research article inflammatory regulation effect and...

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2013 Article ID 340267 10 pageshttpdxdoiorg1011552013340267

Research ArticleInflammatory Regulation Effect andAction Mechanism of Anti-Inflammatory Effective Parts ofHousefly (Musca domestica) Larvae on Atherosclerosis

Fu Jiang Chu12 Xiao Bao Jin12 Yin Ye Xu1 Yan Ma12 Xiao Bo Li12 Xue Mei Lu1

Wen Bin Liu1 and Jia Yong Zhu12

1 Guangdong Provincial Key Laboratory of Pharmaceutical Bioactive Substances Guangdong Pharmaceutical UniversityGuangzhou Higher Education Mega Center 280 Wai Huan Dong Road Guangzhou 510006 China

2 School of Basic Courses Guangdong Pharmaceutical University Guangzhou Higher Education Mega Center280 Wai Huan Dong Road Guangzhou 510006 China

Correspondence should be addressed to Jia Yong Zhu zhujygdpueducn

Received 18 November 2012 Accepted 27 January 2013

Academic Editor Kashmira Nanji

Copyright copy 2013 Fu Jiang Chu et al This 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

The protein-enriched extracts of housefly larvae were segregated by gel-filtration chromatography (GFC) and then anti-inflammatory activity screening in RAW2647 (induced by LPS) was carried out After acquire the anti-inflammatory effectiveparts its anti-atherosclerotic properties in vivo were then evaluated Results showed that the anti-inflammatory effective parts ofhousefly larvae were low-molecular-weight parts After treated with the effective parts oral gavaged for 4 weeks the atheroscleroticlesions of the mouse were significantly decreased The inflammatory and lipid parameters were also reduced (except HDL whichwas increased) Western blot analysis demonstrated that the effective parts exerted potent inhibitory effect on expression of p65in nucleus and cytoplasm The results of immunofluorescence microscopy analysis also showed that the expressions of p65 bothin cytoplasm and nucleus were significantly reduced The hypothesis that the anti-inflammatory effective parts of housefly larvaepossessed anti-atherosclerosis activity in mouse and the possible mechanism could be associated with the inhibition of expressionand nuclear transfer of NF-120581B p65 could be derived

1 Introduction

Insects and insect derivatives have been widely used in folkmedicine across the world since ancient times [1] The use ofinsects is particularly common in China and in many othercountries including Brazil Mexico India Africa and SouthKorea [2] At present there are approximately 300 medicinalinsects distributed in 70 genera 63 families and 14 orders Anestimated 1700 traditional Chinese medicine prescriptionsinclude medicinal insects or insect-derived crude drugs [3]The housefly (Musca domestica) belongs to the order ofDipteraThe housefly larvae have been used clinically to curemalnutritional stagnation decubital necrosis osteomyelitisecthyma and lip boil which was described by Li Shizhen(1518ndash1593 AD) in the pharmaceutical text of Compendiumof Materia Medica [4] Recently effects of antioxidant [5]

antibacterial and in vitro antitumor properties of the proteinextracts of housefly larvae have been reported [6 7] Addi-tionally the oil of housefly larva could be used as a naturalointment to heal burn wound [8] In our earlier studieswe studied whether the protein-enriched fractionextracts ofhousefly larva could potently inhibit multiple proinflamma-tory responses in atherosclerotic lesions The results showedthat the concentrations of TC TG and LDL were lower inthe extracts treatment group than in the negative controlanimal which was treated with cholesterol-enriched dietand LPS (intraperitoneal injection) Also the expressions ofTNF-120572 IL-1120572 and MCP-1 were also decreased after treatedwith the extracts in vitro [9] However the compositionof the protein-enriched extracts of housefly larva is verycomplex and also the specific antiatherosclerotic mechanismis not clear So the objective of this study was to screen

2 Evidence-Based Complementary and Alternative Medicine

the anti-inflammatory effective parts of housefly larvae and toinvestigate inflammatory regulation effect and action mech-anism of those parts on atherosclerosis In the present studythe anti-inflammatory effective parts of housefly larvae wereobtained by gel-filtration chromatography and (macrophagesinduced by LPS) anti-inflammatory activity screening invitro Then the antiatherosclerotic effects in mouse changesof inflammation-related factors and expression and nucleartransfer of NF-120581B p65 in macrophages were also examined

2 Materials and Methods

21 Preparation of Extracts of Housefly Larvae After theprotein-enriched fractionextracts of housefly larva wereharvested [9] the extracts were redissolved in deionized-distilled water (DDW) and the supernatant was collected bycentrifuge at 3000 rmin 4∘C for 10 minutes and concen-trated by solid polyethylene glycol The gel filtration materialused in the experiment was Sephadex G-75 Sephadex G-75exposed in 005molL Tris buffer (pH 75) was incubated inboiling water for 30min to the column (25 cm times 100 cm)The column was equilibrated with 005molL Tris buffer (pH75) at a constant flow rate of 025 mLmin The concentratedextracts were applied to the column and eluted with thesame buffer 15mL fractions were collected at a flow rate of025mLmin Protein profile was monitored by measuringthe absorbance at 280 nm The fractions were combined atthe peaks of elution curves and dialyzed overnight against002molL Tris buffer (pH 85) and then the vacuum freeze-drying was used to remove the solvents for the next step

22 Screening of Housefly Larvae Extracts forAnti-Inflammatory Activity

221 Cell Culture The mouse macrophage cell lineRAW2647 was purchased from Cell Bank Center ofExperimental Animals Sun Yat-Sen University GuangzhouChina RAW2647 cells grown in Dulbeccorsquos modified Eaglersquosmedium (DMEM) were supplemented with 10 (vv) endo-toxin-free fetal calf serum 2mmol glutamineL and100UmL penicillinstreptomycin at 37∘C in atmosphere of10 CO

2and 95 relative humidity

222 Cell Activation and Treatment 1 times 105 RAW2647 cellswere plated in 24-well plates incubated for 24 h and pre-treated with the indicated concentrations of the extracts ofhousefly larvae (40 120583gmL) or a vehicle for another 2 h thenchallenged with LPS (1 120583gmL) for an additional 18 h

