research article the effect of dantonic pill on -catenin...

Hindawi Publishing CorporationJournal of Diabetes ResearchVolume 2013 Article ID 848679 8 pageshttpdxdoiorg1011552013848679

Research ArticleThe Effect of Dantonic Pill on 120573-Catenin Expressionin a Rat Model of Streptozotocin-Induced Early Stage ofDiabetic Nephropathy

Zhou Shuhong Lv Hongjun Cui Bo Xu Li and Shi Bingyin

Department of Endocrinology the First Affiliated Hospital of Xirsquoan Jiaotong University School of Medicine Xirsquoan 710061 China

Correspondence should be addressed to Shi Bingyin shibingy126con

Received 17 January 2013 Accepted 12 March 2013

Academic Editor Weiping Jia

Copyright copy 2013 Zhou Shuhong et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Diabetic nephropathy (DN) is one of the most common causes of end-stage renal failure This study was performed to determinethe effect of Dantonic Pill (DP) treatment on 120573-catenin expression in a rat model of streptozotocin- (STZ-) induced early-stageDN with irbesartan treatment as a positive control Including an analysis of the general metabolic index and renal functionimmunohistochemical staining and reverse transcription real-time PCR for 120573-catenin were performed in the renal cortex of the ratmodels every 4 weeks After the treatments of DP and irbesartan the albuminuria level kidney weightbody weight and thicknessof the glomerular basement membrane were decreased but the expression of 120573-catenin was not downregulated in the renal cortexThe effective drug target of DP to ameliorate albuminuria and renal hypertrophy should not inhibit the upregulated expression of120573-catenin in rats with STZ-induced early-stage diabetic damage

1 Introduction

Diabetes mellitus presents a significant health concernbecause this disorder leads to long-term complicationsthroughout the body involving the renal and other systems[1] Diabetic nephropathy (DN) evolves into a progressivefibrosing kidney disease Wnt pathway components havebeen reported to be associated with various kidney diseasesincluding DN [2] Regulating the 120573-catenin protein levelsto control the activation of Wnt-responsive target genes isreferred to as the canonical Wnt120573-catenin pathway Wntproteins interact with receptor proteins and stabilize thedownstream transcription regulator 120573-catenin by inhibiting120573-catenin phosphorylation which reportedly affects tubuleformation and epithelial differentiation [3] High glucoselevels (HG) increased the phosphorylation of 120573-catenin andreduced the nuclear120573-catenin levelsThe destabilization of120573-catenin was correlated with the increased expression of otherprofibrotic factors in mesangial cells [4] Impaired 120573-cateninsignaling is one prominent pathologic reaction responsiblefor the ECM metabolism induced by HG in mesangial cells[5]

ldquoHerbal medicinesrdquo and ldquoherbal remediesrdquo are inter-changeable terms that are used to refer to treatments contain-ing various mixtures of herbs People choose to take herbalmedicines as alternatives to orthodox medicines due to theirsupposed low levels of toxicity and their ldquonaturalrdquo originsThousands of years ofclinic practice in traditional Chinesemedicine (TCM) have accumulated a considerable number offormulae that exhibit reliable in vivo efficacy and safety TheldquoDantonic Pillrdquo (DP) also known as the ldquoCardiotonic Pillrdquohas shown significant therapeutic benefits in patients withDN [6] Although the DP has been widely used for manyyears systematic scientific evidence and proof of efficacyare generally lacking compared with synthesized chemicalmedicines [7] Chinese herbal formulae consist of multipleherbs and are therefore liable to produce a large number ofmetabolites that may act on multiple targets in the body

The molecular mechanisms that underlie the progressionof DN to end-stage renal failure are not well defined thuslimiting access to potential therapeutic targets Thereforea likely therapeutic strategy may be the modulation of the120573-catenin levels andor function Numerous clinical studieshave defined proteinuria as a major marker of the decline

2 Journal of Diabetes Research

of renal function and treatment with irbesartan whichreduces urinary albumin excretion as a positive controlHowever to date whether DP has an effect on the 120573-cateninlevels in early-stage DN remains unclear This study wasdesigned to establish diabetic animal models to observe theinterventional effects of DP on 120573-catenin expression in early-stage diabetic kidney injury rats and to provide pathogenicand theoretical evidence of DN in these rats

