GATA6 Regulates Bile Acid Synthesis In Primary Biliary Cholangitis Through FXR

Objective : GATA-binding protein 6(GATA6)is a classical transcription factor essential for cell differentiation and organogenesis during vertebrate development.About the liver,most GATA6 studies have focused on its effects on liver development,and little is known about the function of the adult liver.PBC is both an autoimmune liver disease and an intrahepatic cholestatic condition.Clinically,jaundice and itching caused by cholestasis are the main symptoms.The role of GATA6 in the pathogenesis of PBC is unclear.This study intends to investigate the expression of GATA6 in the livers of PBC patients,the association between GATA6 and PBC cholestasis,and the mechanism by which GATA6 regulates the nuclear hormone receptor farnesol X receptor(FXR)and subsequently participates in PBC bile acid metabolism.Methods:1.Collected liver pathological sections and clinical data of patients diagnosed with PBC and control patients at the pathology department of the The Second Affiliated Hospital of Kunming Medical University from January 2019 to December 2021;the expression of GATA6 in the liver pathological sections of the PBC group and the control group was detected by immunohistochemistry.2.Female C57BL/6 mice were injected peritoneally with 2-Octynoic acid bovine serum albumin(2OA-BSA)combined with polyinosine-polycytidylic Acid(poly I:C)to form PBC animal model;RNA was extracted from the liver tissues of PBC mice and control mice,and the m RNA expression levels of GATA6 and FXR in the liver were detected by real-time quantitative PCR(q PCR).3.HepG2 cells were treated with andeoxycholic acid(CDCA)and cholic acid(CA),and the m RNA expression level of GATA6 was detected by q PCR.4.HepG2 cells were treated with the FXR agonist GW4064,and GATA6 m RNA expression in HepG2 cells was detected by q PCR.5.The increase of GATA6 in HepG2 cells is CDCA,CA concentration and time dependent.6.The full-length and segmented truncated sequences of the FXR promoter were constructed,and the mechanism of action between GATA6 and FXR was explored by a double luciferin reporter assay and a chromatin immunoprecipitation(Ch IP)assay.7.HepG2 cells with GATA6 knockdown were treated with the FXR agonist GW4064 to detect the expression of bile acid synthesis-related molecules.Results:1.GATA6 was positively expressed in 47.37% of the liver pathological sections of the PBC group,while GATA6 was negative in the liver of the control group;The levels of alkaline phosphatase(ALP)and γ-glutamyltransferase(GGT)were lower in GATA6-positive PBC patients(both P<0.05).2.In PBC mice,GATA6 first increased and then decreased with the progress of modeling,and the expression trend was consistent with FXR(P<0.01).3.GATA6 rose with time in HepG2 cells treated with CDCA and CA,increasing first and then reducing as concentration increased.4.Treatment of HepG2 cells with GW4064 did not affect GATA6(P>0.05).5.In HepG2 cells,GATA6 knockdown decreases FXR(P<0.01 or P<0.05)and increase bile acid synthesis(P<0.05);overexpression of GATA6 increases FXR(P<0.01)and decreases bile acid synthesis(P<0.01).6.Dual-luciferase reporter tests revealed that the FXR promoter was deleted from-1450 to-990,from-990 to-150,the luciferase activity reduced significantly(P<0.01 or P<0.05);Ch IP showed that GATA6 could Combines with FXR promoter-1233～-1223,-518～-508 sites in vivo.7.Using GW4064 to treat GATA6-knockdown HepG2 cells,the level of bile acid synthesis was restored.Conclusions: The abnormal expression of GATA6 in liver of PBC patients was correlated with the degree of PBC cholestasis.Through activating FXR,GATA6 can inhibit bile acid synthesis and alleviate cholestasis.As an FXR activator,GATA6 could serve as a therapeutic target for PBC cholestasis.