Society of Nephrology vol 20no 8 pp 1744ndash1753 2009

[18] S Bunnag K Allanach G S Jhangri et al ldquoFOXP3 expressionin human kidney transplant biopsies is associatedwith rejectionand time post transplant but not with favorable outcomesrdquoAmerican Journal of Transplantation vol 8 no 7 pp 1423ndash14332008

[19] H Kim G Lee H Lee M Shin M Hong and H Bae ldquoEffectsof Scutellaria barbata on cisplatin induced nephrotoxicity inmicerdquoMolecular and Cellular Toxicology vol 6 no 3 pp 255ndash259 2010

[20] Y Wang Q Cai G Zheng et al ldquoBy homing to the kidneyactivated macrophages potently exacerbate renal injuryrdquo TheAmerican Journal of Pathology vol 172 no 6 pp 1491ndash14992008

[21] S Faubel E C Lewis L Reznikov et al ldquoCisplatin-inducedacute renal failure is associatedwith an increase in the cytokinesinterleukin (IL)-1120573 IL-18 IL-6 and neutrophil infiltrationin the kidneyrdquo Journal of Pharmacology and ExperimentalTherapeutics vol 322 no 1 pp 8ndash15 2007

[22] G Ramesh and W Brian Reeves ldquoTNF-120572mediates chemokineand cytokine expression and renal injury in cisplatin nephro-toxicityrdquoThe Journal of Clinical Investigation vol 110 no 6 pp835ndash842 2002

[23] F Ries and J Klastersky ldquoNephrotoxicity induced by cancerchemotherapy with special emphasis on cisplatin toxicityrdquoAmerican Journal of Kidney Diseases vol 8 no 5 pp 368ndash3791986

[24] N Orsol ldquoBee venom in cancer therapyrdquo Cancer andMetastasisReviews vol 31 no 1-2 pp 173ndash194 2012

[25] N Orsolic L Sver S Verstovsek S Terzic and I BasicldquoInhibition of mammary carcinoma cell proliferation in vitroand tumor growth in vivo by bee venomrdquo Toxicon vol 41 no 7pp 861ndash870 2003

Evidence-Based Complementary and Alternative Medicine 5

0 2412 4836 7260 9684 120

NS

108

150

100

50

0

Perc

ent s

urvi

val

CisCis + BV

Figure 4 Survival test in CD25-depleted mice After CD25 deple-tion mice were treated with 25mg of cisplatinkg intraperitoneallyand followed up for up to 114 h to produce survival curves At 114 hall of the control mice and BV-treated mice had expired (NS 119875 gt005 versus cont 119899 = 8)

every three days (6 times in total) using calipers The volumeof the tumor was calculated as length times width22

211 Statistical Analysis All results are expressed as themeans plusmn SEMThe data were analyzed using two-tailed 119905-testor one-way ANOVA with Tukeyrsquos post hoc test Differenceswere considered significant at 119875 lt 005

3 Results

31 Population of Tregs in Spleen To confirm the immune-modulating effect of BV we isolated splenocytes from sac-rificed Foxp3EGFP mice We treated BV (1120583gmL) or PBS toincubated cells for 3 days The CD4+CD25+Foxp3+ cell pop-ulation was significantly increased in the BV-treated groupcompared to the PBS-treated group in vitro (cont 362 plusmn 017BV 536 plusmn 024 119899 = 5 resp) (Figures 1(a) and 1(b)) We alsoexamined the population of the CD4+CD25+Foxp3+ cells inspleen in vivo The similar increase in Tregs numbers wasobserved in BV-injected mice before cisplatin administration(cont 384 plusmn 031 BV 548 plusmn 025 119899 = 3 resp) (Figure 1(c))Three days after cisplatin administration however the pop-ulation of Tregs markedly increased in splenocytes fromboth of BV- and PBS-injected mice There was no significantdifference in Treg numbers between BV- and PBS-treatedanimals (cont 1369 plusmn 052 BV 1381 plusmn 038 119899 = 4 resp)

32 Survival after Administration of High-Dose CisplatinAfter cisplatin injection (25mgkg ip) the mice weremonitored for 10 days All controlmice had died by 134 h aftercisplatin administration whereas 7 of the 16 BV-treated micesurvived The BV-treated mice that died at early timepointsexpressed features similar to those of the saline-treated miceHowever all of the BV-treated mice that survived to 134 hsurvived until the end of the experiment (Figure 2)

33 Protective Effects of BV on Cisplatin-Induced Nephrotoxic-ity Mice were treated with intraperitoneal (ip) injections ofcisplatin (25mgkg) after BV treatment Blood urea nitrogen(BUN) and serum creatinine levels are common indicatorsof renal function Blood samples were collected at 24 48and 72 h after cisplatin injection and the levels of bloodurea nitrogen (BUN) and creatinine were measured in the48 and 72 h samples Creatinine and BUN were significantlyincreased in the control group compared with BV-treatedgroup (creatinine 48 h cont 016 plusmn 001mgdL Cis 083 plusmn005mgdL Cis + BV 046 plusmn 006mgdL creatinine 72 hcont 016 plusmn 001mgdL Cis 363 plusmn 014mgdL Cis + BV218 plusmn 034mgdL BUN 48 h Cont 2896 plusmn 106mgdL Cis1091plusmn1400mgdL Cis + BV 5538plusmn535mgdL BUN 72 hcont 2259 plusmn 081mgdL Cis 2675 plusmn 1073mgdL Cis + BV1938 plusmn 1812mgdL)

To investigate the effects of BV on cisplatin-induced renaldamage we stained kidney tissue sections with hematoxylinand eosin (HampE) Pretreatmentwith BV significantly reducedthe tubular injury score compared to the score in sectionsfrom control mice treated with cisplatin alone (renal damagescore Cis 2667 plusmn 1556 Cis + BV 1556 plusmn 02318) (Figure 3)

34 Survival in CD25-Depleted Mice After CD25 depletionall mice received an injection of cisplatin (25mgkg ip) andwere evaluated for 114 h All mice in both the control and BV-treated groups had died at 114 h (Figure 4)

35 The Effects of BV in CD25-Depleted Mice To confirmthat the effect of BV on cisplatin-induced nephrotoxicity wasmediated by Tregs we tested the combination treatment ina CD4+CD25+ Treg cell depletion model in vivo by treatingmice with an anti-CD25 antibody (01mgmouse ip) Wemeasured the amount of creatinine and BUN in the serumThese results demonstrated that Treg depletion abolished BV-mediated antinephrotoxic effects (creatinine 48 h Cis 066 plusmn006mgdL Cis + BV 066 plusmn 008mgdL creatinine 72 h Cis257 plusmn 027mgdL Cis + BV 242 plusmn 028mgdL BUN 48 hCis 6113 plusmn 358mgdL Cis + BV 6147 plusmn 473mgdL BUN72 h Cis 2252 plusmn 1116mgdL Cis + BV 2197 plusmn 749mgdL)In addition histological examination revealed that BV hadno protective effect on kidney tissues in CD25-depleted mice(renal damage score Cis 283 plusmn 023 Cis + BV 287 plusmn 019)(Figure 5)

36 Proinflammatory Cytokines in Kidney To evaluate theproinflammatory molecules generated by cisplatin-inducedrenal injury the levels of TNF-120572 and IL-6 proteins in thekidney were measured at 72 h after cisplatin administration

Only cisplatin-treated mice exhibited increased levels ofTNF-120572 and IL-6 at 72 h after cisplatin injection HoweverBV-treated mice showed significantly lower levels of theseproinflammatory cytokines than did control mice while Tregdepletion caused increases in the levels of these cytokines inboth the BV-treated and nontreated groups (Figure 6)

37 Migration of Tregs into Kidney Confocal microscopy wasused with kidney samples obtained from Foxp3EGFP mice

6 Evidence-Based Complementary and Alternative Medicine

2

1

0

3

Cis

NS

NSCrea

tinin

e (m

gdL

)

Cis + BV Cis Cis + BV

48 h 72 h

(a)

250

200

150

100

50

0

NS

NSBUN

(mg

dL)

Cis Cis + BV Cis Cis + BV

48 h 72 h

(b)

Cis Cis + BV

(c)

2

1

0

4

3Tu

bula

r dam

age s

core

NS

Cis Cis + BV

(d)

Figure 5 The inefficacy of bee venom in CD25-depleted mice All mice were treated with anti-CD25 antibody (01mgmouse ip) twicebefore and after BV injection Nephrotoxicity was induced by cisplatin (25mgkg ip) Blood was obtained at 24 48 and 72 h after cisplatininjection (119899 = 12) Renal dysfunction was reflected by the levels of creatinine at 48 h 72 h (a) and BUN at 48 h 72 h (b) Kidney sections fromCD25-depleted mice were stained with HampE at 72 h after cisplatin injection PBS treatment alone (cont) PBS and cisplatin treatments (Cis)BV and cisplatin (Cis + BV) (400x bar = 50 120583m) (c) Tubular injury damage was scored from 0ndash4 (0 none 1 lt10 2 10ndash25 3 25ndash75 4gt75) (d) The values shown indicate the mean plusmn SEM NS 119875 gt 005 versus Cis

that were killed at 6 hours after cisplatin injection MoreFoxp3-positive cells were observed in the kidney in BV-pretreated group compared to the PBS-treated group 6 hoursafter cisplatin injection (Figure 7) suggesting the increasedmigration of Tregs into the kidney in the early phase ofnephrotoxic process caused by cisplatin

38 Effects of BV on Cisplatin-Induced Macrophage Infil-tration Macrophages were counted in the kidney sec-tions at 72 h after cisplatin administration F480-positivemacrophages were rarely detected in BV-treated micecompared with control mice whereas more infiltrationof macrophages was measured in CD25-depleted mice(Figure 8)

39 The Influence of BV on Cisplatin-Induced AnticancerEffects To determine whether the preadministration of BVinfluenced the anticancer effects of cisplatin the experimentwas repeated in a tumor-bearing (EL4 lymphoma) mouse

model with a low dose of cisplatin (5mgkg) Cisplatin wasinjected 3 times at a concentration of 5mgkgThemice wereweighed and the size of the tumor was measured over thewhole experimental period All mice were sacrificed on day21 and the separated tumors were collected

