Function Of Brd4 In Bile Acid Metabolism

Objective:Bile acids,the products of cholesterol catabolism in the liver,have been known to facilitate absorption and transport of lipids,regulate cholesterol metabolism as well as intestinal flora homeostasis.Bile acids are maintained at appropriate levels in multiple tissues through the enterohepatic circulation.Under normal physiological conditions,the concentration of bile acids in the liver is low due to potential cytotoxicity.Although regulation of bile acids has been extensively studied,the molecular mechanisms as to how bile acids maintain low concentration in the liver is not well understood.Bromodomain-containing protein 4(Brd4)is a transcriptional and epigenetic regulator.To determine the in vivo function of Brd4 in the liver,we have generated liver Brd4 knockout(CKO)mice using Mx1 Cre-Lox P system.Compared with wild-type(WT)mice under normal diet,CKO mice showed increased serum bile acid levels,enlarged gallbladder,cholestasis,liver fibrosis and intestinal flora disorder.Under high-fat diet,liver damage in CKO mice was aggravated.Furthermore,RNA-sequencing and subsequent verification experiments revealed that Shp,a critical suppressor of bile acid synthesis,was down-regulated in liver of CKO mice,however,Cyp7a1,the rate-limiting enzyme in the classical pathway of bile acid synthesis,was up-regulated.This study will unveil that the pivotal roles of Brd4 in bile acid synthesis.It will also provide new approaches for pathogenic mechanism,prevention and treatment of cholestatic liver disease.Methods:1.Mice genotyping was identified by PCR and Western Blot;2.Analysis the changes of various biochemical indexes in mice Plasma by Elisa;3.H&E and Masson staining were used to evaluate the liver pathology of mice and the fibrosis area was counted relative quantitatively;4.16 S rDNA sequencing of mice feces was carried out to analyze the changes of intestinal flora after Brd4 knockout;5.Perform transcriptome sequencing on the mice liver,analyzed the changes of genes related to bile acid metabolism,and verified the sequencing results using q PCR and Western blot.Results:1.Increased gallbladder volume and bile acid in plasma increases significantly in Hepatic Brd4 knockout mice;2.Hepatic Brd4 knockout causes impaired liver function,accompanied by significant liver fibrosis;3.Hepatic Brd4 knockout increases bile acid and accelerates the process of liver damage in HFD mice;4.Hepatic Brd4 knockout changes the intestinal flora and raises the ratio of Firmicutes/Bacteroidetes;5.Brd4 may regulate Cyp7a1 expression through the FXR/Shp pathway to modulate bile acid synthesis.Conclusion:Hepatic Brd4 knockout reduces the expression of Shp and increases the expression of Cyp7a1,thereby promoting the synthesis of bile acid.Abnormal increasement of bile acid will cause liver fibrosis and impaired liver function.And hepatic Brd4 knockout could accelerate the liver damage process in mice under high-fat diet.In addition,Hepatic Brd4 knockout changes the intestinal flora and raises the ratio of Firmicutes/Bacteroidetes.Therefore,we consider that Brd4 plays an important role in maintaining bile acid homeostasis.