223 Measurement of TNF-120572 and IL-6 The culture super-natants were collected Supernatant levels of tumor necrosisfactor alpha (TNF-120572) and interleukin 6 (IL-6) in culturedRAW cells were measured by ELISA according to the manu-facturerrsquos instructionsThe requested solutionswere providedwith the ELISA compact kits and additional toolkits Within30 minutes optical density of each well was determined bya microplate reader setting at 450 nm and the correctionwavelength setting at 570 nm

224 Evaluation of Cell Morphology At the indicated timesafter treatment of different extracts of housefly larvae thecell-culture medium were washed with PBS and changes incell morphology were examined using a microscope withcomputer imaging system analysis Cells were counted in fourfields of vision and the total number was determined Thenthe change of cellular morphology was observed and thenumber of pseudopodia (foot like projections) of each cellwas counted

23 Evaluation of Antiatherosclerosis Properties

231 Animals Healthy female C57BL6 mice at age of 6weeks old were purchased from Medical Laboratory Ani-mal Center (Guangzhou Guangdong China) All animalsreceived humane care and the present studywas carried out inaccordance with the guidelines for the humane treatment ofanimals set by theAssociation of Laboratory Animal center atGuangdong Pharmaceutical University Guangzhou ChinaAll mice were housed in individual cages respectively andkept for at least 1 week with commercial solid diet undercontrolled conditions (25plusmn2∘C 12 h lightdark cycle 55plusmn5humidity) with free access to food and water before treat-ment

232 Treatment After acclimatization mice were dividedinto 2 groups natural control group (119899 = 6) andmodel group(119899 = 12) Mice in natural control group were treated withstandard diet whereas mice in model group were treatedwith LPS (2mgkg iv) three times a week and atherogenicsolution (Fat-soluble solution was composed of 20 choles-terol and 80 peanut oil and water-soluble solution wascomposed of 10 glucose 10 sodium cholate and 80distilled water gavage) to induce atherosclerosis All animalshad free access to food and water After the model wasestablished all mice in model group were divided into anti-inflammatory effective parts of housefly larvae treated group(200mgkg gavage 119899 = 6) and negative control group(distilled water equivalent to the same dose gavage 119899 = 6)and received the treatment once a day for 4 weeks

233 Measurement of Blood Biochemical Variables After 4weeks all mice were killed and their serum was analyzedfor elevation of total cholesterol (TC) triglyceride (TG) lowdensity lipoprotein cholesterol (LDL-C) and high densitylipoprotein cholesterol (HDL-C) Serum lipids were mea-sured using commercially available kits Total cholesterolHDL-C and triglycerides were measured by standardizedautomatedmethods andLDL-Cwas calculated by the Friede-wald equation LDL-C = TC minusHDL-C minus TG5 The levels ofL-6 and TNF-120572 were also determined (refer to Section 223)

234 Histology and Morphometric Analysis A section of thethoracic aorta and myocardial tissues were fixed in 100mLLbuffered formalin for 24 hours The tissue was embeddedin paraffin and a 4 120583m section was examined throughhematoxylin and eosin (HE) staining Then the histological

Evidence-Based Complementary and Alternative Medicine 3

slides were imaged under bright field using a digital colorCCD camera

24 Analysis of Expression and Nuclear Transfer ofNuclear Factor 120581B (NF-120581B) p65

241 Cell Culture and Treatment The RAW2647 cells wereplated in 24-well plates incubated for 24 h and pretreatedwith the extracts of housefly larvae (40120583gmL) then chal-lenged with LPS (1mgmL) for additional 18 h

242 Western Blot Analysis of Cytoplasmic and Nuclear Pro-tein Extracts from RAW2647 Cells Cytoplasmic and nuclearextracts were prepared from cells using the NE-PER Nuclearand Cytoplasmic Extraction Reagents kit Protein contentswere measured taking bovine serum albumin as a criterionProtein concentration was determined by a commercialprotein assay reagent Proteins of cytoplasmic and nuclearextracts were separated by sodium dodecyl sulfate polyacry-lamide gel electrophoresis (SDS-PAGE) All gels were electri-cally transferred to PVDF membranes before being blockedfor 1 h at RT in 001 TBSTween containing 3 nonfatmilk powder (TBSTween with nonfat milk) Membraneswere incubated with primary antibodies overnight at 4∘C inTBSTweenwith nonfatmilk and thenwashed three times for5min inTBSTweenVisualization of proteinswas performedvia the addition of a secondary antibody conjugated tohorseradish peroxidase (HRP) which was incubated for 1 h atRT in TBSTweenwith nonfatmilkMembranes were washedthree times for 10min in TBSTween Then protein-antibodycomplexes were visualized using Vectastain ABC and TMB(331015840551015840-tetramethylbenzidine) substrate kits Protein bandsand NF-120581B p65 (65 kDa) and 120573-actin (42KD) scanning andquantification of signal intensities were performed usinga Bio-Rad Gel Doc XR densitometer with Quantity Onesoftware Antibodies were used in the following dilutionsrabbit anti-mouse NF-120581B p65 (dilution 1 500) anti-mouse120573-actin (dilution 1 500) All secondary antibodies were usedat the following dilutions goat anti-rabbit IgG 1 1000 goatanti-mouse IgG 1 1000

243 Immunofluorescence-Microscopy Analysis of NuclearTransfer NF-120581B p65 Briefly cells were fixed in 37 (wtvol)formaldehyde in PBS for 10min and permeatedwith acetone-methanol (1 1 volvol) at minus20∘C for 15min Following a30min blocking in PBS with 3 (wtvol) bovine serum albu-min samples were exposed to rabbit polyclonal anti-p65 (PE-conjugated) Antibodies were used at a 1 100 (volvol) dilutedin blocking solution for 1 h at 20∘C washed with PBS andcounterstained with 410158406-diamidino-2-phenylindole (DAPI)(5min) for the identification of nuclei Coverslips were finallymounted on mounting medium and fluorescent images weretaken under the Laser Confocal Microscope

25 Statistical Analysis All data were expressed as themean plusmn standard error of the mean (SEM) Each measure-ment was performed at least in triplicate The Studentrsquos 119905-testwas applied to compare means of data of two independent

samples For multiple comparisons one-way analysis ofvariance (ANOVA) was performed followed by the DunnettrsquosT 3 (ie equal variance was assumed) or Duncanrsquos 119905 tests(ie equal variance was not assumed) where appropriateDifferences were considered statistically significant when119875 lt 005 All statistical analysis was performed with SPSSstatistical software (version 130 for Windows)