Tumor size in all groups of mice was gradually increasedbefore cisplatin administration (until day 11 in Figure 9(b))There was no significant difference in tumor size beforecisplatin administration between BV and PBS groups Inaddition irrespective of BV pretreatment repeated admin-istrations of cisplatin exerted very potent anticancer effects(a remarkable reduction in tumor size) in EL4 tumor modelresulting in no difference in tumor size between PBS- andBV-preinjected groups (Figures 9(a)ndash9(c)) In contrast tumorsize in controlmice (tumormodel without cisplatin) continu-ously increased after cisplatin administrations (Figure 9(b))The amounts of creatinine and BUN were within the normalrange in all groups (data not shown) These results suggestthat BV has not a big influence on the anticancer effects ofcisplatin

Evidence-Based Complementary and Alternative Medicine 7

5

4

3

2

1

0

TNF

(ng

mg)

lowast NS

Cis CisCis + BV Cis + BVCD25-depleted mice

(a)

120

100

80

60

40

20

0

lowast

NS

Cis CisCis + BV Cis + BV

CD25-depleted mice

IL-6

(pg

mg)

(b)

Figure 6 Proinflammatory cytokines in the kidney Proinflammatory cytokines were measured in kidney tissue from both of normal miceand CD25-depleted mice by ELISA Renal IL-6 and TNF120572were decreased in the BV-treated group (Cis + BV) compared with the PBS-treatedgroup (Cis) However there was no difference between the BV-treated group and PBS-treated group in CD25-depleted mice The valuesshown indicate the mean plusmn SEM lowast119875 lt 005 versus Cis

Cont BV

Cis Cis + BV

(a)

CisCis + BVBV

NS

5

4

3

2

1

0Cont

lowastlowast

Foxp

3-po

sitiv

e cel

ls10

HPF

(b)

Figure 7The number of Foxp3-positive cells in the kidney Tregs in the kidney were detected by EGFP signal under the confocal microscopeimaging Foxp3-positive cells were indicated by arrows (a)There was no difference in the number of Foxp3 positive cells between PBS-treatedgroup and BV-treated group before cisplatin administration However Foxp3-positive cells in the kidney increased 6 hours after cisplatinadministration In BV-pretreated group after cisplatin administration the number of Foxp3-positive cells in the kidney was significantlyhigher than in PBS-pretreated group (b) All data are presented as the mean plusmn SEM NS 119875 gt 005 lowast119875 lt 005 versus cont and

119875 lt 005

versus Cis as determined by one-way ANOVA followed by Tukeyrsquos multiple comparison test

4 Discussion

The development of side effects after cisplatin treatment iscommon and acute renal failure may develop after expo-sure to a single dose [2] Acute renal failure as inducedby cisplatin leads to the generation of danger signals bytubular cells resulting in inflammation mediated by T lym-phocytes and NK cells [15] Tregs are lymphocytes with

immunosuppressive properties and play a pivotal role inthe maintenance of immune tolerance [5] Previous studieshave reported that the anti-inflammatory actions of Tregscan protect against renal injury [16ndash18] Our recent studydemonstrated that CD4+CD25+ Tregs attenuate cisplatin-induced nephrotoxicity in vivo [8] Thus we evaluated theeffects of many candidate agents on CD4+CD25+Foxp3+Tregs in vitro and found that BV potently increases the

8 Evidence-Based Complementary and Alternative Medicine

Cont

(a)

Cis

(b)

Cis + BV

(c)

Cis Cis + BVCis Cis + BV

NS40

30

20

10

0Cont

lowastlowastlowast

F48

0-po

sitiv

e cel

lsfie

ld

CD25-depleted mice

(d)

Cis

CD25-depleted

(e)

Cis + BV

CD25-depleted

(f)

Figure 8 Macrophage infiltration into kidney Macrophage accumulation in the kidney was detected by immunostaining for F480-positivecells at 72 h after cisplatin administration The number of F480-positive cells in each section was counted in 10 fields per slide at an originalmagnification of 200xThe infiltration in BV-treatedmice was compared with those of PBS-treatedmice and CD25-depletedmiceThe valuesshown indicate the mean plusmn SEM lowastlowastlowast119875 lt 0001 versus Cis

Cis

Cis

Cont

+ BV

CisCis

Cis

Cis

Cont

Cont

Cont

(c)

(b)

(a)

Tum

or w

eigh

t (m

g)

+ BV

6000500040003000

3000

2000

2000

10000

50

0

85 11 14 17 20Day

NS

NS

Cis + BV

Cis + BV

Tum

or si

ze (m

m3 )

Figure 9 Influence of BV on the antitumor effect of cisplatin All mice received injections of EL4 lymphoma cells (2 times 10601mL) sc intothe right flank BV (1mgkg) was injected once per day for 5 days from day 5 of tumor inoculation (119899 = 8) Cisplatin was injected 3 times (ondays 11 14 and 17) at a concentration of 5mgkg All mice were sacrificed at day 20 and the tumors were photographed (a) The tumor sizewas measured every three days after BV injection for 16 days After sacrifice the tumors were fully separated from the mice and the size (b)and weight (c) of the tumors were measured All data are presented as the mean plusmn SEM lowastlowastlowast119875 lt 0001 versus cont NS 119875 gt 005 versus Cisas determined by one-way ANOVA followed by Tukeyrsquos test

Evidence-Based Complementary and Alternative Medicine 9

population of CD4+CD25+Foxp3+ Tregs Moreover it wasshown that BV has remarkable renoprotective effects inNZBW lupus mice [12] Based on these lines of evidencewe hypothesized that BV may have therapeutic effects oncisplatin-induced nephrotoxicity through the upregulation ofTregs

In this study we first showed that BV indeed increases thepopulation of CD4+CD25+Foxp3+ Tregs in isolated mousesplenocytes in vitro and in vivo before cisplatin adminis-tration (Figure 1) corroborating the results of our previousstudy [12]Three days after cisplatin administration howeverthe population of Tregs markedly increased in splenocytesfrom both of BV- and PBS-injected mice with no significantdifference in Treg numbers between the two groups Wefurther examined how many Tregs are migrated into thekidney after cisplatin administration We found that moreTregs were migrated into the kidney 6 hours after cisplatininjection in BV-pretreated mice than in PBS-pretreated mice(Figure 7) Next we found that BV has protective effectson cisplatin-induced renal dysfunction and injury SerumBUN and creatinine are typical indicators of renal functionand an increase in these values could indicate renal failure[19] Our data showed that cisplatin treatment significantlyincreased the BUN and serum creatinine levels in the saline-treated control group and that this increase was preventedby BV treatment The histological analysis of the degree ofrenal tubular injury (epithelial necrosis) also suggests thatBV can effectively prevent the structural damage of renaltissuesThe degree of macrophage infiltration into the kidneyis a major parameter used to predict the progression ofrenal disease and activated macrophages express severalinflammatory mediators [20] including TNF-120572 and IL-6which play important roles in the pathogenesis of cisplatin-induced renal injury [21 22] Our data showed that BV treat-ment in cisplatin-injected mice decreased the expression ofproinflammatory cytokines as well asmacrophage infiltrationin the kidney Because renal toxicity is themain cause of deathin cisplatin-injected mice [23] we evaluated the survival ofBV- or saline-treated mice after cisplatin administrationTheBV-treated mice showed a higher survival rate than did thesaline-treated control mice Collectively these results mightsuggest that Tregs in spleen increase in number after BVtreatment before cisplatin administration which cause moremigration of Tregs into the kidney in the early phase ofcisplatin-induced nephrotoxicity Such action might lead tothe protective effects of BV on cisplatin-induced nephrotoxicevents as shown in the present study This is supported byour previous study [8] showing that adoptive transfer of Tregsto mice (iv) caused increased migration of Tregs into thekidney 6 h after cisplatin administration and then attenuatedthe renal injuryThe renoprotective effects of BV in cisplatin-injectedmice in the present data are comparable to the resultsshown in Treg-transfused mice [8]

Since BV is known to have direct anti-inflammatory andantitumor effects [24 25] we cannot rule out the possibleinvolvement of Tregs-independent mechanisms of BV in thepresent results However we demonstrated that depletionof Tregs using anti-CD25 antibody injection completelyinhibited the BV effects on cisplatin-induced nephrotoxic

eventsTherefore our findings strongly suggest that BV couldprevent cisplatin-induced severe side effects in the kidney atleast partly by modulating the Tregs

Acknowledgment

This work was supported by the National Research Founda-tion of Korea (NRF)Grant funded by the Korean government(MEST) (no 2011-006220)

References

[1] K B Meyer and N E Madias ldquoCisplatin nephrotoxicityrdquoMineral and Electrolyte Metabolism vol 20 no 4 pp 201ndash2131994

[2] A H Lau ldquoApoptosis induced by cisplatin nephrotoxic injuryrdquoKidney International vol 56 no 4 pp 1295ndash1298 1999

[3] R Safirstein J Winston M Goldstein et al ldquoCisplatin nephro-toxicityrdquo American Journal of Kidney Diseases vol 8 no 5 pp356ndash367 1986

[4] J D Fontenot M A Gavin and A Y Rudensky ldquoFoxp3 pro-grams the development and function ofCD4+CD25+ regulatoryT cellsrdquo Nature Immunology vol 4 no 4 pp 330ndash336 2003

[5] G Raimondi M S Turner A W Thomson and P A MorelldquoNaturally occurring regulatory T cells recent insights in healthand diseaserdquo Critical Reviews in Immunology vol 27 no 1 pp61ndash95 2007

[6] S Sakaguchi M Ono R Setoguchi et al ldquoFoxp3+CD25+CD4+natural regulatory T cells in dominant self-tolerance andautoimmune diseaserdquo Immunological Reviews vol 212 no 1 pp8ndash27 2006