2 Materials and Methods

21 Materials This study was performed at the Endocrinol-ogy Department Laboratory of the First Hospital Affiliatedto Xirsquoan Jiaotong University from October 2009 to January2011 Male SD rats (weighing 180ndash200 g clean grade) wereselected from the Experimental AnimalCenter of theMedicalCollege of Xirsquoan Jiaotong University and cared for usingstandardized methods The rats were maintained undertemperature-controlled conditions (22∘C plusmn 2∘C) and artificial12 h lightdark cycles with food and water ad libitum Allrats were randomly divided into four groups the nondiabeticcontrol rats with vehicle treatment (control group 119899 = 21)the streptozotocin- (STZ-) induced diabetic nephropathy ratswith vehicle treatment (STZ + vehicle group 119899 = 21) theSTZ-induced diabetic rats with irbesartan treatment (STZ +irbesartan group 119899 = 21) and the STZ-induced diabetic ratswith DP treatment (STZ + DP group 119899 = 21) In each group7 rats were evaluated every 4 weeks until the end of the studywhich involved a 12-week observation period The study wasapproved by the Institutional Animal Ethical Committee

22 Methods

221 Model Establishment and Drug Administration Strep-tozotocin (10 gL) was single-peritoneally injected at a doseof 60mgkg body weight to establish the diabetic modelThe blood glucose levels derived from the caudal vein wererespectively measured at 72 h and on the 6th day after theinjection The model standard was confirmed by both of theblood glucose levels gt166mmoL The rats in the STZ +irbesartan and STZ + DP groups were administered withirbesartan 50mg(kgsdotday) and DP 500mg(kgsdotday) respec-tively starting 4 weeks after model establishment Every 4weeks 24-hour urine was reserved blood was collected fromthe heart and rats were sacrificed under anesthesia Renalcortex tissue (100mg) was used to extract total RNA and theremaining tissues were fixed with 4 paraformaldehyde forpathological examination

222 Renal Biochemical and Functional Detection Theserum creatinine (serum Cr) concentration was determinedusing a commercial assay kit (BeckmanCoulter Miami FLUSA) The urine creatinine concentration was measuredusing an assay based on Jaffersquos reaction according to the man-ufacturerrsquos protocol (Creatinine Assay Kit) The creatinineclearance rate (Ccr) was calculated using the following equa-tion Ccr[ml(minsdotkg body weight)] = [urinary Cr (120583molL)times urinary volume (ml)serum Cr (120583molL)][11440 (min)]

times [1000body weight (g)]) [8] The urine protein level wasdetected by radioimmunoassay following the manufacturerrsquosprotocol The kidney weightbody weight (KWBW ) wasmeasured

223 Renal Pathological Examination The renal glomerulusand renal tubule mesenchymal lesions were observed fol-lowing HE and PAS staining Twenty renal glomeruli wererandomly selected from the cortical area under 400x magni-fication by an experienced pathologist to measure the indexof mesangial expansion The index of mesangial expansionwas scored by a quantitative estimate of the mesangial zonewidth in each glomerulus expressed as a function of thetotal glomerular area [9] 0 normal glomeruli 1 matrixexpansion occurring in up to 50of the glomerulus 2matrixexpansion occurring in 50ndash75 of the glomerulus and 3matrix expansion occurring in 75ndash100 of the glomerulus

224 Renal Electron Microscopy Morphometric EvaluationThe electron microscopy morphometric evaluation was per-formed as described below A portion of the renal tissues wascut into 1mm cubes fixed in 25 glutaraldehyde and post-fixed in 1 osmium tetroxide The samples were dehydratedin a graded alcohol series and embedded in Epon 812 Fourultrathin sections (60 nm) were cut with a diamond knifecontinuously and stained with uranyl acetate and lead citrateThe thickness of glomerular basement membrane (GBM)was examined with a Hitachi H-7650 (80 kV) transmissionelectron microscope (JEM100SX Japan)

225 Determination of 120573-Catenin Protein Expression byImmunohistochemistry (IHC) Paraffin sections were rou-tinely dewaxed hydrated and dipped in a 003 volumefraction of H

2O2and methyl alcohol Then a microwave was

used to repair the antigen and the tissues were blocked withnormal caprine serum and dribbled with attenuated antibodyI (antirabbit) The rat 120573-catenin antibody was diluted at theratio of 1 50 stained with biotin-labeled caprine antirabbitantibody I and diaminobenzidine restained with hematinedehydrated cleared and sealed with glycerin gelatin Stainedrenal glomeruli and tubules that were randomly selectedfrom 20 sights were observed under 400x magnification byan experienced pathologist The positive staining rate wasevaluated as the following le25 positive cells (+) 26ndash50positive cells (++) 51ndash75 positive cells (+++) and gt75positive cells (++++) each scored as 1 2 3 and 4 pointsrespectively for the statistical analysis [10]

226 Determination of 120573-Catenin mRNA Expression byQuantitative Real-Time PCR Total RNA was extracted fromthe renal tissues with the Trizol method according to themanufacturerrsquos protocol cDNA was synthesized by reversetranscription using a kit (TaKaRaCompanyDalian CA)Theexpression of 120573-catenin mRNA was detected by quantitativereal-time polymerase chain reaction (RT-PCR) 120573-actin wasused as the internal reference The total volume of thePCR was 20 120583L with 30 ng of cDNA as the template Afteran initial denaturation step at 95∘C for 3min 40 cycles