3 Results

31 Chromatographic Behavior of Housefly Larvae ExtractsGel chromatography with Sephadex G-75 (separated molec-ules with molecular weights from 3000 to 70000) was usedfor segregation and purification of the extracts of houseflylarvae According to the basic principles of chromatographythe smallmolecules entering into the interior of the gel shouldbe eluted out of the system Molecular weight distributionsand elution curvewere shown in Figure 1The active fractionsof major peaks were pooled concentrated and loadedaccording to the molecular weight (MW) which were recov-ered in three main fractions of the void volume (part I highMW part II middle MW part III lowMW)The percentageof each part was also shown

32 Anti-Inflammation Activity Screening of Gel-FiltrationChromatography Fractions of Housefly Larvae Extracts Theconcentrations of TNF-120572 and IL-6 in the RAW2647 cellsupernatants were measured by ELISA RAW2647 cellstreated with LPS alone resulted in significant increases incytokine production compared to the control group Com-pared with the LPS-treated group part I and part II treatedgroup the levels of TNF-120572 and IL-6 were both significant-ly decreased in part III treated group (119875 lt 001 for all)Although the cytokines inpart II treated group also decreasedcompared with that in LPS-treated group the decrease wasnot as significant as that ofpart III treated group (Table 1)

The cell morphology of the macrophage RAW2647 cellstreated with different fractions of housefly larvae extracts inthe presence or absence of LPS was presented in Figure 2In the non-LPS-stimulated cells the cell morphology gen-erally showed a round form whereas in the LPS-activatedRAW2647 cells the cell morphology changed into an irreg-ular form with pseudopodia developing rapidly The cotreat-ment of LPS with part III inhibited the cell spread and forma-tion of pseudopodia by suppressing cell differentiation But itwas not evident in the other two groups

The above observations indicate that compared withother fractions (part I and II) part III had more signifi-cant anti-inflammatory activity in LPS-stimulatedRAW2647cells In general these results support the hypothesis thatpart III of housefly larvae extracts was the anti-inflammatoryeffective parts

33 Antiatherosclerosis Properties of the Anti-InflammatoryEffective Parts of Housefly Larvae in Mouse After mice weretreated with LPS (iv) and atherogenic solution (gavage)


Table 1 Effect of different gel-filtration chromatography fractions of housefly larvae extracts on the secretion of TNF-120572 and IL-6 in LPS-stimulated RAW 2647 cells (pgmL 119899 = 6)

LPSminus LPS+ LPS+ part I LPS+ part II LPS+ part IIITNF-120572 2565 plusmn 87 18479 plusmn 272 17955 plusmn 271 15790 plusmn 322

lowastlowast

6651 plusmn 129lowastlowast

IL-6 828 plusmn 44 6325 plusmn 82 6247 plusmn 48 5623 plusmn 116lowastlowast


lowastlowast

119875 lt 001 versus LPS group 119875 lt 001 versus part II group

9181716151413121111

040

020

030

010

000

II

I

III

Abso

rban

ce at

280

nm

(a)

0

20

40

60

80

Part I Part IIIPart II

Each

par

t (

)

(b)

Figure 1 Gel filtration profile (Sephadex G-75) of housefly larvae extracts and percentage of each part

LPSminus LPS+ LPS+PartII+LPS+PartI+ LPS+PartIII+

Figure 2Morphological change inmacrophageRAW2647 cells after treatedwithwithout different parts of extracts of housefly larvae (times400)

for 8 weeks the histology and morphometric of thoracicaorta and myocardial tissues (HE staining) and the levelsof TC TG HDL LDL TNF-120572 and IL-6 in serum wereanalyzed Compared with the nature control group the resultof HE staining showed that the aortic walls were thickened(Figure 3) and the myocardial cells became hypertrophicand arranged loosely (Figure 3) Additionally mononuclearinfiltrate both in thoracic aorta and myocardial tissues in themodel groupwere observed (Figure 3)TheTCandTG serumlevels of themodel group were significantly higher than thoseof the normal control group (TC 333plusmn054 versus 131plusmn024TG 328plusmn045 versus 116plusmn031119875 lt 001 for all) (Figure 4)High fat solution reduced HDL-C level (113 plusmn 025 versus068 plusmn 013 119875 lt 001) and increased LDL-C level (142 plusmn 034versus 349 plusmn 041 119875 lt 001) The expression levels of TNF-120572

and IL-6were higher in themodel group (TNF-120572 4655plusmn835versus 25732plusmn1895 IL-6 4747plusmn748 versus 36007plusmn1947119875 lt 001 for all) (Figure 4)

After treatedwith orwithout the anti-inflammatory effec-tive parts for 4 weeks compared with the negative controlgroup the size of atherosclerotic lesions in aortaswas reducedand the inflammatory infiltrates both in thoracic aorta andmyocardial tissues were decreased in effective parts treatedgroup (Figure 5) And also except HDL which was increased(093 plusmn 004 versus 074 plusmn 003 119875 lt 001) the other inflam-matory and lipid parameters associated with atherosclerosiswere reduced (TNF-120572 12632 plusmn 1108 versus 27526 plusmn 1235IL-6 11466plusmn798 versus 31433plusmn1568 TC 352plusmn068 versus612plusmn104 TG 188plusmn042 versus 279plusmn056 LDL 223plusmn025versus 387 plusmn 063 119875 lt 001 for all) (Figure 6)


(A)(a)

(B)

(C) (D)

(b)

Figure 3 Histology and morphology of thoracic aorta and myocardial tissues treated with LPS (iv) and atherogenic solution (gavage) for 8weeks (a) Thoracic aorta in normal control group (times200) (b) Thoracic aorta in model group (times200) (A) Thoracic aorta in normal controlgroup (times400) (B) Thoracic aorta in model group (times400) (C) Myocardial tissues in normal control group (times400) (D) Myocardial tissues inmodel group (times400)

34 Affect of Expression and Nuclear Transfer of NF-120581B p65 ofEffective Parts RAW2647 macrophages were treated with orwithout effective parts and incubated for 18 h with LPS Theprotein expressions of NF-120581B p65 in nucleus and cytoplasmwere measured by western blot assay It was demonstratedthat LPS stimulation resulted in significant increase of p65 innucleus and cytoplasm compared with that of natural controlgroup (119875 lt 001 Figure 7) Effective parts exerted potentinhibitory effect on expression of p65 in nucleus and cyto-plasm compared with that in LPS-treated group (119875 lt 001Figure 7)

