Obeticholic Acid Prevents Carbon Tetrachloride-induced Liver Fibrosis In Mice And Its Mechanisms

Background and objective Liver fibrosis is one of the most important manifestations of chronic liver injury.At present,the mechanism of liver fibrosis has not been elucidated,and there are no effective treatment drugs.Obeticholic acid（OCA）is a farnesyl receptor X（FXR）agonist.Studies have shown that OCA can reduce acute liver injury caused by D-galactosamine/lipopolysaccharide through inflammation and hepatocyte apoptosis inhibition.At the same time,OCA can reduce bleomycin-induced lung fibrosis in rat,but the mechanism is unknown.The purpose of this study was to investigate the role of OCA in carbon tetrachloride（CCl₄）-induced liver fibrosis in mice and to further explore its possible mechanisms.Methods Ninety-six 8-week old male CD-1 mice were divided into four groups:control group,OCA group,CCl₄group,and CCl₄+OCA group,with 24 mice in each group.In control group,mice were administrated with the solvent of OCA and intraperitoneal injected with corn oil.In OCA group,mice were given OCA gavage（OCA 5mg/kg once a day）.In CCl₄group,mice were intraperitoneal injected with CCl₄（CCl₄0.15ml/kg twice a week）.In CCl₄+OCA group,mice were given OCA gavage（OCA 5mg/kg once a day）and intraperitoneal injection with CCl₄（CCl₄0.15ml/kg twice a week）.After CCl₄treatment,12 mice of each group were sacrificed at 3 weeks or 6 weeks,and fasted for 12h before sacrificed.The body weight and liver weight of mice were recorded,and the liver/body weight coefficient was calculated.Serum samples were collected to detect hepatic biochemical indexes like alanine aminotransferase（ALT）,aspartate aminotransferase（AST）and total bile acid（TBA）.Liver tissues were retained,part of which was fixed in 4%paraformaldehyde and then embedded in paraffin sections.Liver pathological tissues were stained with hematoxylin and eosin（H&E）,Sirius red andα-SMA immunohistochemical staining.The rest part of the liver was immediately frozen in liquid nitrogen and transferred to the refrigerator at-80℃for storage for real-time PCR（RT-PCR）detection of timp-1,mmp-2,mmp-9 and Western blot detection of FXR,TIMP-1,MMP-9,α-SMA,p Smad3.The content of hydroxyproline（Hyp）in the liver tissue was also measured.Results Compared with the control group,CCl₄treatment increased liver weight and liver/body weight coefficient of mice at 3 weeks and 6 weeks,while OCA treatment significantly reduced liver/body weight coefficient in both groups.At the same time,CCl₄treatment significantly increased ALT,AST and TBA in the liver at 3 week group and 6 week group,while OCA treatment significantly reduced the elevation of ALT,AST and TBA.Liver H&E staining showed that,compared with the control group,CCl₄treatment resulted in inflammatory cell accumulation,liver fiber deposition and pseudolobule formation in liver at 3 weeks and 6 weeks,significantly damaging the normal hepatic lobule structure of the liver.OCA treatment reduced liver damage and liver fibrosis in both 3 weeks and 6 weeks groups.Sirius red staining and Hyp content detection in liver tissues showed that CCl₄treatment induced fibrin deposition in liver tissues,and OCA treatment reduced liver fibrin content.CCl₄treatment significantly increased the expression of metalloproteinase timp-1,mmp-2,mmp-9 m RNA and TIMP-1 and MMP-9 protein expression.OCA treatment significantly inhibited the increase of timp-1,mmp-2,mmp-9 m RNA and TIMP-1 and MMP-9 protein expression at 6 weeks.CCl₄treatment increased liver expression ofα-SMA and p Smad3,while OCA treatment reduced CCl₄-induced expression ofα-SMA and p Smad3.Further studies showed that OCA increased the expression of FXR protein in the hepatocyte nucleus.The co-immunoprecipitation experiment showed that OCA increased the interaction between FXR and Smad3.Conclusion Under the conditions of this study,FXR agonist OCA increased the interaction between FXR and Smad3 and alleviated CCl₄-induced liver injury and fibrosis.