Journal of Diabetes Research 3

of 95∘C for 30 sec and 60∘C for 30 sec for annealing andextension were run on a CFX384 Dice real-time PCR system(Bio-Rad Laboratories Inc CA) A melting curve analysiswas performed after the amplification was completed ThemRNA levels were normalized to the 120573-actin levels of therespective control and presented as a ratio The primers(designed and synthesized by Dalian Baosen Company) andtheir sequences product lengths and reactive conditions areshown in Table 1

227 Statistical Analysis The measurement data wereexpressed as the mean plusmn SD Significant differences amongthe groups were analyzed using a factorial-designedANOVA The method of simple linear correlation analysiswas employed to measure the correlation between twovariables P values lt 005 were considered significant SPSS160 software was used in this study

3 Results

31 General Group Comparisons There were four groups ofrats in the study and the metabolic indices such as bodyweight food intake water intake urine output and bloodglucose levels for each group are summarized in Table 2Thefood intake water intake urine output and blood glucoselevels in the rats with STZ-induced diabetes were higherthanthose in the normal control rats (119875 lt 005) The bodyweights were lower in the diabetic rats than in the normalrats However there were no significant differences in any ofthe metabolic indices among the diabetic rats treated withvehicle DP and irbesartan during the 12 weeks (119875 gt 005)

32 Effect of DP and Irbesartan on Renal Functional andPathological Examination The renal pathological examina-tion by HE and PAS staining indicated that the mesangialmatrix was increased and the renal tubule was vacuolarlydegenerated in the diabetic ratsThe urine protein levels CcrKWBW mesangial expansion index and GBM thicknesswere significantly higher in the diabetic rats compared withthe normal rats (119875 lt 005) After 12 weeks of treatment withDP and irbesartan compared with vehicle alone the urineprotein levels KWBW and GBM thickness were decreasedin the diabetic rats but Ccr and the mesangial expansionindex were not (119875 lt 005 and 119875 gt 005 resp) The effects ofDP and irbesartan on the renal functional and pathologicalexamination in the diabetic rats are shown in Table 3 andFigure 1

33 Effect of DP and Irbesartan on the mRNA and ProteinExpressions of 120573-Catenin For 12 weeks the 120573-cateninmRNAexpression in the STZ-induced diabetic rats was significantlyupregulated compared with the control group (119875 lt 005)The 120573-catenin mRNA expression in the STZ + DP and STZ+ irbesartan groups was reduced but these changes were notsignificantly different compared with the levels in the STZ +vehicle group (119875 gt 005) Immunohistochemical staining of120573-catenin in the STZ + vehicle group was greater than that ofthe control group (119875 lt 005) but the staining in the STZ +

DP and STZ + irbesartan groups was not less compared withthe STZ + vehicle group (119875 gt 005) The effect of DP andirbesartan on the 120573-catenin mRNA and protein expressionlevels is shown in Figure 2

Correlation Analysis There was no significant correlationbetween the mRNA and protein expression levels of 120573-catenin and the urinary protein level or the KWBW in thediabetic rats (119875 gt 005)

4 Discussion

DN is characterized by definite renal morphological andfunctional alterations The features of early diabetic renalchanges are glomerular hyperfiltration and glomerular andrenal hypertrophyThis disease is characterized by thickeningof GBM and mesangial matrix expansion [11] The datapresented here demonstrate that the long-termmodel of STZ-induced diabetes is associated with the early stage of diabeticnephropathy By 16 weeks renal hypertrophy hyperfiltrationand proteinuria were established in our model At thattime the earliest changes of glomerular mesangial matrixaccumulation but not collapse of the glomerular capillarieswere detectable as previously reported [12] In our experi-ments the DP herbal preparation and irbesartan attenuatedthe early-stage nephropathic symptoms in the diabetic ratswhich were characterized by proteinuria kidney hypertro-phy and thickening of the glomerular basement membraneThere were no effects of the DP and irbesartan treatmentsover the 12 weeks on distinctive metabolic indices especiallyhyperglycemia induced in the diabetic rats Therefore itshould be noted that these treatments possess a protectiveeffect against the diabetic renal damage that is independent oflowering the plasma glucose level which is in agreement withother studies [13] In this regard we can conclude that thetherapeutic dose of DP (500mg(kgsdotday)) is at least equallyeffective as that of irbesartan (50mg(kgsdotday)) in early-stagediabetic nephropathy rats