To confirm that effective parts inhibit LPS-inducednuclear translocation of NF-120581B p65 protein in RAW2647macrophage confocal microscopy was used to visualise the

expression of p65 in cytoplasm and nucleus (Figure 8) Theresults also showed that the expressions of p65 both incytoplasm and nucleus were reduced significantly (red fluo-rescence intensity declined 119875 lt 001 data not show) com-pared to those in the LPS-treated group

4 Discussion

Traditional medicines such as traditional Chinese medicineuse a great array of insects and their products as drugs Insectsldquoare a kind of large unexplored and unexploited sourcescontaining useful compounds for modern medicinerdquo [10]In china housefly (Musca domestica) larvae have been usedclinically to cure inflammation and inflammation-related


lowastlowast

lowastlowast

lowastlowastlowastlowast

TC TG HLD LDL0

1

2

3

4

5

Bloo

d lip

ids (

mm

olL

)

Normal control groupModel group

(a)

100

200

300

400


lowastlowast

lowastlowast

0

lL-6TNF-120572

Infla

mm

ator

y in

dica

tors

(pg

mL)

(b)

Figure 4 Blood biochemical variables inmice treated with LPS (iv) and atherogenic solution (gavage) for 8 weeks lowastlowast119875 lt 001 versus normalcontrol group

diseases such as osteomyelitis decubital necrosis lip boiland ecthyma ever sinceMingDynasty (1368AD) [4]Duringthe past decade due to the antibacterial activity of houseflylarvae and its products they have been usedmainly in preser-vation [11] environmental protection [12] and inhibition ofmultiresistant bacteria [13] In recent years several reportssuggested that atherosclerosis is a chronic inflammatorydisease [14 15] that can be accelerated by microbial infectionin its early phase [16 17] Based on the above informationthe antiatherosclerotic effect of the protein-enriched extractsof housefly larvae had been previously studied On the basisof preliminary studies the present study tested the effect ofanti-inflammatory effective parts on atherosclerosis in vivoand explored the anti-inflammatory mechanism in vitro

In the present work Gel-filtration chromatography(GFC) was used for proteins segregation in order of molec-ular size large to small which is similar to the method usedby purification of a specific 120573-glucosidase from the digestivefluid of larvae of palm weevil (Rhynchophorus palmarum)[18] and small cationic protein from Tobacco Hornworm(Manduca sexta) [19] Compared with other methods suchas organic solvent precipitation [20] salting out precipitation[21] and high pressureperformance liquid chromatography(HPLC) [22] gel-filtration was the most promising methodwith the advantages of high specificity and efficiency Gelfiltration was carried out with fine grade Sephadex G-75which had optimum range of fractionation (3000ndash70000)The crude protein-enriched extracts of housefly larvae weretested by SDS-page in previous studies which showed thatthere were three parts of proteins with similar weight ofmolecule whichwas consistent with the results of the presentstudy In the present study three main parts of extracts wereobtained based on thedifferent molecular weights So in thenext step the anti-inflammatory activity screening of all thethree main parts of extracts was carried out in vitro

The stimulation of macrophages induced by LPS is auseful protocol in vitro screening to identify new anti-inflammatory compounds [23]The release of TNF-120572 and IL-6 could be used as indicators for anti-inflammatory activity[24] In this study LPS-stimulated RAW2647 macrophageswere used and the levels of TNF-120572 and IL-6 were investigatedThe results of anti-inflammatory activity showed that thepart III (small molecular weight) could reduce pseudopodiaformation and the levels of TNF-120572 and IL-6 significantly Sothis part was considered as the anti-inflammatory effectiveparts of housefly larvae

With these in vitro results the antiatherosclerotic prop-erties in mouse of the anti-inflammatory effective parts ofhousefly larvae were exploredThe atherosclerosis model wasestablished by LPS (iv) and atherogenic solution (gavage) for8 weeks Although most mouse strains are highly resistantto atherosclerosis when the atherosclerosis was induced byLPS and atherogenic diet for several weeks the animal modelcould be established [25] In the present study the athero-genic diet was instead by atherogenic solution to guaranteethat every animal could intake the same amount of cholesteroland fat and ensure the atherosclerotic model could be builtquickly Then the atherosclerotic mice were treated withthe anti-inflammatory effective parts for 4 weeks by oralgavage The hypothesis that the anti-inflammatory effectiveparts of housefly larvae had the effect of antiatherosclerosisin vivo was confirmed The small molecular weight partswere initially postulated to be as antimicrobial peptides [26]Although the present study was not aimed at optimizing themetabolic stability approaches were available for the medic-inal chemist to increase the metabolic resistance againstdegradation as described recently [27] So the effect of anti-atherosclerosis in vivo of effective parts could be explained

Today atherosclerosis is recognized as a complex diseasewith serious inflammation Consistent with its key role


(a)

(b)

(A)

(B)

(C) (D)

Figure 5 Histological and morphometric observation of thoracic aorta and myocardial tissues from atherosclerotic mice treated with anti-inflammatory effective parts of housefly larvae (a) Thoracic aorta in anti-inflammatory effective parts of housefly larvae treatment group(times200) (b) Thoracic aorta in negative control group (times200) (A) Thoracic aorta in anti-inflammatory effective parts of housefly larvae treat-ment group (times400) (B) Thoracic aorta in negative control group (times400) (C) Myocardial tissues in anti-inflammatory effective parts ofhousefly larvae treatment group (times400) (D) Myocardial tissues in negative control group (times400)

in coordinating inflammatory responses numerous studieshave suggested that the transcription factor nuclear factor120581B (NF-120581B) is one of the most important proinflammatorypathways in atherogenesis [28 29] It was reported that TNF-120572 expression was mainly regulated by the transcription factorNF-120581B [30] In the present work the level of TNF-120572 wasdecreased in vitro and in vivo suggesting that regulation ofNF-120581B signal pathway might be one of the possible mecha-nisms underlying anti-inflammatory effect of effective partsIn order to examine this possibility effects on expression and

nuclear transfer of NF-120581B p65 of anti-inflammatory effectiveparts of housefly larvae were detected The results indicatedthat the expression and nuclear transfer of NF-120581B p65 wereinhibited In fact entomic NF-120581B activation in response toimmune challenge was homologous or related to moleculesfound inmammalian signal pathways activated during innateimmune defence Complex signal pathways regulate theinnate immune system of insects with NF-120581B transcriptionfactors playing a key role in the activation of antimicrobialpeptides and other immune genes [31]