[7] S Sakaguchi N Sakaguchi M Asano M Itoh and M TodaldquoImmunologic self-tolerance maintained by activated T cellsexpressing IL- 2 receptor 120572-chains (CD25) Breakdown of asingle mechanism of self- tolerance causes various autoimmunediseasesrdquo Journal of Immunology vol 155 no 3 pp 1151ndash11641995

[8] H Lee D Nho H S Chung et al ldquoCD4+CD25+ regulatoryT cells attenuate cisplatin-induced nephrotoxicity in micerdquoKidney International vol 78 no 11 pp 1100ndash1109 2010

[9] M H Park M S Choi D H Kwak et al ldquoAnti-cancer effect ofbee venomin prostate cancer cells through activation of caspasepathway via inactivation of NF-120581Brdquo Prostate vol 71 no 8 pp801ndash812 2011

[10] S Y Yoon Y B Kwon H W Kim et al ldquoBee venominjection produces a peripheral anti-inflammatory effect byactivation of a nitric oxide-dependent spinocoeruleus pathwayrdquoNeuroscience Letters vol 430 no 2 pp 163ndash168 2008

[11] Y B KwonHWKim TWHamet al ldquoThe anti-inflammatoryeffect of bee venom stimulation in a mouse air pouch modelis mediated by adrenal medullary activityrdquo Journal of Neuroen-docrinology vol 15 no 1 pp 93ndash96 2003

[12] H Lee E J Lee H Kim et al ldquoBee venom-associated Th1Th2immunoglobulin class switching results in immune toleranceof NZBW F1 murine lupus nephritisrdquo American Journal ofNephrology vol 34 no 2 pp 163ndash172 2011

[13] J Megyesi R L Safirstein and P M Price ldquoInduction ofp21WAF1CIP1SDI1 in kidney tubule cells affects the courseof cisplatin-induced acute renal failurerdquoThe Journal of ClinicalInvestigation vol 101 no 4 pp 777ndash782 1998

10 Evidence-Based Complementary and Alternative Medicine

[14] K K Meldrum P Metcalfe J A Leslie R Misseri K L Hileand D R Meldrum ldquoTNF-120572 neutralization decreases nuclearfactor-120581B activation and apoptosis during renal obstructionrdquoJournal of Surgical Research vol 131 no 2 pp 182ndash188 2006

[15] A Linkermann N Himmerkus L Rolver et al ldquoRenal tubularFas ligandmediates fratricide in cisplatin-induced acute kidneyfailurerdquo Kidney International vol 79 no 2 pp 169ndash178 2011

[16] T Muthukumar D Dadhania R Ding et al ldquoMessenger RNAfor FOXP3 in the urine of renal-allograft recipientsrdquo The NewEngland Journal of Medicine vol 353 no 22 pp 2342ndash23512005

[17] G R Kinsey R Sharma L Huang et al ldquoRegulatory Tcells suppress innate immunity in kidney ischemia-reperfusioninjuryrdquo Journal of the American Society of Nephrology vol 20no 8 pp 1744ndash1753 2009

[18] S Bunnag K Allanach G S Jhangri et al ldquoFOXP3 expressionin human kidney transplant biopsies is associatedwith rejectionand time post transplant but not with favorable outcomesrdquoAmerican Journal of Transplantation vol 8 no 7 pp 1423ndash14332008

[19] H Kim G Lee H Lee M Shin M Hong and H Bae ldquoEffectsof Scutellaria barbata on cisplatin induced nephrotoxicity inmicerdquoMolecular and Cellular Toxicology vol 6 no 3 pp 255ndash259 2010

[20] Y Wang Q Cai G Zheng et al ldquoBy homing to the kidneyactivated macrophages potently exacerbate renal injuryrdquo TheAmerican Journal of Pathology vol 172 no 6 pp 1491ndash14992008

[21] S Faubel E C Lewis L Reznikov et al ldquoCisplatin-inducedacute renal failure is associatedwith an increase in the cytokinesinterleukin (IL)-1120573 IL-18 IL-6 and neutrophil infiltrationin the kidneyrdquo Journal of Pharmacology and ExperimentalTherapeutics vol 322 no 1 pp 8ndash15 2007

[22] G Ramesh and W Brian Reeves ldquoTNF-120572mediates chemokineand cytokine expression and renal injury in cisplatin nephro-toxicityrdquoThe Journal of Clinical Investigation vol 110 no 6 pp835ndash842 2002

[23] F Ries and J Klastersky ldquoNephrotoxicity induced by cancerchemotherapy with special emphasis on cisplatin toxicityrdquoAmerican Journal of Kidney Diseases vol 8 no 5 pp 368ndash3791986

[24] N Orsol ldquoBee venom in cancer therapyrdquo Cancer andMetastasisReviews vol 31 no 1-2 pp 173ndash194 2012

[25] N Orsolic L Sver S Verstovsek S Terzic and I BasicldquoInhibition of mammary carcinoma cell proliferation in vitroand tumor growth in vivo by bee venomrdquo Toxicon vol 41 no 7pp 861ndash870 2003

6 Evidence-Based Complementary and Alternative Medicine

2

1

0

3

Cis

NS

NSCrea

tinin

e (m

gdL

)

Cis + BV Cis Cis + BV

48 h 72 h

(a)

250

200

150

100

50

0

NS

NSBUN

(mg

dL)

Cis Cis + BV Cis Cis + BV

48 h 72 h

(b)

Cis Cis + BV

(c)

2

1

0

4

3Tu

bula

r dam

age s

core

NS

Cis Cis + BV

(d)

Figure 5 The inefficacy of bee venom in CD25-depleted mice All mice were treated with anti-CD25 antibody (01mgmouse ip) twicebefore and after BV injection Nephrotoxicity was induced by cisplatin (25mgkg ip) Blood was obtained at 24 48 and 72 h after cisplatininjection (119899 = 12) Renal dysfunction was reflected by the levels of creatinine at 48 h 72 h (a) and BUN at 48 h 72 h (b) Kidney sections fromCD25-depleted mice were stained with HampE at 72 h after cisplatin injection PBS treatment alone (cont) PBS and cisplatin treatments (Cis)BV and cisplatin (Cis + BV) (400x bar = 50 120583m) (c) Tubular injury damage was scored from 0ndash4 (0 none 1 lt10 2 10ndash25 3 25ndash75 4gt75) (d) The values shown indicate the mean plusmn SEM NS 119875 gt 005 versus Cis

that were killed at 6 hours after cisplatin injection MoreFoxp3-positive cells were observed in the kidney in BV-pretreated group compared to the PBS-treated group 6 hoursafter cisplatin injection (Figure 7) suggesting the increasedmigration of Tregs into the kidney in the early phase ofnephrotoxic process caused by cisplatin

38 Effects of BV on Cisplatin-Induced Macrophage Infil-tration Macrophages were counted in the kidney sec-tions at 72 h after cisplatin administration F480-positivemacrophages were rarely detected in BV-treated micecompared with control mice whereas more infiltrationof macrophages was measured in CD25-depleted mice(Figure 8)

39 The Influence of BV on Cisplatin-Induced AnticancerEffects To determine whether the preadministration of BVinfluenced the anticancer effects of cisplatin the experimentwas repeated in a tumor-bearing (EL4 lymphoma) mouse

model with a low dose of cisplatin (5mgkg) Cisplatin wasinjected 3 times at a concentration of 5mgkgThemice wereweighed and the size of the tumor was measured over thewhole experimental period All mice were sacrificed on day21 and the separated tumors were collected

Tumor size in all groups of mice was gradually increasedbefore cisplatin administration (until day 11 in Figure 9(b))There was no significant difference in tumor size beforecisplatin administration between BV and PBS groups Inaddition irrespective of BV pretreatment repeated admin-istrations of cisplatin exerted very potent anticancer effects(a remarkable reduction in tumor size) in EL4 tumor modelresulting in no difference in tumor size between PBS- andBV-preinjected groups (Figures 9(a)ndash9(c)) In contrast tumorsize in controlmice (tumormodel without cisplatin) continu-ously increased after cisplatin administrations (Figure 9(b))The amounts of creatinine and BUN were within the normalrange in all groups (data not shown) These results suggestthat BV has not a big influence on the anticancer effects ofcisplatin

Evidence-Based Complementary and Alternative Medicine 7

5

4

3

2

1

0

TNF

(ng

mg)

lowast NS

Cis CisCis + BV Cis + BVCD25-depleted mice

(a)

120

100

80

60

40

20

0

lowast

NS

Cis CisCis + BV Cis + BV

CD25-depleted mice

IL-6

(pg

mg)

(b)

Figure 6 Proinflammatory cytokines in the kidney Proinflammatory cytokines were measured in kidney tissue from both of normal miceand CD25-depleted mice by ELISA Renal IL-6 and TNF120572were decreased in the BV-treated group (Cis + BV) compared with the PBS-treatedgroup (Cis) However there was no difference between the BV-treated group and PBS-treated group in CD25-depleted mice The valuesshown indicate the mean plusmn SEM lowast119875 lt 005 versus Cis

Cont BV

Cis Cis + BV

(a)

CisCis + BVBV

NS

5

4

3

2

1

0Cont

lowastlowast

Foxp

3-po

sitiv

e cel

ls10

HPF

(b)

Figure 7The number of Foxp3-positive cells in the kidney Tregs in the kidney were detected by EGFP signal under the confocal microscopeimaging Foxp3-positive cells were indicated by arrows (a)There was no difference in the number of Foxp3 positive cells between PBS-treatedgroup and BV-treated group before cisplatin administration However Foxp3-positive cells in the kidney increased 6 hours after cisplatinadministration In BV-pretreated group after cisplatin administration the number of Foxp3-positive cells in the kidney was significantlyhigher than in PBS-pretreated group (b) All data are presented as the mean plusmn SEM NS 119875 gt 005 lowast119875 lt 005 versus cont and

119875 lt 005

versus Cis as determined by one-way ANOVA followed by Tukeyrsquos multiple comparison test

