Study On The Effects Of 2-dodecyl-6-methoxycyclohexa-2,5-Diene-1,4-dione Isolated From The Roots Of Averrhoa Carambola On Insulin Resistance In Mice

Objective:To investigate the effects and potential mechanisms of 2-dodecyl-6-methoxycyclohexa-2,5-diene-1,4-dione,(DMDD)isolated from the roots of Averrhoa carambola on high-fat,high-sugar diet induced insulin resistance in mice.Methods:In this study,six to eight weeks old male C57BL/6J mice were randomly divided into two groups:normal diet(ND or Normal)group and high-fat,high-sugar diet(HFSD)group,HFSD group were fed a high-fat,high-sugar diet for 12 weeks,subsequently the mice which body weight were 20%higher than that of the ND group and fasting blood glucose(FBG)≧7.8mmol/L were randomly divided into five groups:model group,metformin group,DMDD low dose(DMDDL)group,DMDD middle dose(DMDDM)group,and DMDD high dose(DMDDH)group.The animals were orally administered DMDD(12.5,25.0,or 50 mg·kg-1 of body weight per day)and metformin(280 mg/kg of body weight per day)once a day for 4 weeks,and the mice in ND and HFSD groups received an equal volume of distilled deionized water.Body weight and FBG were monitored weekly.Intraperitoneal glucose tolerance test(IPGTT)was performed at the end of week 14,Insulin tolerance test(ITT)was conducted during the middle of week 15 and Insulin resistance test(IRT)was performed at the beginning of week 16.After 4 weeks of treatment,the mice were fasted overnight,and blood was then collected from the retro-orbital venous sinus.Subsequently,the mice were executed,and the following indicators were measured:1.Body length and abdominal girth were measured;the livers,pancreas and epididymal fats were collected and weighted;The serum levels of total cholesterol(TC)and triglycerides(TG)were measured with enzyme-linked colorimetry assay;The content of malondialdehyde(MDA),the activities of total superoxide dismutase(T-SOD)and reduced glutathione(GSH-PX)in liver homogenates were determined using commercial assays.2.The serum levels of insulin,Free fatty acids(FFA),Tumor necrosis factor-a(TNF-a),Interleukin-6(IL-6),Leptin(LEP),Adiponectin(ADP)and Resistin were determined by Enzyme-linked immunosorbent assay(ELISA);Insulin resistance index(IRI)and Insulin sensitivity index(ISI)were calculated by formulas;Areas under the curve(AUCs)of IPGTT,ITT,and IRT were calculated by trapezoidal method.3.The pathological changes of liver,pancreas and adipose tissue were observed by regular Hematoxylin-eosin(HE)staining;The protein expression of Toll-like receptor 4(TLR4),Myeloid differentiation factor 88(MyD88),Nuclear factor-kappa B(NF-κB),Cysteinyl aspartate specific proteinase-3(Caspase-3),Cysteinyl aspartate specific proteinase-8(Caspase-8),Cysteinyl aspartate specific proteinase-9(Caspase-9),Bcl-associated X protein(Bax)and B-celllymphoma-2(Bcl-2)in the liver and pancreas tissue were analyzed by immunohistochemistry;Meanwhile,terminal-deoxynucleoitidyl transferase mediated nick end labeling(TUNEL)staining was used to determine the number of positive cells in pancreas samples;4.The mRNA expression of TLR4,MyD88,NF-κB,TNF-α and IL-6 in liver and adipose tissue were evaluated using Real-time quantitative polymerase chain reaction(RTQ-PCR).Results:1.Compared with the model group,the body weight,epididymal fat,the proportion of the liver,FBQ serum level of TC and TG,content of MDA were decreased by DMDD(P<0.01),while the activities of T-SOD and GSH-PX were increased by DMDD.However,body length,abdominal girth,and food intake were not obviously different;2.Compared to the model group,the serum levels of Insulin,FFA,TNF-α,IL-6,LEP,Resistin and IRI were decreased by DMDD;Meanwhile,AUCs of the three tests were decreased by DMDD.However,the level of ADP and ISI were increased by DMDD;3.H&E staining demonstrated that the area of adipose cells were reduced,and there were less fat deposits in the adipocytes of the DMDD and metformin groups;hepatic steatosis was remarkably improved by treatment with metformin and DMDD;Compared to the model group,after the treatments with metformin and with DMDD,the cytoarchitecture of pancreatic islet cells were effectively recovered.Immunohistochemical analysis revealed that the liver and pancreatic tissue expression levels of TLR4,MyD88,NF-κB and the expressions of apoptosis-related regulators(including caspase-3,caspase-8 and caspase-9)and the apoptosis-induced protein Bax were markedly down-regulated by DMDD,whereas the expression of the anti-apoptotic Bcl-2 protein was notably increased,compared with the model group.In addition,the number of TUNEL-positive cells in model group markedly increased compared with that in the normal group,while the mice through the post-treatment showed a significant decrease in cells positively stained with TUNEL when compared with the model group.4.RTQ-PCR analysis revealed that the mRNA expression levels of TLR4,MyD88,NF-κB,TNF-α and IL-6 were upregulated in the liver and adipose of the model group compared with the levels observed in the normal group.Following the DMDD and metformin treatment,the mRNA expression levels of TLR4,MyD88,NF-κB,TNF-α and IL-6 were downregulated.Conclusion:The study shows that DMDD has potential benefts for the treatment of obesity-induced insulin resistance,and its effects may be associated with the improvements of obesity,hyperglycemia,hyperlipidemia,antioxidant,anti-inflammatory,anti-apoptotic.What’s more,the possible mechanism of improving IR may be associated with the TLR4/MyD88/NF-KB signaling pathway.