The principle of formulation in TCM has been adoptedto guide the selection of herbs (herb matching) in the multi-component herbal formulae prescribed for the treatment ofdiseases The most important and effective pharmacologicalingredients identified in DP are danshensu and Panax noto-ginseng [14]Dan Shen as themain herb inDP has been iden-tified as containing lipophilic tanshinones such as tanshinoneIIA and hydrophilic phenolic acids particularly includingdanshensu [15] Danshensu downregulated the expression offibronectin and collagen-1 induced by high glucose levels inhuman peritoneal mesothelial cells [16] In the water extractof San Qi Panax notoginseng downregulates the expressionof 120573-tumor growth factor [17] vascular endothelial growthfactor [18] and connective tissue growth factor (CTGF) [19]and inhibits the overproduction of advanced glycation endproducts [20] to protect the kidney in a diabetic modelThus we confirmed that DP therapy to treat diabetic renalcomplications should be effective

Previous studies revealed that the Wnt pathway wasidentified to be associated with DN The binding of specificWnt proteins to receptorcoreceptor complexes transduces


Table 1 Nucleotide sequence of the primers used in real-time PCR

Gene Primers Nucleotide sequence 51015840-31015840 Length (bp) Temperature (∘C)

120573-catenin Forward AACGGCTTTCGGTTGAGCTG 118 60Reverse TGGCGATATCCAAGGGCTTC

120573-actin Forward TGCCTTTGTGCACTGGTATG 152 60Reverse CTGGAGCAGTTTGACGACAC

Table 2 Metabolic effects of STZ-induced diabetes and treatments in rats

Parameters Body weight (g) Food intake (g) Water intake (mL) Urine output (mL) Blood glucose (mmolL)0w

Control (119899 = 7) 44333 plusmn 3928lowast 2492 plusmn 859lowast 400 plusmn 285lowastlowast 1080 plusmn 545lowastlowast 448 plusmn 015lowastlowast

STZ (119899 = 7) 30329 plusmn 4661 5621 plusmn 525 30003 plusmn 5691 19743 plusmn 4255 2807 plusmn 8914w


STZ + vehicle (n=7) 30329 plusmn 4661 5621 plusmn 525 30000 plusmn 5692 21743 plusmn 4255 2807 plusmn 891STZ + DP (119899 = 7) 30937 plusmn 4457 5028 plusmn 844 22786 plusmn 3806 17143 plusmn 2387 2828 plusmn 718STZ + irbesartan (119899 = 7) 34683 plusmn 3928 5225 plusmn 980 25343 plusmn 4440 18414 plusmn 3451 2497 plusmn 103

8wControl (119899 = 7) 52171 plusmn 4259lowast 2314 plusmn 581lowast 3000 plusmn 1100lowastlowast 1493 plusmn 246lowastlowast 413 plusmn 041lowastlowast

STZ + vehicle (119899 = 7) 33188 plusmn 7139 5081 plusmn 827 22750 plusmn 4175 15650 plusmn 2074 2888 plusmn 832STZ + DP (119899 = 7) 29629 plusmn 2512 5901 plusmn 736 23233 plusmn 3369 17100 plusmn 2582 2833 plusmn 653STZ + irbesartan (119899 = 7) 31100 plusmn 5178 5225 plusmn 808 24125 plusmn 5436 17875 plusmn 4452 2570 plusmn 128

12 wControl (119899 = 7) 58233 plusmn 8535lowast 2233 plusmn 505lowast 4000 plusmn 1095lowastlowast 1235 plusmn 473lowastlowast 416 plusmn 054lowastlowast


lowast

119875 lt 005 versus STZ + vehicle rats lowastlowast119875 lt 001 versus STZ + vehicle rats

Table 3 Renal functional detection and pathological changes of STZ-induced diabetes and treatments in rats

Parameters Urinary protein (mg24 h) Ccr (mLminsdotkg) KWBW () Mesangial expansion index0w

Control (n = 7) 012 plusmn 007lowast 290 plusmn 047lowast 065 plusmn 009lowast 022 plusmn 002lowast

STZ (n = 7) 032 plusmn 008 2643 plusmn 750 130 plusmn 003 060 plusmn 0024w


STZ + vehicle (n = 7) 039 plusmn 002 1398 plusmn 299 140 plusmn 003 064 plusmn 004STZ + DP (n = 7) 034 plusmn 002lowast 1415 plusmn 331 126 plusmn 002lowast 046 plusmn 005STZ + irbesartan (n = 7) 020 plusmn 003lowast 1351 plusmn 308 125 plusmn 004lowast 056 plusmn 002

8wControl (n = 7) 018 plusmn 002lowast 188 plusmn 046lowast 065 plusmn 004lowast 038 plusmn 008lowast




lowastP lt 005 versus STZ + vehicle rats


(B) (C) (D)

(F) (G) (H)

(J)

10120583m

10120583m

10nm

(Alowast)

(Elowast)