4 Discussion

The development of side effects after cisplatin treatment iscommon and acute renal failure may develop after expo-sure to a single dose [2] Acute renal failure as inducedby cisplatin leads to the generation of danger signals bytubular cells resulting in inflammation mediated by T lym-phocytes and NK cells [15] Tregs are lymphocytes with

immunosuppressive properties and play a pivotal role inthe maintenance of immune tolerance [5] Previous studieshave reported that the anti-inflammatory actions of Tregscan protect against renal injury [16ndash18] Our recent studydemonstrated that CD4+CD25+ Tregs attenuate cisplatin-induced nephrotoxicity in vivo [8] Thus we evaluated theeffects of many candidate agents on CD4+CD25+Foxp3+Tregs in vitro and found that BV potently increases the

8 Evidence-Based Complementary and Alternative Medicine

Cont

(a)

Cis

(b)

Cis + BV

(c)

Cis Cis + BVCis Cis + BV

NS40

30

20

10

0Cont

lowastlowastlowast

F48

0-po

sitiv

e cel

lsfie

ld

CD25-depleted mice

(d)

Cis

CD25-depleted

(e)

Cis + BV

CD25-depleted

(f)

Figure 8 Macrophage infiltration into kidney Macrophage accumulation in the kidney was detected by immunostaining for F480-positivecells at 72 h after cisplatin administration The number of F480-positive cells in each section was counted in 10 fields per slide at an originalmagnification of 200xThe infiltration in BV-treatedmice was compared with those of PBS-treatedmice and CD25-depletedmiceThe valuesshown indicate the mean plusmn SEM lowastlowastlowast119875 lt 0001 versus Cis

Cis

Cis

Cont

+ BV

CisCis

Cis

Cis

Cont

Cont

Cont

(c)

(b)

(a)

Tum

or w

eigh

t (m

g)

+ BV

6000500040003000

3000

2000

2000

10000

50

0

85 11 14 17 20Day

NS

NS

Cis + BV

Cis + BV

Tum

or si

ze (m

m3 )

Figure 9 Influence of BV on the antitumor effect of cisplatin All mice received injections of EL4 lymphoma cells (2 times 10601mL) sc intothe right flank BV (1mgkg) was injected once per day for 5 days from day 5 of tumor inoculation (119899 = 8) Cisplatin was injected 3 times (ondays 11 14 and 17) at a concentration of 5mgkg All mice were sacrificed at day 20 and the tumors were photographed (a) The tumor sizewas measured every three days after BV injection for 16 days After sacrifice the tumors were fully separated from the mice and the size (b)and weight (c) of the tumors were measured All data are presented as the mean plusmn SEM lowastlowastlowast119875 lt 0001 versus cont NS 119875 gt 005 versus Cisas determined by one-way ANOVA followed by Tukeyrsquos test

Evidence-Based Complementary and Alternative Medicine 9

population of CD4+CD25+Foxp3+ Tregs Moreover it wasshown that BV has remarkable renoprotective effects inNZBW lupus mice [12] Based on these lines of evidencewe hypothesized that BV may have therapeutic effects oncisplatin-induced nephrotoxicity through the upregulation ofTregs

In this study we first showed that BV indeed increases thepopulation of CD4+CD25+Foxp3+ Tregs in isolated mousesplenocytes in vitro and in vivo before cisplatin adminis-tration (Figure 1) corroborating the results of our previousstudy [12]Three days after cisplatin administration howeverthe population of Tregs markedly increased in splenocytesfrom both of BV- and PBS-injected mice with no significantdifference in Treg numbers between the two groups Wefurther examined how many Tregs are migrated into thekidney after cisplatin administration We found that moreTregs were migrated into the kidney 6 hours after cisplatininjection in BV-pretreated mice than in PBS-pretreated mice(Figure 7) Next we found that BV has protective effectson cisplatin-induced renal dysfunction and injury SerumBUN and creatinine are typical indicators of renal functionand an increase in these values could indicate renal failure[19] Our data showed that cisplatin treatment significantlyincreased the BUN and serum creatinine levels in the saline-treated control group and that this increase was preventedby BV treatment The histological analysis of the degree ofrenal tubular injury (epithelial necrosis) also suggests thatBV can effectively prevent the structural damage of renaltissuesThe degree of macrophage infiltration into the kidneyis a major parameter used to predict the progression ofrenal disease and activated macrophages express severalinflammatory mediators [20] including TNF-120572 and IL-6which play important roles in the pathogenesis of cisplatin-induced renal injury [21 22] Our data showed that BV treat-ment in cisplatin-injected mice decreased the expression ofproinflammatory cytokines as well asmacrophage infiltrationin the kidney Because renal toxicity is themain cause of deathin cisplatin-injected mice [23] we evaluated the survival ofBV- or saline-treated mice after cisplatin administrationTheBV-treated mice showed a higher survival rate than did thesaline-treated control mice Collectively these results mightsuggest that Tregs in spleen increase in number after BVtreatment before cisplatin administration which cause moremigration of Tregs into the kidney in the early phase ofcisplatin-induced nephrotoxicity Such action might lead tothe protective effects of BV on cisplatin-induced nephrotoxicevents as shown in the present study This is supported byour previous study [8] showing that adoptive transfer of Tregsto mice (iv) caused increased migration of Tregs into thekidney 6 h after cisplatin administration and then attenuatedthe renal injuryThe renoprotective effects of BV in cisplatin-injectedmice in the present data are comparable to the resultsshown in Treg-transfused mice [8]

Since BV is known to have direct anti-inflammatory andantitumor effects [24 25] we cannot rule out the possibleinvolvement of Tregs-independent mechanisms of BV in thepresent results However we demonstrated that depletionof Tregs using anti-CD25 antibody injection completelyinhibited the BV effects on cisplatin-induced nephrotoxic

eventsTherefore our findings strongly suggest that BV couldprevent cisplatin-induced severe side effects in the kidney atleast partly by modulating the Tregs

Acknowledgment

This work was supported by the National Research Founda-tion of Korea (NRF)Grant funded by the Korean government(MEST) (no 2011-006220)

References

[1] K B Meyer and N E Madias ldquoCisplatin nephrotoxicityrdquoMineral and Electrolyte Metabolism vol 20 no 4 pp 201ndash2131994

[2] A H Lau ldquoApoptosis induced by cisplatin nephrotoxic injuryrdquoKidney International vol 56 no 4 pp 1295ndash1298 1999

[3] R Safirstein J Winston M Goldstein et al ldquoCisplatin nephro-toxicityrdquo American Journal of Kidney Diseases vol 8 no 5 pp356ndash367 1986

[4] J D Fontenot M A Gavin and A Y Rudensky ldquoFoxp3 pro-grams the development and function ofCD4+CD25+ regulatoryT cellsrdquo Nature Immunology vol 4 no 4 pp 330ndash336 2003

[5] G Raimondi M S Turner A W Thomson and P A MorelldquoNaturally occurring regulatory T cells recent insights in healthand diseaserdquo Critical Reviews in Immunology vol 27 no 1 pp61ndash95 2007

[6] S Sakaguchi M Ono R Setoguchi et al ldquoFoxp3+CD25+CD4+natural regulatory T cells in dominant self-tolerance andautoimmune diseaserdquo Immunological Reviews vol 212 no 1 pp8ndash27 2006

[7] S Sakaguchi N Sakaguchi M Asano M Itoh and M TodaldquoImmunologic self-tolerance maintained by activated T cellsexpressing IL- 2 receptor 120572-chains (CD25) Breakdown of asingle mechanism of self- tolerance causes various autoimmunediseasesrdquo Journal of Immunology vol 155 no 3 pp 1151ndash11641995

[8] H Lee D Nho H S Chung et al ldquoCD4+CD25+ regulatoryT cells attenuate cisplatin-induced nephrotoxicity in micerdquoKidney International vol 78 no 11 pp 1100ndash1109 2010

[9] M H Park M S Choi D H Kwak et al ldquoAnti-cancer effect ofbee venomin prostate cancer cells through activation of caspasepathway via inactivation of NF-120581Brdquo Prostate vol 71 no 8 pp801ndash812 2011

[10] S Y Yoon Y B Kwon H W Kim et al ldquoBee venominjection produces a peripheral anti-inflammatory effect byactivation of a nitric oxide-dependent spinocoeruleus pathwayrdquoNeuroscience Letters vol 430 no 2 pp 163ndash168 2008

[11] Y B KwonHWKim TWHamet al ldquoThe anti-inflammatoryeffect of bee venom stimulation in a mouse air pouch modelis mediated by adrenal medullary activityrdquo Journal of Neuroen-docrinology vol 15 no 1 pp 93ndash96 2003

[12] H Lee E J Lee H Kim et al ldquoBee venom-associated Th1Th2immunoglobulin class switching results in immune toleranceof NZBW F1 murine lupus nephritisrdquo American Journal ofNephrology vol 34 no 2 pp 163ndash172 2011

[13] J Megyesi R L Safirstein and P M Price ldquoInduction ofp21WAF1CIP1SDI1 in kidney tubule cells affects the courseof cisplatin-induced acute renal failurerdquoThe Journal of ClinicalInvestigation vol 101 no 4 pp 777ndash782 1998

10 Evidence-Based Complementary and Alternative Medicine

[14] K K Meldrum P Metcalfe J A Leslie R Misseri K L Hileand D R Meldrum ldquoTNF-120572 neutralization decreases nuclearfactor-120581B activation and apoptosis during renal obstructionrdquoJournal of Surgical Research vol 131 no 2 pp 182ndash188 2006

[15] A Linkermann N Himmerkus L Rolver et al ldquoRenal tubularFas ligandmediates fratricide in cisplatin-induced acute kidneyfailurerdquo Kidney International vol 79 no 2 pp 169ndash178 2011

[16] T Muthukumar D Dadhania R Ding et al ldquoMessenger RNAfor FOXP3 in the urine of renal-allograft recipientsrdquo The NewEngland Journal of Medicine vol 353 no 22 pp 2342ndash23512005

[17] G R Kinsey R Sharma L Huang et al ldquoRegulatory Tcells suppress innate immunity in kidney ischemia-reperfusioninjuryrdquo Journal of the American Society of Nephrology vol 20no 8 pp 1744ndash1753 2009