Effective parts treatment groupNegative control group

lowastlowast

lowastlowast

lowastlowast

lowastlowast8

6

4

2

0

TC TG HDL LDL

Bloo

d lip

ids

(mm

olL

)

(a)


lowastlowast

lowastlowast

TNF-120572 lL-60

100

200

300

400

Infla

mm

ator

y in

dica

tors

(pg

mL)

(b)

Figure 6 Blood biochemical variables in anti-inflammatory effective parts of housefly larvae treated group and negative control grouplowastlowast

119875 lt 001 versus negative control group

1

2

3

M

LPS treatment groupEffective parts treatment groupNormal control group

lowastlowast

4

1 2 3

p65 expression in cytoplasm

40 kD

50 kD

70 kD

42 kD

65 kD

(a)

lowastlowast

12

10

08

06

1 2 3

15KD

65KD

P65 expression in nucleus


14

(b)

Figure 7 Effects of anti-inflammatory effective parts of housefly larvae on protein expression of NF-120581B p65 in LPS-induced RAW2647macrophages measured by Western blot (a) Anti-inflammatory effective parts of housefly larvae decreased LPS-induced p65 expression incytoplasm 120573-actin was set as control normalization LaneM proteinmark lane 1 LPS-treated group lane 2 anti-inflammatory effective partsof housefly larvae treated group lane 3 natural control group (b) Anti-inflammatory effective parts of housefly larvae inhibited LPS-inducedp65 expression in nucleus Histon H 31 was set as control normalization Lane 1 LPS-treated group lane 2 anti-inflammatory effective partstreatedgroup lane 3 natural control group lowastlowast119875 lt 005 versus LPS-treated group

In a further study the composition and structure ofthe anti-inflammatory effective parts of housefly larvae willbe explored by high-performance liquid chromatographytandem mass spectrometry (HPLC-MSMS) sequencingand bioinformatics analysis

5 Conclusion

Anti-inflammatory effective parts of housefly larvae havethe function of antiatherosclerosis in the mouse There isa possibility that the mechanism could be associated with


DAPI blue Overlay

Nor

mal

cont

rol g

roup

LPS

treat

men

t gro

upEff

ectiv

e par

tstre

atm

ent g

roup

p65 (PE-conjugated) red

Figure 8 Immunofluorescent images of NF-120581B p65 under the laser scanning confocal microscopy in anti-inflammatory effective parts ofhousefly larvae treated group negative control group and normal control group (1000x) NF-120581B p65 was stained with PE (red) and nucleiwere stained with DAPI (blue)

the inhibition of expression and nuclear transfer of NF-120581Bp65

Authorsrsquo Contribution

F J Chu X B Jin and J Y Zhu equally contributed to thiswork

Conflict of Interests

The authors declare that there is no conflict of interests

Acknowledgments

This study was supported by Core Technology ResearchProjects of Strategic Emerging Industries GuangdongProvince China (Grant no 2012A080800016) Science andTechnology Plan Projects Panyu district Guangzhou CityGuangdong Province China (Grant no 2010-Special-12-3)and National Natural Science Foundation of China (Grantno 81274061)

References

[1] E P Cherniack ldquoBugs as drugs part 1 insects The ldquonewrdquoalternative medicine for the 21st centuryrdquo Alternative MedicineReview vol 15 no 2 pp 124ndash135 2010

[2] N A Ratcliffe C BMello E S Garcia TM Butt and P Azam-buja ldquoInsect natural products and processes new treatments forhuman diseaserdquo Insect Biochemistry and Molecular Biology vol41 no 10 pp 747ndash769 2011

[3] Y Feng M Zhao Z He Z Chen and L Sun ldquoResearchand utilization of medicinal insects in Chinardquo EntomologicalResearch vol 39 no 5 pp 313ndash316 2009

[4] L Shizhen ldquoDepartment of insectrdquo in Compendium of MateriaMedica L Shenzhen Ed pp 2289ndash2291 Peoplersquos MedicalPublishing House Beijing China 1981

[5] H Ai F Wang and C Lei ldquoAntioxidant activities of protein-enriched fraction from the larvae of houseflyMusca domesticardquoNatural Product Research vol 22 no 6 pp 507ndash515 2008

[6] L Hou Y Shi P Zhai and G Le ldquoAntibacterial activity andin vitro anti-tumor activity of the extract of the larvae of thehousefly (Musca domestica)rdquo Journal of Ethnopharmacologyvol 111 no 2 pp 227ndash231 2007

[7] S O Park J H Shin W K Choi B S Park J S Oh and AJang ldquoAntibacterial activity of house fly-maggot extracts againstMRSA (Methicillin-resistant Staphylococcus aureus) and VRE(Vancomycin-resistant enterococci)rdquo Journal of EnvironmentalBiology vol 31 no 5 pp 865ndash871 2010

[8] X Feng G Cheng S Y Chen H Yang and W HuangldquoEvaluation of the burn healing properties of oil extraction fromhousefly larva in micerdquo Journal of Ethnopharmacology vol 130no 3 pp 586ndash592 2010

[9] F J Chu X B Jin and J Y Zhu ldquoHousefly maggots (Muscadomestica) protein-enriched fractionextracts (PE) inhibit


lipopolysaccharide-induced atherosclerosis pro-inflammatoryresponsesrdquo Journal of Atherosclerosis andThrombosis vol 18 no4 pp 282ndash290 2011

[10] R W Pemberton ldquoInsects and other arthropods used as drugsin Korean traditional medicinerdquo Journal of Ethnopharmacologyvol 65 no 3 pp 207ndash216 1999

[11] Y Wang X Dang X Zheng J Wang and W Zhang ldquoEffectof extracted housefly pupae peptide mixture on chilled porkpreservationrdquo Journal of Food Science vol 75 no 6 pp M383ndashM388 2010

[12] HCickovaMKozanek IMoravek andP Takac ldquoAbehavioralmethod for separation of house fly (Diptera Muscidae) larvaefrom processed pig manurerdquo Journal of Economic Entomologyvol 105 no 1 pp 62ndash66 2012

