Mechanism Of FXR Regulating PLIN5 And Bile Acids In The Process Of Fatty Liver Fibrosis

Non-alcoholic fatty liver disease(NAFLD)has become an increasingly serious global public health problem.There is currently no effective treatment for NAFLD because of its extremely complex pathogenesis.Liver fibrosis is a key process in the progression of NAFLD,and develops into cirrhosis,even hepatocellular carcinoma finally.Therefore,the treatment of liver fibrosis plays an important role in NAFLD.The activation of hepatic stellate cells is an important event in the process of liver fibrosis,for which inhibiting or reversing the activation of hepatic stellate cells is an important approach for treating liver fibrosis.Objective: This study aims to reveal(1)the regulatory mechanism of farnesoid X receptor(FXR)in alleviating liver fibrosis by regulating the perilipin5(PLIN5);(2)the mechanism of action of bile acids in the process of liver fibrosis.Methods:(1)An activated human hepatic stellate cell LX-2 model was constructed by induction of TGF-β1;(2)a quiescent LX-2 model was constructed by induction of retinol and oleic acid;(3)FXR response elements(FXRE)in the upstream region of PLIN5 gene were predicted by the bioinformatics,which were confirmed by a dual luciferase reporter gene system;(4)m RNA and protein levels of α-smooth muscle actin(α-SMA)and Collagen Ⅰ was detected by q PCR and western blot after over-expression of FXR or PLIN5;(5)the formation of lipid droplets was detected by Oil red O staining,and the content of triglyceride(TG)was detected;(6)the changes of liver fibrosis marker genes and lipid droplets in LX-2 cells treated with different bile acids were detected;(7)the activation level of FXR induced by different bile acids was detected by the dual luciferase reporter gene system;(8)the changes of liver fibrosis marker genes in LX-2 cells after over-expression of FXR,over-expression of PLIN5,and formation of lipid droplets were detected;(9)JNK and ERK1/2 phosphorylation of LX-2 treated with bile acids were detected.Results:(1)A reverse repeats-1(IR-1)in the promoter region of the PLIN5 gene was determined;(2)the gene expression of PLIN5 in LX-2 cells can be up-regulated by the FXR activation(p<0.01);(3)over-expression of PLIN5 can promote the formation of lipid droplets and significantly reduce the expression of fibrosis gene induced by TGF-β1(p<0.05);(4)the activation of LX-2 cells cannot be inhibited by the activation of FXR;(5)the activation of LX-2(p<0.05)and FXR(p<0.05)can be induced significantly by 100 μM Chenodeoxycholic acid(CDCA)and Deoxycholic acid(DCA)treatments;(6)the activation of LX-2 cells cannot be inhibited by the activation of FXR induced by CDCA and DCA;(7)the expression of fibrosis genes induced by CDCA and DCA can be reduced significantly by the over-expression of PLIN5 and formation of exogenous lipid droplet;(8)The ERK1/2 phosphorylation in LX-2cells was promoted by CDCA and DCA treatment.Conclusion:(1)A FXRE site in the upstream region of PLIN5 gene was determined,which can bind to FXR,and the over-expression of PLIN5 can promote the formation of lipid droplets and inhibit activation of LX-2 cells,for which FXR may inhibit liver fibrosis partially by regulating the expression of PLIN5;(2)the activation of LX-2 cells and FXR can be promoted by 100 μM CDCA and DCA,but the activation of LX-2 cannot be inhibited by the activation of FXR induced by the above two bile acids.(3)the activation of LX-2 cells induced by CDCA and DCA can be inhibited by over-expression of PLIN5 and formation of exogenous lipid droplets.