[18] S Bunnag K Allanach G S Jhangri et al ldquoFOXP3 expressionin human kidney transplant biopsies is associatedwith rejectionand time post transplant but not with favorable outcomesrdquoAmerican Journal of Transplantation vol 8 no 7 pp 1423ndash14332008

[19] H Kim G Lee H Lee M Shin M Hong and H Bae ldquoEffectsof Scutellaria barbata on cisplatin induced nephrotoxicity inmicerdquoMolecular and Cellular Toxicology vol 6 no 3 pp 255ndash259 2010

[20] Y Wang Q Cai G Zheng et al ldquoBy homing to the kidneyactivated macrophages potently exacerbate renal injuryrdquo TheAmerican Journal of Pathology vol 172 no 6 pp 1491ndash14992008

[21] S Faubel E C Lewis L Reznikov et al ldquoCisplatin-inducedacute renal failure is associatedwith an increase in the cytokinesinterleukin (IL)-1120573 IL-18 IL-6 and neutrophil infiltrationin the kidneyrdquo Journal of Pharmacology and ExperimentalTherapeutics vol 322 no 1 pp 8ndash15 2007

[22] G Ramesh and W Brian Reeves ldquoTNF-120572mediates chemokineand cytokine expression and renal injury in cisplatin nephro-toxicityrdquoThe Journal of Clinical Investigation vol 110 no 6 pp835ndash842 2002

[23] F Ries and J Klastersky ldquoNephrotoxicity induced by cancerchemotherapy with special emphasis on cisplatin toxicityrdquoAmerican Journal of Kidney Diseases vol 8 no 5 pp 368ndash3791986

[24] N Orsol ldquoBee venom in cancer therapyrdquo Cancer andMetastasisReviews vol 31 no 1-2 pp 173ndash194 2012

[25] N Orsolic L Sver S Verstovsek S Terzic and I BasicldquoInhibition of mammary carcinoma cell proliferation in vitroand tumor growth in vivo by bee venomrdquo Toxicon vol 41 no 7pp 861ndash870 2003

Evidence-Based Complementary and Alternative Medicine 7

5

4

3

2

1

0

TNF

(ng

mg)

lowast NS

Cis CisCis + BV Cis + BVCD25-depleted mice

(a)

120

100

80

60

40

20

0

lowast

NS

Cis CisCis + BV Cis + BV

CD25-depleted mice

IL-6

(pg

mg)

(b)

Figure 6 Proinflammatory cytokines in the kidney Proinflammatory cytokines were measured in kidney tissue from both of normal miceand CD25-depleted mice by ELISA Renal IL-6 and TNF120572were decreased in the BV-treated group (Cis + BV) compared with the PBS-treatedgroup (Cis) However there was no difference between the BV-treated group and PBS-treated group in CD25-depleted mice The valuesshown indicate the mean plusmn SEM lowast119875 lt 005 versus Cis

Cont BV

Cis Cis + BV

(a)

CisCis + BVBV

NS

5

4

3

2

1

0Cont

lowastlowast

Foxp

3-po

sitiv

e cel

ls10

HPF

(b)

Figure 7The number of Foxp3-positive cells in the kidney Tregs in the kidney were detected by EGFP signal under the confocal microscopeimaging Foxp3-positive cells were indicated by arrows (a)There was no difference in the number of Foxp3 positive cells between PBS-treatedgroup and BV-treated group before cisplatin administration However Foxp3-positive cells in the kidney increased 6 hours after cisplatinadministration In BV-pretreated group after cisplatin administration the number of Foxp3-positive cells in the kidney was significantlyhigher than in PBS-pretreated group (b) All data are presented as the mean plusmn SEM NS 119875 gt 005 lowast119875 lt 005 versus cont and

119875 lt 005

versus Cis as determined by one-way ANOVA followed by Tukeyrsquos multiple comparison test

4 Discussion

The development of side effects after cisplatin treatment iscommon and acute renal failure may develop after expo-sure to a single dose [2] Acute renal failure as inducedby cisplatin leads to the generation of danger signals bytubular cells resulting in inflammation mediated by T lym-phocytes and NK cells [15] Tregs are lymphocytes with

immunosuppressive properties and play a pivotal role inthe maintenance of immune tolerance [5] Previous studieshave reported that the anti-inflammatory actions of Tregscan protect against renal injury [16ndash18] Our recent studydemonstrated that CD4+CD25+ Tregs attenuate cisplatin-induced nephrotoxicity in vivo [8] Thus we evaluated theeffects of many candidate agents on CD4+CD25+Foxp3+Tregs in vitro and found that BV potently increases the

8 Evidence-Based Complementary and Alternative Medicine

Cont

(a)

Cis

(b)

Cis + BV

(c)

Cis Cis + BVCis Cis + BV

NS40

30

20

10

0Cont

lowastlowastlowast

F48

0-po

sitiv

e cel

lsfie

ld

CD25-depleted mice

(d)

Cis

CD25-depleted

(e)

Cis + BV

CD25-depleted

(f)

Figure 8 Macrophage infiltration into kidney Macrophage accumulation in the kidney was detected by immunostaining for F480-positivecells at 72 h after cisplatin administration The number of F480-positive cells in each section was counted in 10 fields per slide at an originalmagnification of 200xThe infiltration in BV-treatedmice was compared with those of PBS-treatedmice and CD25-depletedmiceThe valuesshown indicate the mean plusmn SEM lowastlowastlowast119875 lt 0001 versus Cis

Cis

Cis

Cont

+ BV

CisCis

Cis

Cis

Cont

Cont

Cont

(c)

(b)

(a)

Tum

or w

eigh

t (m

g)

+ BV

6000500040003000

3000

2000

2000

10000

50

0

85 11 14 17 20Day

NS

NS

Cis + BV

Cis + BV

Tum

or si

ze (m

m3 )

Figure 9 Influence of BV on the antitumor effect of cisplatin All mice received injections of EL4 lymphoma cells (2 times 10601mL) sc intothe right flank BV (1mgkg) was injected once per day for 5 days from day 5 of tumor inoculation (119899 = 8) Cisplatin was injected 3 times (ondays 11 14 and 17) at a concentration of 5mgkg All mice were sacrificed at day 20 and the tumors were photographed (a) The tumor sizewas measured every three days after BV injection for 16 days After sacrifice the tumors were fully separated from the mice and the size (b)and weight (c) of the tumors were measured All data are presented as the mean plusmn SEM lowastlowastlowast119875 lt 0001 versus cont NS 119875 gt 005 versus Cisas determined by one-way ANOVA followed by Tukeyrsquos test

Evidence-Based Complementary and Alternative Medicine 9

population of CD4+CD25+Foxp3+ Tregs Moreover it wasshown that BV has remarkable renoprotective effects inNZBW lupus mice [12] Based on these lines of evidencewe hypothesized that BV may have therapeutic effects oncisplatin-induced nephrotoxicity through the upregulation ofTregs

In this study we first showed that BV indeed increases thepopulation of CD4+CD25+Foxp3+ Tregs in isolated mousesplenocytes in vitro and in vivo before cisplatin adminis-tration (Figure 1) corroborating the results of our previousstudy [12]Three days after cisplatin administration howeverthe population of Tregs markedly increased in splenocytesfrom both of BV- and PBS-injected mice with no significantdifference in Treg numbers between the two groups Wefurther examined how many Tregs are migrated into thekidney after cisplatin administration We found that moreTregs were migrated into the kidney 6 hours after cisplatininjection in BV-pretreated mice than in PBS-pretreated mice(Figure 7) Next we found that BV has protective effectson cisplatin-induced renal dysfunction and injury SerumBUN and creatinine are typical indicators of renal functionand an increase in these values could indicate renal failure[19] Our data showed that cisplatin treatment significantlyincreased the BUN and serum creatinine levels in the saline-treated control group and that this increase was preventedby BV treatment The histological analysis of the degree ofrenal tubular injury (epithelial necrosis) also suggests thatBV can effectively prevent the structural damage of renaltissuesThe degree of macrophage infiltration into the kidneyis a major parameter used to predict the progression ofrenal disease and activated macrophages express severalinflammatory mediators [20] including TNF-120572 and IL-6which play important roles in the pathogenesis of cisplatin-induced renal injury [21 22] Our data showed that BV treat-ment in cisplatin-injected mice decreased the expression ofproinflammatory cytokines as well asmacrophage infiltrationin the kidney Because renal toxicity is themain cause of deathin cisplatin-injected mice [23] we evaluated the survival ofBV- or saline-treated mice after cisplatin administrationTheBV-treated mice showed a higher survival rate than did thesaline-treated control mice Collectively these results mightsuggest that Tregs in spleen increase in number after BVtreatment before cisplatin administration which cause moremigration of Tregs into the kidney in the early phase ofcisplatin-induced nephrotoxicity Such action might lead tothe protective effects of BV on cisplatin-induced nephrotoxicevents as shown in the present study This is supported byour previous study [8] showing that adoptive transfer of Tregsto mice (iv) caused increased migration of Tregs into thekidney 6 h after cisplatin administration and then attenuatedthe renal injuryThe renoprotective effects of BV in cisplatin-injectedmice in the present data are comparable to the resultsshown in Treg-transfused mice [8]

Since BV is known to have direct anti-inflammatory andantitumor effects [24 25] we cannot rule out the possibleinvolvement of Tregs-independent mechanisms of BV in thepresent results However we demonstrated that depletionof Tregs using anti-CD25 antibody injection completelyinhibited the BV effects on cisplatin-induced nephrotoxic

eventsTherefore our findings strongly suggest that BV couldprevent cisplatin-induced severe side effects in the kidney atleast partly by modulating the Tregs

Acknowledgment

This work was supported by the National Research Founda-tion of Korea (NRF)Grant funded by the Korean government(MEST) (no 2011-006220)

References

[1] K B Meyer and N E Madias ldquoCisplatin nephrotoxicityrdquoMineral and Electrolyte Metabolism vol 20 no 4 pp 201ndash2131994

