The Molecular Mechanisms That Underlie The Liver Fibrosis And Bile Acid Metabolism Of Biliary Atresia

AimsHepatic stallate cells(HSCs)and cholangiocyte proliferation play vital roles in liver fibrosis of biliary atresia.Micro RNAs(mi RNAs)are endogenous small(~22 nt)non-coding RNAs that aberrant mi RNA expression is associated with the development of multiple human diseases.It has been observed recently that mi R-200 s increased and mi R-124 decreased in liver tissues from BA paitents.However,the roles and underlying mechanism of these mi RNAs in hepatic fibrogenesis remains unknown.Here,we investigate the role of mi R-200 b in the activation of immortalized human hepatic stallate cells(HSCs),LX-2 cells.In this study,we futher examined the expression level of mi R-124 in bile duct ligation(BDL)rats and tested its effects on cholangiocytes proliferation in vitro and in vivo.Additionally,we also explored the underlying mechanism of the function of mi R-124 in cholangiocyte proliferation.Cholestasis plays an important role in HSCs activation and cholangiocyte proliferation.The adjuvant glucocorticoids(GCs)therapy in BA appears to be beneficial for jaundice clearance but their effects are not definitive and controversial.The aim of this study is further to provide a mechanistic basis for effects of glucocorticoids on bile acid homeostasis in vivo.MethodsThe cell migration assays were performed with wound-scratch assay and transwell migration assay.Expression of IL-6,Foxa2,and phosphorylated signal transducer activator of transcription 3(STAT3)was anlyszed with western-blot and immunohistochemical assay in patients with biliary atresia(BA)and in rats subjected to bile duct ligation(BDL).Mi RNA expression was determined in BA patients and BDL rats,with mi RNA array and quantitative realtime PCR.Biological functions of mi RNAs were studied using immunoblot,immunohistochemical and proliferation assays.Luciferase reporter assays and Western blots were performed to identify mi RNA targets.Male rats were treated with methylprednisolone for 7 days with sow-release osmotic pumps under physiological and cholestatic status that induced by bile duct-ligation(BDL).Expression of glucocorticoids receptor(GR),FOXA2,and the genes related to bile acid metablism was investigated in patients with BA and in rats with methylprednisolone treatment.Promoter activity was studied in luciferase reporter assays.ResultsOur results indicated that the expression of mi R-200 b was unregulated in the biliary atresia(BA)and associated with liver fibrotic progression.We found that upregulation of mi R-200 b significantly enhanced proliferation and migration of the immortalized human HSCs,LX-2 cells.Our findings also showed that mi R-200 b activates the pathway of phosphatidyl-inositol 3-Kinase(PI3K)/Akt.FOG2 directly binds to p85? and inhibits the activation of the PI3K/Akt pathway.It showed that FOG2 protein levels in LX-2 cells were suppressed significantly by mi R-200 b mimics.FOG2 knockdown by si RNAs activated the PI3K/Akt signaling,which increased cell growth and migration that mimicked the effect of mi R-200 b.Conversely,LY294002,a highly selective inhibitor of PI3 K,could block phosphorylation of Akt and effect of mi R-200 b.We next indicated hepatic interleukin-6(IL-6)expression was significantly elevated in BA patients and BDL rats,while the expression of mi R-124 was dramatically decreased in comparison to controls.Moreover,m RNA levels of STAT3 and IL-6 receptor(IL-6R)were inversely correlated with those of mi R-124.Ectopic expression of mi R-124 inhibited IL-6-mediated cholangiocyte proliferation in vitro and cholangiocyte hyperplasia in vivo,through a mechanism involving direct targeting of the 3'-untranslated region(3'-UTR)of STAT3 and IL-6R.We further demonstrated that mi R-200 family members were significantly upregulated in cholestasis and inhibited FOXA2 expression in cholangiocytes,which further enhanced the expression of IL-6.We lastly show that sustained GCs treatment in rats disrupts the normal changes in systemic bile acid distribution by elevating plasma bile acid levels and reducing fecal bile acid loss.GCs treatment stimulated bile acid absorption in the ileum by increasing expression of the apical sodium-dependent bile acid transporter(Asbt,Slc10a2).Concomitantly,GC enhances liver bile acid uptake through increasing expression of the major hepatocyte basolateral bile transporter(Ntcp,Slc10a1).As evident from reduced a bile acid synthesized rate-controlling enzyme Cyp7a1 expression,GC treatment suppresses hepatic bile acid synthesis.Taken together,GCs can enlarge enterohepatic bile acid circulation,which suggests plasma bile acid levels should be monitored during GCs treating patients with BA.ConclusionsThe expression of mi R-200 b associated with liver fibrotic progression suggests that mi R-200 b might be a potential marker for monitoring the liver fibrosis progress of BA paitens.It suggests a potential mechanism for Akt activation through FOG2 down-regulation by mi R-200 b that can lead to HSC growth and migration.In view of the putative pathogenic role of mi R-200 b in HSCs,mi R-200 b may constitute a potential marker for HSC activation and liver fibrosis progression.We also suggest that mi R-124 inhibits bile duct proliferation in vitro and in vivo by disrupting IL-6/STAT3 signalling,which suggests mi R-124 is a potential target for anti-fibrosis progression of BA.Our results identified a potential molecular mechanism by which FOXA2 depletion might account for the activation of the IL-6R/STAT3 pathway in the cholestatic liver.Our study further provides evidence that GC can enlarge enterohepatic bile acid circulation,which suggests GCs therapy has no benefit for jaundice and bile acid clearance in BA patients.