[13] X Lu J Shen X Jin et al ldquoBactericidal activity of Muscadomestica cecropin (Mdc) on multidrug-resistant clinical iso-late of Escherichia colirdquo Applied Microbiology and Biotechnol-ogy vol 95 no 4 pp 939ndash945 2012

[14] R Ross ldquoAtherosclerosismdashan inflammatory diseaserdquo New Eng-land Journal of Medicine vol 340 no 2 pp 115ndash126 1999

[15] P Libby Y Okamoto V Z Rocha and E Folco ldquoInflammationin atherosclerosis transition from theory to practicerdquo Circula-tion Journal vol 74 no 2 pp 213ndash220 2010

[16] A Maniar C L Ellis D Asmuth R Pollard and J RutledgeldquoHIV infection and atherosclerosis evaluating the drivers ofinflammationrdquo European Journal of Preventive Cardiology 2012

[17] F C Gibson T Ukai and C A Genco ldquoEngagement of specificinnate immune signaling pathways during Porphyromonasgingivalis induced chronic inflammation and atherosclerosisrdquoFrontiers in Bioscience vol 13 no 6 pp 2041ndash2059 2008

[18] D Y A Yapi D Gnakri S L Niamke and L P KouameldquoPurification and biochemical characterization of a specific 120573rdquo-glucosidase from the digestive fluid of larvae of the palmweevilRhynchophorus palmarumrdquo Journal of Insect Science vol 9article 4 pp 1ndash13 2009

[19] E Ling X J Rao J Q Ao and X Q Yu ldquoPurification andcharacterization of a small cationic protein from the tobaccohornworm Manduca sextardquo Insect Biochemistry and MolecularBiology vol 39 no 4 pp 263ndash271 2009

[20] R Peng J Lin and D Wei ldquoPurification and characterizationof an organic solvent-tolerant lipase from Pseudomonas aerug-inosa CS-2rdquo Applied Biochemistry and Biotechnology vol 162no 3 pp 733ndash743 2010

[21] K Tsumoto M Umetsu I Kumagai D Ejima J S Philo and TArakawa ldquoRole of arginine in protein refolding solubilizationand purificationrdquo Biotechnology Progress vol 20 no 5 pp 1301ndash1308 2004

[22] L R Masterson N Bortone T Yu et al ldquoExpression andpurification of isotopically labeled peptide inhibitors and sub-strates of cAMP-dependant protein kinase A forNMR analysisrdquoProtein Expression and Purification vol 64 no 2 pp 231ndash2362009

[23] A I Medeiros R C Gandolfi A Secatto et al ldquo11-Oxoaerothi-onin isolated from the marine sponge Aplysina fistularis showsanti-inflammatory activity in LPS-stimulated macrophagesrdquoImmunopharmacol and Immunotoxicol vol 34 no 6 pp 919ndash924 2012

[24] B Hernandez-Ledesma C C Hsieh and B O de LumenldquoAntioxidant and anti-inflammatory properties of cancer pre-ventive peptide lunasin in RAW 2647 macrophagesrdquo Biochem-ical and Biophysical Research Communications vol 390 no 3pp 803ndash808 2009

[25] S J Kim J H Park K H Kim W R Lee K S Kimand K K Park ldquoMelittin inhibits atherosclerosis in LPShigh-fat treated mice through atheroprotective actionsrdquo Journal ofAtherosclerosis andThrombosis vol 18 no 12 pp 1117ndash1126 2011

[26] Y Liang J X Wang X F Zhao X J Du and J F XueldquoMolecular cloning and characterization of cecropin from thehousefly (Musca domestica) and its expression in Escherichiacolirdquo Developmental and Comparative Immunology vol 30 no3 pp 249ndash257 2006

[27] J Svenson V Vergote R Karstad C Burvenich J S Svendsenand B de Spiegeleer ldquoMetabolic fate of lactoferricin-basedantimicrobial peptides effect of truncation and incorporationof amino acid analogs on the in vitrometabolic stabilityrdquo Journalof Pharmacology and Experimental Therapeutics vol 332 no 3pp 1032ndash1039 2010

[28] R Gareus E Kotsaki S Xanthoulea et al ldquoEndothelial cell-specific NF-120581B inhibition protects mice from atherosclerosisrdquoCell Metabolism vol 8 no 5 pp 372ndash383 2008

[29] R G Baker M S Hayden and S Ghosh ldquoNF-120581B inflamma-tion and metabolic diseaserdquo Cell Metabolism vol 13 no 1 pp11ndash22 2011

[30] M D Zoysa C Nikapitiya C Oh et al ldquoMolecular evidencefor the existence of lipopolysaccharide-induced TNF-120572 factor(LITAF) and RelNF-kB pathways in disk abalone (Haliotisdiscus discus)rdquo Fish and Shellfish Immunology vol 28 no 5-6pp 754ndash763 2010

[31] J A Hoffmann ldquoThe immune response of DrosophilardquoNaturevol 426 no 6962 pp 33ndash38 2003

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


the anti-inflammatory effective parts of housefly larvae and toinvestigate inflammatory regulation effect and action mech-anism of those parts on atherosclerosis In the present studythe anti-inflammatory effective parts of housefly larvae wereobtained by gel-filtration chromatography and (macrophagesinduced by LPS) anti-inflammatory activity screening invitro Then the antiatherosclerotic effects in mouse changesof inflammation-related factors and expression and nucleartransfer of NF-120581B p65 in macrophages were also examined

2 Materials and Methods

21 Preparation of Extracts of Housefly Larvae After theprotein-enriched fractionextracts of housefly larva wereharvested [9] the extracts were redissolved in deionized-distilled water (DDW) and the supernatant was collected bycentrifuge at 3000 rmin 4∘C for 10 minutes and concen-trated by solid polyethylene glycol The gel filtration materialused in the experiment was Sephadex G-75 Sephadex G-75exposed in 005molL Tris buffer (pH 75) was incubated inboiling water for 30min to the column (25 cm times 100 cm)The column was equilibrated with 005molL Tris buffer (pH75) at a constant flow rate of 025 mLmin The concentratedextracts were applied to the column and eluted with thesame buffer 15mL fractions were collected at a flow rate of025mLmin Protein profile was monitored by measuringthe absorbance at 280 nm The fractions were combined atthe peaks of elution curves and dialyzed overnight against002molL Tris buffer (pH 85) and then the vacuum freeze-drying was used to remove the solvents for the next step

