| Objective:In this study,we investigated the ameliorative effects and mechanisms of Dangfei Liganning Tablets on non-alcoholic fatty liver disease through in vivo and in vitro experiments based on FXR-related signaling pathway-mediated lipid and bile acid metabolism.Methods:1.In vivo experiments:(1)During 24 weeks of high-fat chow feeding,we established an in vivo model of NAFLD in SD rats and treated them with high,medium and low doses of Dangfei Liganning Tablets and Metformin at 23 weeks,respectively,and recorded the changes in body weight of each group of rats weekly.(2)Differences in fat vacuoles and inflammation in the liver of each group of rats were observed by HE staining.(3)Biochemical kits were used to determine the serum TG,TCHO,HDL,LDL,INS,HOMA-IR,AST,ALT,TBA,TNF-α,IL-1βand IL-6 in each group of rats.(4)Western Blot was performed to determine the hepatic intestinal FXR and hepatic BSEP protein expression in each group of rats.The expression of FXR in the liver and intestine,SHP,BSEP,NTCP and ppar-αm RNA in the liver of different groups of rats was assessed by q RT-PCR.(6)A targeted bile acid metabolomics approach was used to analyse differential bile acid metabolites.2.In vitro experiments:(1)To determine the effect of different concentrations of Dangfei Liganning Tablets-containing serum on the activity of Hep G2 cells.(2)To study the effect of Dangfei Liganning on Hep G2 cells,we established a lipid accumulation model using free fatty acids and intervened with different concentrations of Metformin and Dangfei Liganning Tablets-containing serum.The oil red O staining method and kit allowed us to study the cellular lipid deposition and to accurately measure the changes in TG and TCHO in each group of cells.(3)Western Blot method was employed to measure FXR and BSEP protein expression levels in each Hep G2 cell group.(4)q RT-PCR was then employed to detect FXR,SHP,BSEP,and NTCP m RNA expression levels in the same groups.Results:1.In vivo experiments:(1)The body weight of SD rats fed high-fat chow continued to increase,while that of rats fed normal chow changed less.The body mass of rats given Dangfei Liganning Tablets and Metformin tended to decrease compared to the modeling group.(2)Compared with the normal group,the liver of rats in the NAFLD model group showed obvious fat vacuolation,lipid degeneration and inflammatory lesions,and the liver pathological status of rats after the administration of Dangfei Liganning Tablets and Metformin improved to some extent.(3)After serum biochemical tests,the results showed that the levels of TG,TCHO,LDL,INS,HOMA-IR,AST,ALT,TBA,TNF-α,IL-1βand IL-6 were significantly increased(P<0.05,P<0.01)and HDL was significantly decreased(P<0.01)in the NAFLD model group.There were significant improvements in all serum indices in the high,medium and low dose groups of Dangfei Liganning Tablets and Metformin group compared with the model group(P<0.05,P<0.01).(4)Western Blot showed that hepatic and intestinal FXR and hepatic BSEP protein levels were significantly down-regulated in the NAFLD model group compared with the normal group(P<0.01),and that hepatic and intestinal FXR protein levels and hepatic BSEP protein expression were significantly up-regulated in all dose groups of Dangfei Liganning Tablets and Metformin group(P<0.05,P<0.01).(5)The expression levels of FXR,SHP,BSEP,NTCP and ppar-αm RNA were decreased in the liver in the NAFLD model group after q RT-PCR(P<0.01).In the treated group of Dangfei Liganning Tablets,the expression levels of these genes were elevated in the high-dose group compared to the model group,with a highly significant difference(P<0.01).SHP and BSEP m RNA expression levels were significantly upregulated in the mid-dose group(P<0.01),and SHP,BSEP,NTCP and ppar-αm RNA expression levels were significantly increased in the low-dose group(P<0.01).It was shown that in intestinal tissues,the expression levels of FXR m RNA were reduced compared to the normal group with a highly significant difference(P<0.01),when the expression levels of FXR m RNA were significantly improved(P<0.01)in the medium and low doses of FELI Heparin tablets as well as in the Metformin group.(6)By targeted bile acid metabolomics analysis,the results showed that the relative contents of CA,CDCA and DCA were down-regulated,and the relative contents of TCDCA,TUDCA,UDCA and T-α-MCA were significantly increased(P<0.05),and the relative contents of LCA,THDCA,α-MCA andβ-MCA were also up-regulated in the liver samples of rats in the model group compared with the normal group.When the relative levels of CA,CDCA,UDCA and DCA were increased differently in the Dangfei Liganning Tablets administration group,the relative levels of LCA,TCDCA,THDCA,TUDCA,T-α-MCA,α-MCA andβ-MCA were also modulated back,in addition to the relative levels of CA,CDCA,THDCA,TUDCA,UDCA,α-MCA andβ-MCA in the Metformin group.MCA relative levels also showed a tendency to be retraced.And there was no significant correlation between the NAFLD model group and the normal group by PCA(R~2X=0.752).2.In vitro experiments:(1)Compared to the normal group,there was essentially no effect on Hep G2 cell activity by all concentrations of drug-containing serum in Dangfei Liganning Tablets.(2)In the Hep G2 cell-modelling group,including a significant increase in the number of lipid droplets,degree of staining,TG and TCHO(P<0.01),the treatment group of Dangfei Liganning Tablets and Metformin showed a significant improvement,exhibiting a significant decrease in the degree of lipid droplet aggregation,TG and TCHO(P<0.05,P<0.01).(3)A significant decrease in FXR and BSEP protein levels was found in lipid-accumulated Hep G2 cells compared to the control group by WB assay(P<0.01).In contrast,FXR and BSEP protein levels were significantly increased in the high and medium dose groups of Dangfei Liganning Tablets(P<0.05,P<0.01),and the low dose significantly increased BSEP protein levels(P<0.05).Intracellular FXR protein expression levels were similarly significantly increased after Metformin treatment(P<0.01).(4)After q RT-PCR,the results showed that the levels of FXR,SHP and BSEP m RNA in lipid-accumulated Hep G2 cells were significantly decreased(P<0.01,P<0.05),while the expression of NTCP m RNA was significantly increased(P<0.05)compared with the control group.Compared with the model group,high dose of Dangfei Liganning Tablets could significantly elevate the expression of FXR and SHP m RNA in Hep G2 cells(P<0.05,P<0.01),medium dose could significantly elevate the expression of BSEP and SHP m RNA(P<0.05),low dose group could also significantly elevate the expression of SHP and BSEP m RNA(P<0.01),The Metformin group significantly upregulated FXR and BSEP m RNA expression in Hep G2 cells(P<0.01,P<0.05),in addition,all dosing groups significantly downregulated NTCP m RNA levels(P<0.05).Conclusion:1.The overall level experiment showed that Dangfei Liganning Tablets can improve lipid degeneration and inflammation in the liver of NAFLD rats,regulate abnormal serum metabolism,modulate FXR,SHP,BSEP,NTCP and ppar-αtargets,and affect the levels of bile acids such as CA,CDCA,DCA,THDCA,TUDCA,α-MCA,β-MCA and T-α-MCA in the liver,improving NAFLD through the above pathways.2.Cellular level experiments show that Dangfei Liganning Tablets reduced the lipid accumulation in FFAs-induced Hep G2 cells and modulated intracellular FXR,SHP,BSEP and NTCP targets,suggesting that Dangfei Liganning Tablets may affect cellular lipid metabolism through FXR-related pathways. |
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