(Ilowast) (Klowast) (Llowast)

(a)

0

100

200

300

400

500

600

700

0

01

02

03

04

05

06

07

08

Control STZ + vehicle STZ + DP STZ + irbesartan Control STZ + DPSTZ +vehicle

STZ +irbesartan

lowast lowastlowast

lowast

Mes

angi

al ex

pans

ion

inde

x

Thic

knes

s of G

BM (n

m)

(A) (B)

(b)

Figure 1 Effect of DP and irbesartan treatments on the renal pathological changes (a) Renal pathological changes of mesangial expansionwere examined by histological staining (HE and PAS) and thickness of the GBM was determined by electron microscopy as described inthe materials and methods section (A)(E)(I) Normal renal cortex in the control group (B)(F)(J) STZ + vehicle group (C)(G)(K) STZ + DP(500mg(kgsdotday) group (D)(H)(L) STZ + irbesartan (50mg(kgsdotday)) group (b) Thickness of the GBM (A) and mesangial expansion index(B) expressed as a quantitative estimate score All values are the means plusmn SD (119899 = 7) 119875 lt 005 compared with the STZ + vehicle group

intracellular signals through either 120573-catenin-dependent or120573-catenin-independent pathways There are generally twopools of 120573-catenin in cells one that is tightly associatedwith cadherins at cell-cell junctions and the other that isldquofreerdquo in the cytosolnucleus The latter pool is involved ingene transcription regulation In the resting state cytoso-licnuclear 120573-catenin must be maintained at a very low level

through the rapid turnover of free 120573-catenin [21] IncludingtheWnt proteins120573-catenin activity is also regulated by tumornecrosis factor [22] N-cadherin and matrix metallopro-teinase (MMP) [23] Moreover ROS liberate the cadherin-sequestered pool of 120573-catenin to promote signaling [24] Thetarget genes of 120573-catenin are known to mediate inflamma-tion angiogenesis and fibrosis through the upregulation of


0

01

01

02

02

03

03

04

04

05

05

0

05

1

15

2

25

3

120573-c

aten

in m

RNA

of a

ctin

Control STZ + vehicle STZ + DP STZ + irbesartan

120573-c

aten

in p

rote

in ex

pres

sion

(IH

C)


lowast

lowast

(A) (B)

(a)

(B) (C) (D)

10120583m

(Alowast)

(b)

Figure 2 Effect of DP and irbesartan treatments on the renal expressions of 120573-catenin in the rats (a) Effect of DP and irbesartan treatmentson the renal mRNA and protein expressions of 120573-catenin as described in the materials and methods section (A) The relative levels of the120573-catenin mRNA were assessed by real-time PCR and the results were normalized to 120573-actin (B) The expression of the 120573-catenin proteinexpressed as a quantitative estimate score (b) The protein expression specific to 120573-catenin ((A)ndash(D)) detected by immunohistochemistry(400x magnification) (A) normal glomerulus in the control (B) STZ + vehicle group (C) STZ + irbesartan (50mg(kgsdotday)) group and (D)STZ + DP (500mg(kgsdotday)) group All values are the means plusmn SD (119899 = 7) lowast119875 lt 005 compared with the STZ + vehicle group

intercellular adhesion molecule [25] plasminogen activatorinhibitor-1 [26] and CTGF [27] which are important factorsparticipating in the pathogenesis and progression of DN [28]Therefore the development of STZ-induced diabetic renaldisease appears to be a complex process involving 120573-catenin

Immunohistochemistry and real-time PCR analysesshowed that the expression of 120573-catenin in the renal cortexwas upregulated in our untreated diabetic rats comparedwith the nondiabetic control rats during the 16 weeks ofthis study A similar study also showed upregulated 120573-catenin levels in both the renal cytosol and nuclei of thestreptozotocin-induced diabetic rats [29] After 12 weeks oftreatment with either DP or irbesartan the decrease in the120573-catenin expression levels was too slight to be significantlydifferent compared with the diabetic control rats Theseresults suggested that DP and irbesartan decrease urinaryalbumin and renal hypertrophy to prevent renal injury inthe early stage of DN through a mechanism that doesnot involve the inhibition of 120573-catenin The explanation is

that in experimental models the kinetics concerning thedevelopment of fibrosis is faster (weeks or a few monthsat best) compared with that of humans (years) Thereforethe treatment in animals might have to be started relativelyearly before the irreversible destruction of the renal structure(ie before reaching a point of no return) The molecularmechanisms that underlie the progression of DN to end-stage renal failure are not well defined thereby limiting accessto potential therapeutic targets Because albuminuria causedby diabetic nephropathy is an independent risk factor forcardiovascular events and death [30] a strategy to protectthe kidney by reducing the effects of early diabetic kidneydisease is the first step in treating this disease To the bestof our knowledge there has been no study to investigate thetherapeutic mechanism of DP on DN via the regulation of 120573-catenin

