Study On The Role And Mechanism Of Transcription Factor RUNX1 High Expression Against Cholestatic Liver Injury

Background and purpose:Cholestasis is a common clinical syndrome that can be caused by a variety of diseases,including genetic disorders,and acquired diseases such as primary biliary cholangitis(PBC),primary sclerosing cholangitis(PSC),viral hepatitis,and biliary obstruction.Regardless of the etiology,elevated levels of bile acids in the blood and liver are characteristic of all forms of cholestasis.Long-term chronic cholestasis can lead to liver fibrosis,cirrhosis,hepatocellular carcinoma,and even liver failure.Treatment for cholestasis is limited,with the only drugs currently approved for treatment being ursodeoxycholic acid(UDCA)and obeticholic acid(OCA);however,one-third of patients respond incompletely or are intolerant to UDCA.Obeticholic acid is approved as a second-line treatment for PBC because of its high price and adverse effects such as pruritus that limit its use.Therefore,it is particularly important to develop new therapeutic strategies for the treatment of cholestatic liver injury.The pathogenesis of cholestasis is not fully understood,and a better understanding of the pathogenic role of bile acids in the development of cholestatic liver injury will provide new strategies for the treatment of cholestatic liver injury.Recent studies have shown that inflammation plays a crucial role in the progression of cholestatic liver disease.When bile acids accumulate in the liver,they cause oxidative stress and mitochondrial damage,which leads to the release of pro-inflammatory cytokines and triggers an inflammatory response.The recruited neutrophils and other immune cells can damage hepatic parenchymal cells,leading to the development of cholestatic liver injury.The Janus kinase/signal transducer and activator of transcription 3(JAK/STAT3)signaling pathway is an essential signaling pathway participating in a broad range of biological processes such as inflammation,apoptosis,aging,and liver regeneration.STAT3 signaling has been shown to be activated in animal models of cholestasis,and its activation attenuates sepsis-induced liver injury in rats,suggesting its role in the hepatic inflammatory response protective effect.However,it is not clear whether JAK/STAT3 signaling mediates bile acid-induced liver inflammation in cholestasis.Runt-related transcription factor 1(RUNX1),widely expressed in cells and organs,is highly conserved in vertebrates.RUNX1 plays a key transcription factor role in the regulation of embryonic development,hematopoiesis,angiogenesis,tumorigenesis,immune response,and especially inflammatory response.Recently,it has been reported that RUNX1 inhibits the activation of NF-κB signaling in respiratory epithelial cells by binding to IKKβ to form protein complexes in the cytoplasm.Genetic deletion of RUNX1 in alveolar epithelial cells enhances lung inflammation after lipopolysaccharide(LPS)treatment,suggesting that RUNX1 acts as a suppressor of the inflammatory response.In contrast,RUNX1 in hepatic sinusoidal endothelial cells promotes the expression of angiogenic and adhesion molecules(e.g.,PECAM1 and VCAM1),and knockdown of RUNX1 in endothelial cells attenuates liver inflammation in a mouse model of nonalcoholic fatty liver disease(NASH),thus suggesting that RUNX1 is a positive regulator of inflammation.The differences in RUNX1 function in these studies suggest that RUNX1 may play different roles in different cells/tissues and diseases.However,the characterization of RUNX1 in hepatocytes is poorly understood and the expression,functional role and regulatory mechanisms in cholestatic liver disease are not yet reported in the literature.Methods:1.Detection of hepatic expression of RUNX1 in patients with cholestasis and mouse models.(1)Reanalysis of single-cell sequencing data from the liver of normal controls and PBC patients to clarify the changes in RUNX1 expression in various types of cells in the liver.(2)Detection of RUNX1 mRNA and protein expression changes in livers of obstructive cholestasis patients(OC)using RT-PCR,Western Blot,and Multiplex fluorescence immunohistochemistry.(3)Detection of RUNX1 expression changes in livers of various cholestasis mouse models,such as BDL,1% CA,0.1% DDC,Abcb4-/-,LPS models,using RT-PCR,Western Blot,Multiplex fluorescence immunohistochemistry and other techniques.2.We introduced Runx1 liver-specific knockout mice and constructed cholestasis mouse models to clarify the functional role of RUNX1.(1)Construct Runx1 liver-specific knockout mice,and construct 1% CA,BDL cholestasis mouse models.(2)Collect sera from cholestatic mouse models,send serum biochemical indexes(liver function indexes,jaundice indexes),and compare the differences in values between groups.