Mechanism Of CSE Involved In Cholestatic Liver Injury

Background and objective: Cholestatic liver injury(CLI)is a group of caused by the accumulation of abnormal bile production,secretion,and excretion.It can damage hepatocytes and cholangiocytes and eventually progress to liver fibrosis and cirrhosis which can lead to life-threatening liver failure.Cystathionine-γ-lyase(CSE)is a key enzyme in the last step of the reverse sulfur pathway in mammals.Studies have suggested that CSE plays a critical role in acute liver failure,non-alcoholic steatohepatitis,and other liver diseases.However,the role of CSE in CLI remains unclear.This study intends to explore the role and mechanism of CSE in CLI so as to provide new ideas and novel targets for the treatment of CLI.Methods:1.Analysis of CSE expression characteristics in patients with CLIDetection of CSE expression in liver tissues of patients with primary biliary cholangitis(PBC),primary sclerosing cholangitis(PSC),and cholestatic liver fibrosis by immunohistochemical staining(IHC)and reverse transcription quantitative real-time polymerase chain reaction(RTq PCR),and clarify the cellular localization of CSE in liver tissue by immunofluorescence technique.2.Analysis of CSE expression characteristics in CLI animal modelsThe expression characteristics of CSE in the liver tissues of bile duct ligation(BDL),3,5-diethoxycarbonyl-1,4-dihydrocollidine(DDC)feeding-induced CLI animal models were detected by IHC,RT-q PCR,and Western Blot,and the cellular localization of CSE was performed by immunofluorescence.3.Analysis of the expression characteristics of CSE in hepatocytes cultured with bile acids in vitroBile acids exposure in AML12 hepatocytes and primary mouse hepatocyte was performed,and the expression of CSE was detected by RTq PCR.4.Exploration of the role of high expression of CSE in CLI1)Mouse models of CLI induced by BDL-1W and 2W,DDC feeding2 W and 3W were constructed.Meanwhile,those mouses were daily intraperitoneally injected with D,L-Propargylglycine(PAG),an inhibitors of CSE,until the end of the experiment.Hematoxylin and eosin(HE),IHC and serum liver function tests were used to investigate the indicators of injury in CLI models.Liver fibrosis was evaluated by Masson’s Trichrome,Sirius Red staining,IHC,Western Blot and RT-q PCR.2)CSE was overexpressed in AML12 hepatocytes using gene recombination technology,the CCK8 assay was used to detect cell viability of AML12 exposed to bile acid after CSE overexpression.5.Exploration of the mechanism of CSE involved in CLI1)Differentially expressed genes in the sham operation group,BDL group,and BDL+PAG group were identified using RNA sequencing analysis.In addition,to detect the signaling molecules and pathways of CSE involved in CLI,gene ontology and pathway enrichment analysis of differentially expressed genes of the three groups were also performed.2)RT-qPCR,Western Blot,and other methods were done for verifying the changed signaling molecules in RNA sequencing analysis.3)The potential downstream signaling pathways of the altered signaling molecules in RNA sequencing were detected by Western Blot to further explore the mechanism of CSE involved in CLI.Results:1.Compared with the control group,the expression of m RNA and protein levels of CSE in the liver tissue of patients with PBC,PSC,and cholestatic liver fibrosis was significantly increased,and the elevated CSE was mainly expressed in hepatocytes.2.The expression of CSE in liver tissue was markedly up-regulated in CLI mouse models induced by BDL-1W and 2W,DDC-2W and 3W.Moreover,the elevated CSE was mainly located in hepatocytes.3.The expression of CSE was dose-and time-dependently elevated in AML12 hepatocytes exposed to chenodeoxycholic acid,and the m RNA level of CSE in primary hepatocytes of mice was also significantly increased compared with the control group after bile acid culture.4.The CSE inhibitor PAG reduced hepatic inflammatory cell infiltration and collagen deposition,and improved liver function and fibrosis indicators in animal models of CLI,in addition,the cell viability of AML12 exposed to bile acid after CSE overexpression was decreased.5.In the RNA sequencing analysis,GO and KEGG enrichment analyses indicated that CSE inhibitor PAG could improved CLI was related to inflammatory pathways,and GSEA enrichment analysis indicated that CSE inhibitor PAG improved CLI that was related to oxidative stress.Notably,the above results were verified in animal models of CLI,PAG could inhibit the expression of inflammatory factors(IL-1β,IL-6,TNF-α)and chemokines(CXCL1 and CXCL2)induced by BDL and DDC feeding.In addition,PAG could also inhibit the expression of total antioxidant capacity and malondialdehyde in BDL and DDC feeding models,and promote the expression of superoxide dismutase in BDL and DDC feeding models.4.In terms of detection of possible downstream signaling pathways of inflammation and oxidative stress,PAG inhibited the expression of phosphorylated mixed lineage kinase domain-like protein(p-MLKL)and phosphorylated receptor interacting protein kinase 3(p-RIP3)at the protein level in BDL animal model,and also inhibited the expression of p-MLKL at the protein level in DDC feeding animal model,indicating that PAG can improve the hepatic necroptosis in CLI.In addition,overexpression of CSE in AML12 cells exacerbated necroptosis after exposure to bile acids.Conclusions:1.CSE is mainly expressed in hepatocytes,and its expression is significantly increased in patients with CLI.2.High expression of CSE is involved in the development of CLI.3.High expression of CSE can promote necroptosis of hepatocytes.4.The mechanism of CSE involved in CLI is related to inflammation and oxidative stress.