In summary DP may contribute to the further under-standing of the mechanisms of DN and to the developmentof novel strategies for both its prevention and management


However our understanding of the specificities of 120573-cateninis very limited in DN due to the lack of reliable approachesto selectively assess its function Such studies are importantnot only for the quality control of herbal medicine but alsofor the development of new pharmaceutical products in theprevention and management of diabetic nephropathy

Conflict of Interests

The authors report no conflict of interests

References

[1] D Daneman ldquoType 1 diabetesrdquo The Lancet vol 367 no 9513pp 847ndash858 2006

[2] K Pulkkinen S Murugan and S Vainio ldquoWnt signaling inkidney development and diseaserdquo Organogenesis vol 4 no 2pp 55ndash59 2008

[3] S Kuure A Popsueva M Jakobson K Sainio and H SariolaldquoGlycogen synthase kinase-3 inactivation and stabilization of 120573-catenin induce nephron differentiation in isolated mouse andrat kidney mesenchymesrdquo Journal of the American Society ofNephrology vol 18 no 4 pp 1130ndash1139 2007

[4] C L Lin J YWang Y THuang et al ldquoWnt120573-catenin signalingmodulates survival of high glucose-stressed glomerular mesan-gial cellsrdquo Journal of the American Society of Nephrology vol 17no 10 pp 2812ndash2820 2006

[5] C L Lin J Y Wang J Y Ko et al ldquoDickkopf-1 promoteshyperglycemia-induced accumulation of mesangial matrix andrenal dysfunctionrdquo Journal of the American Society of Nephrol-ogy vol 21 no 1 pp 124ndash135 2010

[6] X J Zhou L Li L J Zhou et al ldquoEffect of compound Danshendropping pills on microalbuminuria in type 2 diabetic patientswith nephropathyrdquo Chinese Journal of New Drugs vol 18 no 15pp 1427ndash1429 2009

[7] P A De Smet ldquoDrug therapy herbal remediesrdquo The NewEngland Journal of Medicine vol 347 no 25 pp 2046ndash20562002

[8] I M Liu T F Tzeng S S Liou and C J Chang ldquoTheamelioration of streptozotocin diabetes-induced renal damageby Wu-Ling-San (Hoelen Five Herb Formula) a traditionalChinese prescriptionrdquo Journal of Ethnopharmacology vol 124no 2 pp 211ndash218 2009

[9] F Zheng Y J Zeng A R Plati et al ldquoCombinedAGE inhibitionand ACEi decreases the progression of established diabeticnephropathy in B6 dbdb micerdquo Kidney International vol 70no 3 pp 507ndash514 2006

[10] X P Zhang J Jiang Y P Yu Q H Cheng and B ChenldquoEffect of Danshen on apoptosis and NF-120581B protein expressionof the intestinal mucosa of rats with severe acute pancreatitisor obstructive jaundicerdquo Hepatobiliary and Pancreatic DiseasesInternational vol 9 no 5 pp 537ndash546 2010

[11] R D Sonawane S L Vishwakarma S Lakshmi M RajaniH Padh and R K Goyal ldquoAmelioration of STZ-induced type1 diabetic nephropathy by aqueous extract of EnicostemmalittoraleBlume and swertiamarin in ratsrdquoMolecular andCellularBiochemistry vol 340 no 1-2 pp 1ndash6 2010

[12] A B Fogo ldquoDiabetic nephropathy itrsquos in the numbersrdquo KidneyInternational vol 61 no 6 pp 2274ndash2275 1998

[13] M Shimamura H Nakagami T Shimosato et al ldquoIrbesar-tan improves endothelial dysfunction abnormal lipid profile

proteinuria and liver dysfunction in Zucker diabetic fatty ratsindependent of glucose and insulin levelsrdquo Experimental andTherapeutic Medicine vol 2 no 5 pp 957ndash961 2011

[14] Chinese Pharmacopoeia Commission Pharmacopoeia of thePeoplersquos Republic of China 2005 (Volumes I) vol 528 2005

[15] J L Zhang M Cui Y He H L Yu and D A Guo ldquoChemicalfingerprint and metabolic fingerprint analysis of Dansheninjection by HPLC-UV and HPLC-MS methodsrdquo Journal ofPharmaceutical and Biomedical Analysis vol 36 no 5 pp 1029ndash1035 2005

[16] H Zhang Y Xu J Wang et al ldquoEffect of Danshensu onfibronectin and collagen-1 secretion induced by high glucosein human peritoneal mesotheial cellsrdquo Zhong Nan Da Xue XueBaoYi Xue Ban vol 36 no 1 pp 44ndash50 2011