[2] A H Lau ldquoApoptosis induced by cisplatin nephrotoxic injuryrdquoKidney International vol 56 no 4 pp 1295ndash1298 1999

[3] R Safirstein J Winston M Goldstein et al ldquoCisplatin nephro-toxicityrdquo American Journal of Kidney Diseases vol 8 no 5 pp356ndash367 1986

[4] J D Fontenot M A Gavin and A Y Rudensky ldquoFoxp3 pro-grams the development and function ofCD4+CD25+ regulatoryT cellsrdquo Nature Immunology vol 4 no 4 pp 330ndash336 2003

[5] G Raimondi M S Turner A W Thomson and P A MorelldquoNaturally occurring regulatory T cells recent insights in healthand diseaserdquo Critical Reviews in Immunology vol 27 no 1 pp61ndash95 2007

[6] S Sakaguchi M Ono R Setoguchi et al ldquoFoxp3+CD25+CD4+natural regulatory T cells in dominant self-tolerance andautoimmune diseaserdquo Immunological Reviews vol 212 no 1 pp8ndash27 2006

[7] S Sakaguchi N Sakaguchi M Asano M Itoh and M TodaldquoImmunologic self-tolerance maintained by activated T cellsexpressing IL- 2 receptor 120572-chains (CD25) Breakdown of asingle mechanism of self- tolerance causes various autoimmunediseasesrdquo Journal of Immunology vol 155 no 3 pp 1151ndash11641995

[8] H Lee D Nho H S Chung et al ldquoCD4+CD25+ regulatoryT cells attenuate cisplatin-induced nephrotoxicity in micerdquoKidney International vol 78 no 11 pp 1100ndash1109 2010

[9] M H Park M S Choi D H Kwak et al ldquoAnti-cancer effect ofbee venomin prostate cancer cells through activation of caspasepathway via inactivation of NF-120581Brdquo Prostate vol 71 no 8 pp801ndash812 2011

[10] S Y Yoon Y B Kwon H W Kim et al ldquoBee venominjection produces a peripheral anti-inflammatory effect byactivation of a nitric oxide-dependent spinocoeruleus pathwayrdquoNeuroscience Letters vol 430 no 2 pp 163ndash168 2008

[11] Y B KwonHWKim TWHamet al ldquoThe anti-inflammatoryeffect of bee venom stimulation in a mouse air pouch modelis mediated by adrenal medullary activityrdquo Journal of Neuroen-docrinology vol 15 no 1 pp 93ndash96 2003

[12] H Lee E J Lee H Kim et al ldquoBee venom-associated Th1Th2immunoglobulin class switching results in immune toleranceof NZBW F1 murine lupus nephritisrdquo American Journal ofNephrology vol 34 no 2 pp 163ndash172 2011

[13] J Megyesi R L Safirstein and P M Price ldquoInduction ofp21WAF1CIP1SDI1 in kidney tubule cells affects the courseof cisplatin-induced acute renal failurerdquoThe Journal of ClinicalInvestigation vol 101 no 4 pp 777ndash782 1998

10 Evidence-Based Complementary and Alternative Medicine

[14] K K Meldrum P Metcalfe J A Leslie R Misseri K L Hileand D R Meldrum ldquoTNF-120572 neutralization decreases nuclearfactor-120581B activation and apoptosis during renal obstructionrdquoJournal of Surgical Research vol 131 no 2 pp 182ndash188 2006

[15] A Linkermann N Himmerkus L Rolver et al ldquoRenal tubularFas ligandmediates fratricide in cisplatin-induced acute kidneyfailurerdquo Kidney International vol 79 no 2 pp 169ndash178 2011

[16] T Muthukumar D Dadhania R Ding et al ldquoMessenger RNAfor FOXP3 in the urine of renal-allograft recipientsrdquo The NewEngland Journal of Medicine vol 353 no 22 pp 2342ndash23512005

[17] G R Kinsey R Sharma L Huang et al ldquoRegulatory Tcells suppress innate immunity in kidney ischemia-reperfusioninjuryrdquo Journal of the American Society of Nephrology vol 20no 8 pp 1744ndash1753 2009

[18] S Bunnag K Allanach G S Jhangri et al ldquoFOXP3 expressionin human kidney transplant biopsies is associatedwith rejectionand time post transplant but not with favorable outcomesrdquoAmerican Journal of Transplantation vol 8 no 7 pp 1423ndash14332008

[19] H Kim G Lee H Lee M Shin M Hong and H Bae ldquoEffectsof Scutellaria barbata on cisplatin induced nephrotoxicity inmicerdquoMolecular and Cellular Toxicology vol 6 no 3 pp 255ndash259 2010

[20] Y Wang Q Cai G Zheng et al ldquoBy homing to the kidneyactivated macrophages potently exacerbate renal injuryrdquo TheAmerican Journal of Pathology vol 172 no 6 pp 1491ndash14992008

[21] S Faubel E C Lewis L Reznikov et al ldquoCisplatin-inducedacute renal failure is associatedwith an increase in the cytokinesinterleukin (IL)-1120573 IL-18 IL-6 and neutrophil infiltrationin the kidneyrdquo Journal of Pharmacology and ExperimentalTherapeutics vol 322 no 1 pp 8ndash15 2007

[22] G Ramesh and W Brian Reeves ldquoTNF-120572mediates chemokineand cytokine expression and renal injury in cisplatin nephro-toxicityrdquoThe Journal of Clinical Investigation vol 110 no 6 pp835ndash842 2002

[23] F Ries and J Klastersky ldquoNephrotoxicity induced by cancerchemotherapy with special emphasis on cisplatin toxicityrdquoAmerican Journal of Kidney Diseases vol 8 no 5 pp 368ndash3791986

[24] N Orsol ldquoBee venom in cancer therapyrdquo Cancer andMetastasisReviews vol 31 no 1-2 pp 173ndash194 2012

[25] N Orsolic L Sver S Verstovsek S Terzic and I BasicldquoInhibition of mammary carcinoma cell proliferation in vitroand tumor growth in vivo by bee venomrdquo Toxicon vol 41 no 7pp 861ndash870 2003

8 Evidence-Based Complementary and Alternative Medicine

Cont

(a)

Cis

(b)

Cis + BV

(c)

Cis Cis + BVCis Cis + BV

NS40

30

20

10

0Cont

lowastlowastlowast

F48

0-po

sitiv

e cel

lsfie

ld

CD25-depleted mice

(d)

Cis

CD25-depleted

(e)

Cis + BV

CD25-depleted

(f)

Figure 8 Macrophage infiltration into kidney Macrophage accumulation in the kidney was detected by immunostaining for F480-positivecells at 72 h after cisplatin administration The number of F480-positive cells in each section was counted in 10 fields per slide at an originalmagnification of 200xThe infiltration in BV-treatedmice was compared with those of PBS-treatedmice and CD25-depletedmiceThe valuesshown indicate the mean plusmn SEM lowastlowastlowast119875 lt 0001 versus Cis

Cis

Cis

Cont

+ BV

CisCis

Cis

Cis

Cont

Cont

Cont

(c)

(b)

(a)

Tum

or w

eigh

t (m

g)

+ BV

6000500040003000

3000

2000

2000

10000

50

0

85 11 14 17 20Day

NS

NS

Cis + BV

Cis + BV

Tum

or si

ze (m

m3 )

Figure 9 Influence of BV on the antitumor effect of cisplatin All mice received injections of EL4 lymphoma cells (2 times 10601mL) sc intothe right flank BV (1mgkg) was injected once per day for 5 days from day 5 of tumor inoculation (119899 = 8) Cisplatin was injected 3 times (ondays 11 14 and 17) at a concentration of 5mgkg All mice were sacrificed at day 20 and the tumors were photographed (a) The tumor sizewas measured every three days after BV injection for 16 days After sacrifice the tumors were fully separated from the mice and the size (b)and weight (c) of the tumors were measured All data are presented as the mean plusmn SEM lowastlowastlowast119875 lt 0001 versus cont NS 119875 gt 005 versus Cisas determined by one-way ANOVA followed by Tukeyrsquos test

Evidence-Based Complementary and Alternative Medicine 9

population of CD4+CD25+Foxp3+ Tregs Moreover it wasshown that BV has remarkable renoprotective effects inNZBW lupus mice [12] Based on these lines of evidencewe hypothesized that BV may have therapeutic effects oncisplatin-induced nephrotoxicity through the upregulation ofTregs

In this study we first showed that BV indeed increases thepopulation of CD4+CD25+Foxp3+ Tregs in isolated mousesplenocytes in vitro and in vivo before cisplatin adminis-tration (Figure 1) corroborating the results of our previousstudy [12]Three days after cisplatin administration howeverthe population of Tregs markedly increased in splenocytesfrom both of BV- and PBS-injected mice with no significantdifference in Treg numbers between the two groups Wefurther examined how many Tregs are migrated into thekidney after cisplatin administration We found that moreTregs were migrated into the kidney 6 hours after cisplatininjection in BV-pretreated mice than in PBS-pretreated mice(Figure 7) Next we found that BV has protective effectson cisplatin-induced renal dysfunction and injury SerumBUN and creatinine are typical indicators of renal functionand an increase in these values could indicate renal failure[19] Our data showed that cisplatin treatment significantlyincreased the BUN and serum creatinine levels in the saline-treated control group and that this increase was preventedby BV treatment The histological analysis of the degree ofrenal tubular injury (epithelial necrosis) also suggests thatBV can effectively prevent the structural damage of renaltissuesThe degree of macrophage infiltration into the kidneyis a major parameter used to predict the progression ofrenal disease and activated macrophages express severalinflammatory mediators [20] including TNF-120572 and IL-6which play important roles in the pathogenesis of cisplatin-induced renal injury [21 22] Our data showed that BV treat-ment in cisplatin-injected mice decreased the expression ofproinflammatory cytokines as well asmacrophage infiltrationin the kidney Because renal toxicity is themain cause of deathin cisplatin-injected mice [23] we evaluated the survival ofBV- or saline-treated mice after cisplatin administrationTheBV-treated mice showed a higher survival rate than did thesaline-treated control mice Collectively these results mightsuggest that Tregs in spleen increase in number after BVtreatment before cisplatin administration which cause moremigration of Tregs into the kidney in the early phase ofcisplatin-induced nephrotoxicity Such action might lead tothe protective effects of BV on cisplatin-induced nephrotoxicevents as shown in the present study This is supported byour previous study [8] showing that adoptive transfer of Tregsto mice (iv) caused increased migration of Tregs into thekidney 6 h after cisplatin administration and then attenuatedthe renal injuryThe renoprotective effects of BV in cisplatin-injectedmice in the present data are comparable to the resultsshown in Treg-transfused mice [8]