(3)Paraffin-embedded and sectioned livers of the cholestasis mouse model were stained and scored pathologically to compare the differences in inflammation,fibrosis,necrosis and bile duct proliferation among the groups.(4)RT-PCR,Western Blot and immunofluorescence were used to detect the differences of chemokines,inflammatory cytokines and immune cell markers among the groups.3.Primary mouse hepatocytes and human hepatocellular carcinoma PLC/RPF/5-ASBT cell lines were used for in vitro experimental mechanism studies.(1)Using RT-PCR,Western Blot,overexpression plasmid transfection,primary mouse hepatocyte isolation and culture,we detected the expression of RUNX1,CCL2,CXCL2 mRNA and nuclear protein RUNX1,and clarified whether bile acid could promote the expression of RUNX1,and whether the increase or decrease of RUNX1 expression could decrease the expression of chemokines CCL2 and CXCL2 expression.(2)TCA stimulation of PLC/RPF/5-ASBT cells to detect changes in RUNX1 nuclear protein.(3)Chromatin immunoprecipitation to confirm whether bile acids promote enhanced binding activity of RUNX1 to the CCL2 and CXCL2 promoters.(4)Detection of whether bile acids stimulate activation of the JAK-STAT3 signaling pathway using Western Blot,chromatin immunoprecipitation,and inhibitor assays.(5)Chromatin immunoprecipitation,dual luciferase reporter gene(truncator,mutant)to clarify the minimal reaction element of STAT3 binding to RUNX1.4.Mechanistic studies in Runx1 liver-specific knockout mouse cholestasis model in vivo.(1)Using Western Blot technique,the expression of Runx1 nuclear protein was detected to clarify that bile acids stimulate the expression of Runx1 nucleus.(2)Detection of nuclear protein Stat3 expression using Western Blot technique to clarify whether bile acids can stimulate JAK-STAT3 signaling pathway activation.(3)Chromatin immunoprecipitation to clarify whether bile acids can promote enhanced binding activity of Runx1 to Cccl2 and Cxcl2 promoters;(4)Chromatin immunoprecipitation to clarify whether bile acids can stimulate the activation of JAK-STAT3 signaling pathway.(4)Chromatin immunoprecipitation to clarify whether bile acids can promote enhanced binding activity of Stat3 to the Runx1 promoter.5.Validate the findings in human obstructive cholestasis liver samples in hepatocyte lines and cholestasis mouse models.(1)To detect whether the JAK-STAT3 signaling pathway is activated using Western Blot technique.(2)Detection of mRNA expression of chemokines(CCL2,CXCL2)and neutrophil markers(ELANE,MPO)in human liver samples with obstructive cholestasis using RT-PCR.(3)Chromatin immunoprecipitation clarified that bile acids promote the binding activity of STAT3 to the RUNX1 promoter and RUNX1 to the CCL2/CXCL2 promoter.Results:1.Hepatic RUNX1 expression was markedly increased in cholestatic patients and mouse models of cholestasis(1)Single-cell sequencing reanalysis revealed that RUNX1 was widely expressed in hepatocytes,including hepatocytes,cholangiocytes,endothelial cells,and immune cells,and that RUNX1 expression was significantly elevated in hepatocytes from PBC patients compared to controls.(2)RUNX1 expression was elevated in the livers of patients with obstructive cholestasis.(3)RUNX1 expression was elevated in the livers of various cholestasis model mice,such as BDL,1% CA,0.1% DDC,Abcb4-/-,LPS models.2.Up-regulation of RUNX1 expression has an effect against cholestatic liver injury(1)Runx1 liver-specific knockout exacerbates liver inflammation and cholestatic liver injury in the BDL,1% CA mouse model.(2)Increased chemokine production in the liver of Runx1 liver-specific knockout mice in a BDL mouse model.(3)Increased neutrophil infiltration in the liver of Runx1 liver-specific knockout mice in a BDL mouse model.3.Transcription factor RUNX1 ameliorates bile acid-induced liver inflammation via JAK-STAT3 signaling pathway in cholestasis(1)Bile acid induces an increase in RUNX1 expression,and overexpression of RUNX1 reduces the expression of chemokines CCL2 and CXCL2 in hepatocytes.(2)Bile acids enhance nuclear expression of RUNX1 and enhance the activity of RUNX1 with the CCL2 and CXCL2 promoters.(3)Bile acid activates JAK-STAT3 signaling pathway activation and enhances STAT3 binding activity to the RUNX1 promoter.(4)JAK-STAT3 signaling pathway is activated in human obstructive cholestasis liver,and STAT3 binds to RUNX1 promoter,and RUNX1 binds to CCL2 and CXCL2 promoters with enhanced activity.Conclusions:This study was validated by in vivo and in vitro experiments,as well as in human obstructive cholestasis livers,and led to the following conclusions.1.elevated hepatic RUNX1 protein expression induced by cholestasis plays a protective role in cholestasis.2.activation of the JAK-STAT3 signaling pathway in cholestasis promotes up-regulation of RUNX1 expression.3.upregulation of RUNX1 expression counteracts inflammation-mediated cholestatic liver injury by suppressing the expression of chemokines CCL2 and CXCL2.