[17] W Sun L Y Feng Z J Zhao et al ldquoStudy on antioxidant effectsand inhibition of podocyte apoptosis of PNS on DN ratrdquo ChinaJournal of Traditional Chinese Medicine and Pharmacy vol 26no 5 pp 1061ndash1067 2011

[18] Q N Tu and Y Shi ldquoProtective effect of panax notoginosideon rats with type 1 diabetic nephropathyrdquo Journal of InternalIntensive Medicine vol 13 no 5 pp 241ndash242 2007

[19] F U Zhenchun X U Gang G E Ting et al ldquoEffects of PNS onCTGF in diabetic ratsrdquo Chinese Archives of Traditional ChineseMedicine vol 26 no 5 pp 1042ndash1045 2008

[20] G Xu M L Liu Z C Fu et al ldquoEffect of treatment with panaxnotoginseng saponins and aminoguanidine on nonenzymaticglycosylation in kidney of diabetic ratsrdquo Chinese Journal ofClinicians vol 4 no 4 pp 414ndash420 2010

[21] D Wu and W Pan ldquoGSK3 a multifaceted kinase in Wntsignalingrdquo Trends in Biochemical Sciences vol 35 no 3 pp 161ndash168 2010

[22] Z Al-Aly J S Shao C F Lai et al ldquoAortic Msx2-Wntcalcification cascade is regulated by TNF-120572-dependent signalsin diabetic Ldlrminusminus micerdquo Arteriosclerosis Thrombosis andVascular Biology vol 27 no 12 pp 2589ndash2596 2007

[23] H Quasnichka S C Slater C A Beeching M Boehm G BSala-Newby and S J George ldquoRegulation of smoothmuscle cellproliferation by 120573-cateninT-cell factor signaling involves mod-ulation of cyclin D1 and p21 expressionrdquo Circulation Researchvol 99 no 12 pp 1329ndash1337 2006

[24] C F Lai V Seshadri K Huang et al ldquoAn osteopontin-NADPHoxidase signaling cascade promotes pro-matrix metallopro-teinase 9 activation in aortic mesenchymal cellsrdquo CirculationResearch vol 98 no 12 pp 1479ndash1489 2006

[25] T Zhou Y Hu Y Chen et al ldquoThe pathogenic role of thecanonical Wnt pathway in age-related macular degenerationrdquoInvestigative Ophthalmology amp Visual Science vol 51 no 9 pp4371ndash4379 2010

[26] W He R Tan C Dai et al ldquoPlasminogen activator inhibitor-1is a transcriptional target of the canonical pathway of Wnt120573-catenin signalingrdquo Journal of Biological Chemistry vol 285 no32 pp 24665ndash24675 2010

[27] W Si Q Kang H H Luu et al ldquoCCN1Cyr61 is regulated bythe canonicalWnt signal and plays an important role inWnt3A-induced osteoblast differentiation of mesenchymal stem cellsrdquoMolecular and Cellular Biology vol 26 no 8 pp 2955ndash29642006

[28] F Chiarelli S Gaspari andM LMarcovecchio ldquoRole of growthfactors in diabetic kidney diseaserdquo Hormone and MetabolicResearch vol 41 no 8 pp 585ndash593 2009


[29] T Zhou X He R Cheng et al ldquoImplication of dysregulationof the canonical wingless-type MMTV integration site (WNT)pathway in diabetic nephropathyrdquo Diabetologia vol 55 no 1pp 255ndash266 2012

[30] T Ninomiya V Perkovic B E de Galan et al ldquoAlbuminuriaand kidney function independently predict cardiovascular andrenal outcomes in diabetesrdquo Journal of the American Society ofNephrology vol 20 no 8 pp 1813ndash1821 2009

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Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


of renal function and treatment with irbesartan whichreduces urinary albumin excretion as a positive controlHowever to date whether DP has an effect on the 120573-cateninlevels in early-stage DN remains unclear This study wasdesigned to establish diabetic animal models to observe theinterventional effects of DP on 120573-catenin expression in early-stage diabetic kidney injury rats and to provide pathogenicand theoretical evidence of DN in these rats

2 Materials and Methods

21 Materials This study was performed at the Endocrinol-ogy Department Laboratory of the First Hospital Affiliatedto Xirsquoan Jiaotong University from October 2009 to January2011 Male SD rats (weighing 180ndash200 g clean grade) wereselected from the Experimental AnimalCenter of theMedicalCollege of Xirsquoan Jiaotong University and cared for usingstandardized methods The rats were maintained undertemperature-controlled conditions (22∘C plusmn 2∘C) and artificial12 h lightdark cycles with food and water ad libitum Allrats were randomly divided into four groups the nondiabeticcontrol rats with vehicle treatment (control group 119899 = 21)the streptozotocin- (STZ-) induced diabetic nephropathy ratswith vehicle treatment (STZ + vehicle group 119899 = 21) theSTZ-induced diabetic rats with irbesartan treatment (STZ +irbesartan group 119899 = 21) and the STZ-induced diabetic ratswith DP treatment (STZ + DP group 119899 = 21) In each group7 rats were evaluated every 4 weeks until the end of the studywhich involved a 12-week observation period The study wasapproved by the Institutional Animal Ethical Committee