Since BV is known to have direct anti-inflammatory andantitumor effects [24 25] we cannot rule out the possibleinvolvement of Tregs-independent mechanisms of BV in thepresent results However we demonstrated that depletionof Tregs using anti-CD25 antibody injection completelyinhibited the BV effects on cisplatin-induced nephrotoxic

eventsTherefore our findings strongly suggest that BV couldprevent cisplatin-induced severe side effects in the kidney atleast partly by modulating the Tregs

Acknowledgment

This work was supported by the National Research Founda-tion of Korea (NRF)Grant funded by the Korean government(MEST) (no 2011-006220)

References

[1] K B Meyer and N E Madias ldquoCisplatin nephrotoxicityrdquoMineral and Electrolyte Metabolism vol 20 no 4 pp 201ndash2131994

[2] A H Lau ldquoApoptosis induced by cisplatin nephrotoxic injuryrdquoKidney International vol 56 no 4 pp 1295ndash1298 1999

[3] R Safirstein J Winston M Goldstein et al ldquoCisplatin nephro-toxicityrdquo American Journal of Kidney Diseases vol 8 no 5 pp356ndash367 1986

[4] J D Fontenot M A Gavin and A Y Rudensky ldquoFoxp3 pro-grams the development and function ofCD4+CD25+ regulatoryT cellsrdquo Nature Immunology vol 4 no 4 pp 330ndash336 2003

[5] G Raimondi M S Turner A W Thomson and P A MorelldquoNaturally occurring regulatory T cells recent insights in healthand diseaserdquo Critical Reviews in Immunology vol 27 no 1 pp61ndash95 2007

[6] S Sakaguchi M Ono R Setoguchi et al ldquoFoxp3+CD25+CD4+natural regulatory T cells in dominant self-tolerance andautoimmune diseaserdquo Immunological Reviews vol 212 no 1 pp8ndash27 2006

[7] S Sakaguchi N Sakaguchi M Asano M Itoh and M TodaldquoImmunologic self-tolerance maintained by activated T cellsexpressing IL- 2 receptor 120572-chains (CD25) Breakdown of asingle mechanism of self- tolerance causes various autoimmunediseasesrdquo Journal of Immunology vol 155 no 3 pp 1151ndash11641995

[8] H Lee D Nho H S Chung et al ldquoCD4+CD25+ regulatoryT cells attenuate cisplatin-induced nephrotoxicity in micerdquoKidney International vol 78 no 11 pp 1100ndash1109 2010

[9] M H Park M S Choi D H Kwak et al ldquoAnti-cancer effect ofbee venomin prostate cancer cells through activation of caspasepathway via inactivation of NF-120581Brdquo Prostate vol 71 no 8 pp801ndash812 2011

[10] S Y Yoon Y B Kwon H W Kim et al ldquoBee venominjection produces a peripheral anti-inflammatory effect byactivation of a nitric oxide-dependent spinocoeruleus pathwayrdquoNeuroscience Letters vol 430 no 2 pp 163ndash168 2008

[11] Y B KwonHWKim TWHamet al ldquoThe anti-inflammatoryeffect of bee venom stimulation in a mouse air pouch modelis mediated by adrenal medullary activityrdquo Journal of Neuroen-docrinology vol 15 no 1 pp 93ndash96 2003

[12] H Lee E J Lee H Kim et al ldquoBee venom-associated Th1Th2immunoglobulin class switching results in immune toleranceof NZBW F1 murine lupus nephritisrdquo American Journal ofNephrology vol 34 no 2 pp 163ndash172 2011

[13] J Megyesi R L Safirstein and P M Price ldquoInduction ofp21WAF1CIP1SDI1 in kidney tubule cells affects the courseof cisplatin-induced acute renal failurerdquoThe Journal of ClinicalInvestigation vol 101 no 4 pp 777ndash782 1998

10 Evidence-Based Complementary and Alternative Medicine

[14] K K Meldrum P Metcalfe J A Leslie R Misseri K L Hileand D R Meldrum ldquoTNF-120572 neutralization decreases nuclearfactor-120581B activation and apoptosis during renal obstructionrdquoJournal of Surgical Research vol 131 no 2 pp 182ndash188 2006

[15] A Linkermann N Himmerkus L Rolver et al ldquoRenal tubularFas ligandmediates fratricide in cisplatin-induced acute kidneyfailurerdquo Kidney International vol 79 no 2 pp 169ndash178 2011

[16] T Muthukumar D Dadhania R Ding et al ldquoMessenger RNAfor FOXP3 in the urine of renal-allograft recipientsrdquo The NewEngland Journal of Medicine vol 353 no 22 pp 2342ndash23512005

[17] G R Kinsey R Sharma L Huang et al ldquoRegulatory Tcells suppress innate immunity in kidney ischemia-reperfusioninjuryrdquo Journal of the American Society of Nephrology vol 20no 8 pp 1744ndash1753 2009

[18] S Bunnag K Allanach G S Jhangri et al ldquoFOXP3 expressionin human kidney transplant biopsies is associatedwith rejectionand time post transplant but not with favorable outcomesrdquoAmerican Journal of Transplantation vol 8 no 7 pp 1423ndash14332008

[19] H Kim G Lee H Lee M Shin M Hong and H Bae ldquoEffectsof Scutellaria barbata on cisplatin induced nephrotoxicity inmicerdquoMolecular and Cellular Toxicology vol 6 no 3 pp 255ndash259 2010

[20] Y Wang Q Cai G Zheng et al ldquoBy homing to the kidneyactivated macrophages potently exacerbate renal injuryrdquo TheAmerican Journal of Pathology vol 172 no 6 pp 1491ndash14992008

[21] S Faubel E C Lewis L Reznikov et al ldquoCisplatin-inducedacute renal failure is associatedwith an increase in the cytokinesinterleukin (IL)-1120573 IL-18 IL-6 and neutrophil infiltrationin the kidneyrdquo Journal of Pharmacology and ExperimentalTherapeutics vol 322 no 1 pp 8ndash15 2007

[22] G Ramesh and W Brian Reeves ldquoTNF-120572mediates chemokineand cytokine expression and renal injury in cisplatin nephro-toxicityrdquoThe Journal of Clinical Investigation vol 110 no 6 pp835ndash842 2002

[23] F Ries and J Klastersky ldquoNephrotoxicity induced by cancerchemotherapy with special emphasis on cisplatin toxicityrdquoAmerican Journal of Kidney Diseases vol 8 no 5 pp 368ndash3791986

[24] N Orsol ldquoBee venom in cancer therapyrdquo Cancer andMetastasisReviews vol 31 no 1-2 pp 173ndash194 2012

[25] N Orsolic L Sver S Verstovsek S Terzic and I BasicldquoInhibition of mammary carcinoma cell proliferation in vitroand tumor growth in vivo by bee venomrdquo Toxicon vol 41 no 7pp 861ndash870 2003

Evidence-Based Complementary and Alternative Medicine 9

population of CD4+CD25+Foxp3+ Tregs Moreover it wasshown that BV has remarkable renoprotective effects inNZBW lupus mice [12] Based on these lines of evidencewe hypothesized that BV may have therapeutic effects oncisplatin-induced nephrotoxicity through the upregulation ofTregs

In this study we first showed that BV indeed increases thepopulation of CD4+CD25+Foxp3+ Tregs in isolated mousesplenocytes in vitro and in vivo before cisplatin adminis-tration (Figure 1) corroborating the results of our previousstudy [12]Three days after cisplatin administration howeverthe population of Tregs markedly increased in splenocytesfrom both of BV- and PBS-injected mice with no significantdifference in Treg numbers between the two groups Wefurther examined how many Tregs are migrated into thekidney after cisplatin administration We found that moreTregs were migrated into the kidney 6 hours after cisplatininjection in BV-pretreated mice than in PBS-pretreated mice(Figure 7) Next we found that BV has protective effectson cisplatin-induced renal dysfunction and injury SerumBUN and creatinine are typical indicators of renal functionand an increase in these values could indicate renal failure[19] Our data showed that cisplatin treatment significantlyincreased the BUN and serum creatinine levels in the saline-treated control group and that this increase was preventedby BV treatment The histological analysis of the degree ofrenal tubular injury (epithelial necrosis) also suggests thatBV can effectively prevent the structural damage of renaltissuesThe degree of macrophage infiltration into the kidneyis a major parameter used to predict the progression ofrenal disease and activated macrophages express severalinflammatory mediators [20] including TNF-120572 and IL-6which play important roles in the pathogenesis of cisplatin-induced renal injury [21 22] Our data showed that BV treat-ment in cisplatin-injected mice decreased the expression ofproinflammatory cytokines as well asmacrophage infiltrationin the kidney Because renal toxicity is themain cause of deathin cisplatin-injected mice [23] we evaluated the survival ofBV- or saline-treated mice after cisplatin administrationTheBV-treated mice showed a higher survival rate than did thesaline-treated control mice Collectively these results mightsuggest that Tregs in spleen increase in number after BVtreatment before cisplatin administration which cause moremigration of Tregs into the kidney in the early phase ofcisplatin-induced nephrotoxicity Such action might lead tothe protective effects of BV on cisplatin-induced nephrotoxicevents as shown in the present study This is supported byour previous study [8] showing that adoptive transfer of Tregsto mice (iv) caused increased migration of Tregs into thekidney 6 h after cisplatin administration and then attenuatedthe renal injuryThe renoprotective effects of BV in cisplatin-injectedmice in the present data are comparable to the resultsshown in Treg-transfused mice [8]