22 Screening of Housefly Larvae Extracts forAnti-Inflammatory Activity

221 Cell Culture The mouse macrophage cell lineRAW2647 was purchased from Cell Bank Center ofExperimental Animals Sun Yat-Sen University GuangzhouChina RAW2647 cells grown in Dulbeccorsquos modified Eaglersquosmedium (DMEM) were supplemented with 10 (vv) endo-toxin-free fetal calf serum 2mmol glutamineL and100UmL penicillinstreptomycin at 37∘C in atmosphere of10 CO

2and 95 relative humidity

222 Cell Activation and Treatment 1 times 105 RAW2647 cellswere plated in 24-well plates incubated for 24 h and pre-treated with the indicated concentrations of the extracts ofhousefly larvae (40 120583gmL) or a vehicle for another 2 h thenchallenged with LPS (1 120583gmL) for an additional 18 h

223 Measurement of TNF-120572 and IL-6 The culture super-natants were collected Supernatant levels of tumor necrosisfactor alpha (TNF-120572) and interleukin 6 (IL-6) in culturedRAW cells were measured by ELISA according to the manu-facturerrsquos instructionsThe requested solutionswere providedwith the ELISA compact kits and additional toolkits Within30 minutes optical density of each well was determined bya microplate reader setting at 450 nm and the correctionwavelength setting at 570 nm

224 Evaluation of Cell Morphology At the indicated timesafter treatment of different extracts of housefly larvae thecell-culture medium were washed with PBS and changes incell morphology were examined using a microscope withcomputer imaging system analysis Cells were counted in fourfields of vision and the total number was determined Thenthe change of cellular morphology was observed and thenumber of pseudopodia (foot like projections) of each cellwas counted

23 Evaluation of Antiatherosclerosis Properties

231 Animals Healthy female C57BL6 mice at age of 6weeks old were purchased from Medical Laboratory Ani-mal Center (Guangzhou Guangdong China) All animalsreceived humane care and the present studywas carried out inaccordance with the guidelines for the humane treatment ofanimals set by theAssociation of Laboratory Animal center atGuangdong Pharmaceutical University Guangzhou ChinaAll mice were housed in individual cages respectively andkept for at least 1 week with commercial solid diet undercontrolled conditions (25plusmn2∘C 12 h lightdark cycle 55plusmn5humidity) with free access to food and water before treat-ment

232 Treatment After acclimatization mice were dividedinto 2 groups natural control group (119899 = 6) andmodel group(119899 = 12) Mice in natural control group were treated withstandard diet whereas mice in model group were treatedwith LPS (2mgkg iv) three times a week and atherogenicsolution (Fat-soluble solution was composed of 20 choles-terol and 80 peanut oil and water-soluble solution wascomposed of 10 glucose 10 sodium cholate and 80distilled water gavage) to induce atherosclerosis All animalshad free access to food and water After the model wasestablished all mice in model group were divided into anti-inflammatory effective parts of housefly larvae treated group(200mgkg gavage 119899 = 6) and negative control group(distilled water equivalent to the same dose gavage 119899 = 6)and received the treatment once a day for 4 weeks

233 Measurement of Blood Biochemical Variables After 4weeks all mice were killed and their serum was analyzedfor elevation of total cholesterol (TC) triglyceride (TG) lowdensity lipoprotein cholesterol (LDL-C) and high densitylipoprotein cholesterol (HDL-C) Serum lipids were mea-sured using commercially available kits Total cholesterolHDL-C and triglycerides were measured by standardizedautomatedmethods andLDL-Cwas calculated by the Friede-wald equation LDL-C = TC minusHDL-C minus TG5 The levels ofL-6 and TNF-120572 were also determined (refer to Section 223)

234 Histology and Morphometric Analysis A section of thethoracic aorta and myocardial tissues were fixed in 100mLLbuffered formalin for 24 hours The tissue was embeddedin paraffin and a 4 120583m section was examined throughhematoxylin and eosin (HE) staining Then the histological









3 Results









lowastlowast




lowastlowast


9181716151413121111

040

020

030

010

000

II

I

III

Abso

rban

ce at

280

nm

(a)

0

20

40

60

80


Each

par

t (

)

(b)








(A)(a)

(B)

(C) (D)

(b)





4 Discussion



lowastlowast

lowastlowast


TC TG HLD LDL0

1

2

3

4

5

Bloo

d lip

ids (

mm

olL

)


(a)

100

200

300

400


lowastlowast

lowastlowast

0

lL-6TNF-120572

Infla

mm

ator

y in

dica

tors

(pg

mL)

(b)








(a)

(b)

(A)

(B)

(C) (D)






lowastlowast

lowastlowast

lowastlowast

lowastlowast8

6

4

2

0

TC TG HDL LDL

Bloo

d lip

ids

(mm

olL

)

(a)


lowastlowast

lowastlowast

TNF-120572 lL-60

100

200

300

400

Infla

mm

ator

y in

dica

tors

(pg

mL)

(b)



1

2

3

M


lowastlowast

4

1 2 3


40 kD

50 kD

70 kD

42 kD

65 kD

(a)

lowastlowast

12

10

08

06

1 2 3

15KD

65KD



14

(b)



5 Conclusion



DAPI blue Overlay

Nor

mal

cont

rol g

roup

LPS

treat

men

t gro

upEff

ectiv

e par

tstre

atm

ent g

roup








Acknowledgments


References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
























3 Results









lowastlowast




lowastlowast


9181716151413121111

040

020

030

010

000

II

I

III

Abso

rban

ce at

280

nm

(a)

0

20

40

60

80


Each

par

t (

)

(b)








(A)(a)

(B)

(C) (D)

(b)





4 Discussion



lowastlowast

lowastlowast


TC TG HLD LDL0

1

2

3

4

5

Bloo

d lip

ids (

mm

olL

)


(a)

100

200

300

400


lowastlowast

lowastlowast

0

lL-6TNF-120572

Infla

mm

ator

y in

dica

tors

(pg

mL)

(b)








(a)

(b)

(A)

(B)

(C) (D)






lowastlowast

lowastlowast

lowastlowast

lowastlowast8

6

4

2

0

TC TG HDL LDL

Bloo

d lip

ids

(mm

olL

)