22 Methods

221 Model Establishment and Drug Administration Strep-tozotocin (10 gL) was single-peritoneally injected at a doseof 60mgkg body weight to establish the diabetic modelThe blood glucose levels derived from the caudal vein wererespectively measured at 72 h and on the 6th day after theinjection The model standard was confirmed by both of theblood glucose levels gt166mmoL The rats in the STZ +irbesartan and STZ + DP groups were administered withirbesartan 50mg(kgsdotday) and DP 500mg(kgsdotday) respec-tively starting 4 weeks after model establishment Every 4weeks 24-hour urine was reserved blood was collected fromthe heart and rats were sacrificed under anesthesia Renalcortex tissue (100mg) was used to extract total RNA and theremaining tissues were fixed with 4 paraformaldehyde forpathological examination

222 Renal Biochemical and Functional Detection Theserum creatinine (serum Cr) concentration was determinedusing a commercial assay kit (BeckmanCoulter Miami FLUSA) The urine creatinine concentration was measuredusing an assay based on Jaffersquos reaction according to the man-ufacturerrsquos protocol (Creatinine Assay Kit) The creatinineclearance rate (Ccr) was calculated using the following equa-tion Ccr[ml(minsdotkg body weight)] = [urinary Cr (120583molL)times urinary volume (ml)serum Cr (120583molL)][11440 (min)]

times [1000body weight (g)]) [8] The urine protein level wasdetected by radioimmunoassay following the manufacturerrsquosprotocol The kidney weightbody weight (KWBW ) wasmeasured

223 Renal Pathological Examination The renal glomerulusand renal tubule mesenchymal lesions were observed fol-lowing HE and PAS staining Twenty renal glomeruli wererandomly selected from the cortical area under 400x magni-fication by an experienced pathologist to measure the indexof mesangial expansion The index of mesangial expansionwas scored by a quantitative estimate of the mesangial zonewidth in each glomerulus expressed as a function of thetotal glomerular area [9] 0 normal glomeruli 1 matrixexpansion occurring in up to 50of the glomerulus 2matrixexpansion occurring in 50ndash75 of the glomerulus and 3matrix expansion occurring in 75ndash100 of the glomerulus

224 Renal Electron Microscopy Morphometric EvaluationThe electron microscopy morphometric evaluation was per-formed as described below A portion of the renal tissues wascut into 1mm cubes fixed in 25 glutaraldehyde and post-fixed in 1 osmium tetroxide The samples were dehydratedin a graded alcohol series and embedded in Epon 812 Fourultrathin sections (60 nm) were cut with a diamond knifecontinuously and stained with uranyl acetate and lead citrateThe thickness of glomerular basement membrane (GBM)was examined with a Hitachi H-7650 (80 kV) transmissionelectron microscope (JEM100SX Japan)

225 Determination of 120573-Catenin Protein Expression byImmunohistochemistry (IHC) Paraffin sections were rou-tinely dewaxed hydrated and dipped in a 003 volumefraction of H

2O2and methyl alcohol Then a microwave was

used to repair the antigen and the tissues were blocked withnormal caprine serum and dribbled with attenuated antibodyI (antirabbit) The rat 120573-catenin antibody was diluted at theratio of 1 50 stained with biotin-labeled caprine antirabbitantibody I and diaminobenzidine restained with hematinedehydrated cleared and sealed with glycerin gelatin Stainedrenal glomeruli and tubules that were randomly selectedfrom 20 sights were observed under 400x magnification byan experienced pathologist The positive staining rate wasevaluated as the following le25 positive cells (+) 26ndash50positive cells (++) 51ndash75 positive cells (+++) and gt75positive cells (++++) each scored as 1 2 3 and 4 pointsrespectively for the statistical analysis [10]

226 Determination of 120573-Catenin mRNA Expression byQuantitative Real-Time PCR Total RNA was extracted fromthe renal tissues with the Trizol method according to themanufacturerrsquos protocol cDNA was synthesized by reversetranscription using a kit (TaKaRaCompanyDalian CA)Theexpression of 120573-catenin mRNA was detected by quantitativereal-time polymerase chain reaction (RT-PCR) 120573-actin wasused as the internal reference The total volume of thePCR was 20 120583L with 30 ng of cDNA as the template Afteran initial denaturation step at 95∘C for 3min 40 cycles




3 Results






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