Since BV is known to have direct anti-inflammatory andantitumor effects [24 25] we cannot rule out the possibleinvolvement of Tregs-independent mechanisms of BV in thepresent results However we demonstrated that depletionof Tregs using anti-CD25 antibody injection completelyinhibited the BV effects on cisplatin-induced nephrotoxic

eventsTherefore our findings strongly suggest that BV couldprevent cisplatin-induced severe side effects in the kidney atleast partly by modulating the Tregs

Acknowledgment

This work was supported by the National Research Founda-tion of Korea (NRF)Grant funded by the Korean government(MEST) (no 2011-006220)

References

[1] K B Meyer and N E Madias ldquoCisplatin nephrotoxicityrdquoMineral and Electrolyte Metabolism vol 20 no 4 pp 201ndash2131994

[2] A H Lau ldquoApoptosis induced by cisplatin nephrotoxic injuryrdquoKidney International vol 56 no 4 pp 1295ndash1298 1999

[3] R Safirstein J Winston M Goldstein et al ldquoCisplatin nephro-toxicityrdquo American Journal of Kidney Diseases vol 8 no 5 pp356ndash367 1986

[4] J D Fontenot M A Gavin and A Y Rudensky ldquoFoxp3 pro-grams the development and function ofCD4+CD25+ regulatoryT cellsrdquo Nature Immunology vol 4 no 4 pp 330ndash336 2003

[5] G Raimondi M S Turner A W Thomson and P A MorelldquoNaturally occurring regulatory T cells recent insights in healthand diseaserdquo Critical Reviews in Immunology vol 27 no 1 pp61ndash95 2007

[6] S Sakaguchi M Ono R Setoguchi et al ldquoFoxp3+CD25+CD4+natural regulatory T cells in dominant self-tolerance andautoimmune diseaserdquo Immunological Reviews vol 212 no 1 pp8ndash27 2006

[7] S Sakaguchi N Sakaguchi M Asano M Itoh and M TodaldquoImmunologic self-tolerance maintained by activated T cellsexpressing IL- 2 receptor 120572-chains (CD25) Breakdown of asingle mechanism of self- tolerance causes various autoimmunediseasesrdquo Journal of Immunology vol 155 no 3 pp 1151ndash11641995

[8] H Lee D Nho H S Chung et al ldquoCD4+CD25+ regulatoryT cells attenuate cisplatin-induced nephrotoxicity in micerdquoKidney International vol 78 no 11 pp 1100ndash1109 2010

[9] M H Park M S Choi D H Kwak et al ldquoAnti-cancer effect ofbee venomin prostate cancer cells through activation of caspasepathway via inactivation of NF-120581Brdquo Prostate vol 71 no 8 pp801ndash812 2011

[10] S Y Yoon Y B Kwon H W Kim et al ldquoBee venominjection produces a peripheral anti-inflammatory effect byactivation of a nitric oxide-dependent spinocoeruleus pathwayrdquoNeuroscience Letters vol 430 no 2 pp 163ndash168 2008

[11] Y B KwonHWKim TWHamet al ldquoThe anti-inflammatoryeffect of bee venom stimulation in a mouse air pouch modelis mediated by adrenal medullary activityrdquo Journal of Neuroen-docrinology vol 15 no 1 pp 93ndash96 2003

[12] H Lee E J Lee H Kim et al ldquoBee venom-associated Th1Th2immunoglobulin class switching results in immune toleranceof NZBW F1 murine lupus nephritisrdquo American Journal ofNephrology vol 34 no 2 pp 163ndash172 2011

[13] J Megyesi R L Safirstein and P M Price ldquoInduction ofp21WAF1CIP1SDI1 in kidney tubule cells affects the courseof cisplatin-induced acute renal failurerdquoThe Journal of ClinicalInvestigation vol 101 no 4 pp 777ndash782 1998

10 Evidence-Based Complementary and Alternative Medicine

[14] K K Meldrum P Metcalfe J A Leslie R Misseri K L Hileand D R Meldrum ldquoTNF-120572 neutralization decreases nuclearfactor-120581B activation and apoptosis during renal obstructionrdquoJournal of Surgical Research vol 131 no 2 pp 182ndash188 2006

[15] A Linkermann N Himmerkus L Rolver et al ldquoRenal tubularFas ligandmediates fratricide in cisplatin-induced acute kidneyfailurerdquo Kidney International vol 79 no 2 pp 169ndash178 2011

[16] T Muthukumar D Dadhania R Ding et al ldquoMessenger RNAfor FOXP3 in the urine of renal-allograft recipientsrdquo The NewEngland Journal of Medicine vol 353 no 22 pp 2342ndash23512005

[17] G R Kinsey R Sharma L Huang et al ldquoRegulatory Tcells suppress innate immunity in kidney ischemia-reperfusioninjuryrdquo Journal of the American Society of Nephrology vol 20no 8 pp 1744ndash1753 2009

[18] S Bunnag K Allanach G S Jhangri et al ldquoFOXP3 expressionin human kidney transplant biopsies is associatedwith rejectionand time post transplant but not with favorable outcomesrdquoAmerican Journal of Transplantation vol 8 no 7 pp 1423ndash14332008

[19] H Kim G Lee H Lee M Shin M Hong and H Bae ldquoEffectsof Scutellaria barbata on cisplatin induced nephrotoxicity inmicerdquoMolecular and Cellular Toxicology vol 6 no 3 pp 255ndash259 2010

[20] Y Wang Q Cai G Zheng et al ldquoBy homing to the kidneyactivated macrophages potently exacerbate renal injuryrdquo TheAmerican Journal of Pathology vol 172 no 6 pp 1491ndash14992008

[21] S Faubel E C Lewis L Reznikov et al ldquoCisplatin-inducedacute renal failure is associatedwith an increase in the cytokinesinterleukin (IL)-1120573 IL-18 IL-6 and neutrophil infiltrationin the kidneyrdquo Journal of Pharmacology and ExperimentalTherapeutics vol 322 no 1 pp 8ndash15 2007

[22] G Ramesh and W Brian Reeves ldquoTNF-120572mediates chemokineand cytokine expression and renal injury in cisplatin nephro-toxicityrdquoThe Journal of Clinical Investigation vol 110 no 6 pp835ndash842 2002

[23] F Ries and J Klastersky ldquoNephrotoxicity induced by cancerchemotherapy with special emphasis on cisplatin toxicityrdquoAmerican Journal of Kidney Diseases vol 8 no 5 pp 368ndash3791986

[24] N Orsol ldquoBee venom in cancer therapyrdquo Cancer andMetastasisReviews vol 31 no 1-2 pp 173ndash194 2012

[25] N Orsolic L Sver S Verstovsek S Terzic and I BasicldquoInhibition of mammary carcinoma cell proliferation in vitroand tumor growth in vivo by bee venomrdquo Toxicon vol 41 no 7pp 861ndash870 2003

10 Evidence-Based Complementary and Alternative Medicine

[14] K K Meldrum P Metcalfe J A Leslie R Misseri K L Hileand D R Meldrum ldquoTNF-120572 neutralization decreases nuclearfactor-120581B activation and apoptosis during renal obstructionrdquoJournal of Surgical Research vol 131 no 2 pp 182ndash188 2006

[15] A Linkermann N Himmerkus L Rolver et al ldquoRenal tubularFas ligandmediates fratricide in cisplatin-induced acute kidneyfailurerdquo Kidney International vol 79 no 2 pp 169ndash178 2011

[16] T Muthukumar D Dadhania R Ding et al ldquoMessenger RNAfor FOXP3 in the urine of renal-allograft recipientsrdquo The NewEngland Journal of Medicine vol 353 no 22 pp 2342ndash23512005

[17] G R Kinsey R Sharma L Huang et al ldquoRegulatory Tcells suppress innate immunity in kidney ischemia-reperfusioninjuryrdquo Journal of the American Society of Nephrology vol 20no 8 pp 1744ndash1753 2009

[18] S Bunnag K Allanach G S Jhangri et al ldquoFOXP3 expressionin human kidney transplant biopsies is associatedwith rejectionand time post transplant but not with favorable outcomesrdquoAmerican Journal of Transplantation vol 8 no 7 pp 1423ndash14332008

[19] H Kim G Lee H Lee M Shin M Hong and H Bae ldquoEffectsof Scutellaria barbata on cisplatin induced nephrotoxicity inmicerdquoMolecular and Cellular Toxicology vol 6 no 3 pp 255ndash259 2010

[20] Y Wang Q Cai G Zheng et al ldquoBy homing to the kidneyactivated macrophages potently exacerbate renal injuryrdquo TheAmerican Journal of Pathology vol 172 no 6 pp 1491ndash14992008

[21] S Faubel E C Lewis L Reznikov et al ldquoCisplatin-inducedacute renal failure is associatedwith an increase in the cytokinesinterleukin (IL)-1120573 IL-18 IL-6 and neutrophil infiltrationin the kidneyrdquo Journal of Pharmacology and ExperimentalTherapeutics vol 322 no 1 pp 8ndash15 2007

[22] G Ramesh and W Brian Reeves ldquoTNF-120572mediates chemokineand cytokine expression and renal injury in cisplatin nephro-toxicityrdquoThe Journal of Clinical Investigation vol 110 no 6 pp835ndash842 2002

[23] F Ries and J Klastersky ldquoNephrotoxicity induced by cancerchemotherapy with special emphasis on cisplatin toxicityrdquoAmerican Journal of Kidney Diseases vol 8 no 5 pp 368ndash3791986

[24] N Orsol ldquoBee venom in cancer therapyrdquo Cancer andMetastasisReviews vol 31 no 1-2 pp 173ndash194 2012

[25] N Orsolic L Sver S Verstovsek S Terzic and I BasicldquoInhibition of mammary carcinoma cell proliferation in vitroand tumor growth in vivo by bee venomrdquo Toxicon vol 41 no 7pp 861ndash870 2003