(a)


lowastlowast

lowastlowast

TNF-120572 lL-60

100

200

300

400

Infla

mm

ator

y in

dica

tors

(pg

mL)

(b)



1

2

3

M


lowastlowast

4

1 2 3


40 kD

50 kD

70 kD

42 kD

65 kD

(a)

lowastlowast

12

10

08

06

1 2 3

15KD

65KD



14

(b)



5 Conclusion



DAPI blue Overlay

Nor

mal

cont

rol g

roup

LPS

treat

men

t gro

upEff

ectiv

e par

tstre

atm

ent g

roup








Acknowledgments


References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















lowastlowast




lowastlowast


9181716151413121111

040

020

030

010

000

II

I

III

Abso

rban

ce at

280

nm

(a)

0

20

40

60

80


Each

par

t (

)

(b)








(A)(a)

(B)

(C) (D)

(b)





4 Discussion



lowastlowast

lowastlowast


TC TG HLD LDL0

1

2

3

4

5

Bloo

d lip

ids (

mm

olL

)


(a)

100

200

300

400


lowastlowast

lowastlowast

0

lL-6TNF-120572

Infla

mm

ator

y in

dica

tors

(pg

mL)

(b)








(a)

(b)

(A)

(B)

(C) (D)






lowastlowast

lowastlowast

lowastlowast

lowastlowast8

6

4

2

0

TC TG HDL LDL

Bloo

d lip

ids

(mm

olL

)

(a)


lowastlowast

lowastlowast

TNF-120572 lL-60

100

200

300

400

Infla

mm

ator

y in

dica

tors

(pg

mL)

(b)



1

2

3

M


lowastlowast

4

1 2 3


40 kD

50 kD

70 kD

42 kD

65 kD

(a)

lowastlowast

12

10

08

06

1 2 3

15KD

65KD



14

(b)



5 Conclusion



DAPI blue Overlay

Nor

mal

cont

rol g

roup

LPS

treat

men

t gro

upEff

ectiv

e par

tstre

atm

ent g

roup








Acknowledgments


References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(A)(a)

(B)

(C) (D)

(b)





4 Discussion



lowastlowast

lowastlowast


TC TG HLD LDL0

1

2

3

4

5

Bloo

d lip

ids (

mm

olL

)


(a)

100

200

300

400


lowastlowast

lowastlowast

0

lL-6TNF-120572

Infla

mm

ator

y in

dica

tors

(pg

mL)

(b)








(a)

(b)

(A)

(B)

(C) (D)






lowastlowast

lowastlowast

lowastlowast

lowastlowast8

6

4

2

0

TC TG HDL LDL

Bloo

d lip

ids

(mm

olL

)

(a)


lowastlowast

lowastlowast

TNF-120572 lL-60

100

200

300

400

Infla

mm

ator

y in

dica

tors

(pg

mL)

(b)



1

2

3

M


lowastlowast

4

1 2 3


40 kD

50 kD

70 kD

42 kD

65 kD

(a)

lowastlowast

12

10

08

06

1 2 3

15KD

65KD



14

(b)



5 Conclusion



DAPI blue Overlay

Nor

mal

cont

rol g

roup

LPS

treat

men

t gro

upEff

ectiv

e par

tstre

atm

ent g

roup








Acknowledgments


References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















lowastlowast

lowastlowast


TC TG HLD LDL0

1

2

3

4

5

Bloo

d lip

ids (

mm

olL

)


(a)

100

200

300

400


lowastlowast

lowastlowast

0

lL-6TNF-120572

Infla

mm

ator

y in

dica

tors

(pg

mL)

(b)








(a)

(b)

(A)

(B)

(C) (D)






lowastlowast

lowastlowast

lowastlowast

lowastlowast8

6

4

2

0

TC TG HDL LDL

Bloo

d lip

ids

(mm

olL

)

(a)


lowastlowast

lowastlowast

TNF-120572 lL-60

100

200

300

400

Infla

mm

ator

y in

dica

tors

(pg

mL)

(b)



1

2

3

M


lowastlowast

4

1 2 3


40 kD

50 kD

70 kD

42 kD

65 kD

(a)

lowastlowast

12

10

08

06

1 2 3

15KD

65KD



14

(b)



5 Conclusion



DAPI blue Overlay

Nor

mal

cont

rol g

roup

LPS

treat

men

t gro

upEff

ectiv

e par

tstre

atm

ent g

roup








Acknowledgments


References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a)

(b)

(A)

(B)

(C) (D)






lowastlowast

lowastlowast

lowastlowast

lowastlowast8

6

4

2

0

TC TG HDL LDL

Bloo

d lip

ids

(mm

olL

)

(a)


lowastlowast

lowastlowast

TNF-120572 lL-60

100

200

300

400

Infla

mm

ator

y in

dica

tors

(pg

mL)

(b)



1

2

3

M


lowastlowast

4

1 2 3


40 kD

50 kD

70 kD

42 kD

65 kD

(a)

lowastlowast

12

10

08

06

1 2 3

15KD

65KD



14

(b)



5 Conclusion



DAPI blue Overlay

Nor

mal

cont

rol g

roup

LPS

treat

men

t gro

upEff

ectiv

e par

tstre

atm

ent g

roup








Acknowledgments


References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















lowastlowast

lowastlowast

lowastlowast

lowastlowast8

6

4

2

0

TC TG HDL LDL

Bloo

d lip

ids

(mm

olL

)

(a)


lowastlowast

lowastlowast

TNF-120572 lL-60

100

200

300

400

Infla

mm

ator

y in

dica

tors

(pg

mL)

(b)



1

2

3

M


lowastlowast

4

1 2 3


40 kD

50 kD

70 kD

42 kD

65 kD

(a)

lowastlowast

12

10

08

06

1 2 3

15KD

65KD



14

(b)



5 Conclusion



DAPI blue Overlay

Nor

mal

cont

rol g

roup

LPS

treat

men

t gro

upEff

ectiv

e par

tstre

atm

ent g

roup








Acknowledgments


References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















DAPI blue Overlay

Nor

mal

cont

rol g

roup

LPS

treat

men

t gro

upEff

ectiv

e par

tstre

atm

ent g

roup








Acknowledgments


References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article inflammatory regulation effect and...

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