| Objective:The present study was to investigate the mechanisms of gypenosides significantly improved the abnormal lipid profile in mice induced by high fat diet.A new pathway describing the gypenosides effect on lipid homeostasis via FXR and its binding sites would be revealed after this study.Methods:1.Pharmacodynamics Study on Gypenosides: The C57BL/6 male mice were divided into four groups,including normal diet group(ND),high dat diet group(HFD),gypenosides group(HFD+GP),and simvastatin group(HFD+ST).Mice in HFD,HFD+GP,and HFD+ST group were fed with high fat diet for 38 weeks and treated with 0.1% CMC-Na,gypenosides(250 mg/kg,qd),and simvastatin(20 mg/kg,qd),respectively,from 16 weeks to 38 weeks.ND group,mice were fed with normal diet for 38 weeks.In our previous pilot study,the hypercholesterolemia model could be established at week 16 after HFD treatment,we used additional two groups of mice to control the animal model.At the end point,anesthetize mice and collect blood,liver and duodenum tissues.Mice livers were isolated for HE staining.Serum TC and LDLC levels were measured by commercially available kit.2.The transcriptome analysis on hepatic gene regulation of hypercholesterolemic mice by gypenosides: RNA-Seq technology and analysis of differential gene expression can contribute to understanding of the possible molecular mechanisms of gypenosides regulating lipid metabolism.3.Effects of gypenosides on FXR-mediated regulation of bile acid pathway genes: Expression levels of genes in ND,HFD,and HFD+GP group were checked by the quantitative real-time-PCR.4.Effects of gypenosides on relative changes of bile acids(BAs)in mice liver: The LC-MS/MS was applied to quantify 14 BAs in ND,HFD,and HFD+GP group mice livers.5.Chromatin immunoprecipitation analysized the function of FXR after treamenting with ginsenoside: This study aimed to further investigate the regulating mechanism of FXR using chromatin immunoprecipitation to compare the difference amount of the target DNA binding of FXR.Results:1.Effects of gypenosides on the regulation of lipid metabolism: The high-fat diet treatment significantly increased mouse body weight,TC and LDL levels after 16 weeks(P <0.05).In mouse livers,fat accumulation was also observed.In a word,the hypercholesterolemia model was confirmed for this study.Gypenosides and simvastatin attenuated the abnormally elevated TC and LDL-C levels(P <0.05).In addition,histology data showed that the lipid accumulation in mouse hepatic cells was obviously observed,but disappeared in mouse livers treated by gypenosides and simvastatin.Thus,gypenosides showed a role on lipid regulation,in particular on cholesterol reducing.2.The data of transcriptomics showed that gypenosides could significantly regulate the whole genome,bile acid system and the related genes of drug metabolism system,and the significant genes were analyzed.Gypenosides upregulated the expression of Cyp7a1,Nr1h4(Fxr),Slco1a4,Cyp4a14 and Cyp4a31(P <0.05),but significantly down-regulated the expression of Nr0b2(Shp)and Slc25a4(P <0.05).3.RT-PCR found the gene expression level of Shp was down-regulated while of Cyp7a1,Cyp8b1,Fxr,Lrh1,Jnk2,and Erk1/2 were up-regulated by gypenosides(P<0.05).4.Indicated by LC-MS/MS,gypenosides decreased hepatic levels of TUDCA,GCDCA,and GDCA,but increased that of CDCA,DCA,and TDCA(P<0.05).5.CHIP-qPCR data showed that the binding of FXR to the corresponding regions of Sr-Bi,Ostβ and Bsep were significantly increased(P <0.05)after treated with gypenosides.Conclusion:Through the hypercholesterolemia model,the pharmacological effect of gypenosides on regulating lipid metabolism was verified.It was found that gypenosides can regulate the bile acid metabolic pathway from the level of genes and bile acids.The data suggest that FXR may be the target of gypenosides regulating lipid metabolism. |
