The Role And Mechanism Of O-GlcNAcylation In Nonalcoholic Steatohepatitis

Backgrounds:Nonalcoholic fatty liver disease(NAFLD)is a commonly encountered liver metabolic disease.Nonalcoholic steatohepatitis(NASH)is the severe form and inflammatory subtype of NAFLD,which is histologically characterized by hepatic steatosis,inflammation,hepatocyte ballooning and fibrosis.It was reported that more than 20%of NASH patients will develop fibrosis,cirrhosis,and even liver cancer.At the same time,NASH is also an independent risk factor for metabolic diseases such as obesity and diabetes,as well as cardiovascular and cerebrovascular diseases.However,the progression from simple steatosis to NASH involves a multitude of factors.Hepatic steatosis and lipotoxicity caused by lipid metabolism disorder,accompanied by cellular stress response and cell death,release of damage associated molecular patterns,inflammatory cell activation and other factors,promote NAFLD from simple steatosis to NASH,but the underlying mechanism still need further exploration.O-GlcNAcylation is one of the most important post-translational modifications,which attaches O-GlcNAc moieties to serine or threonine residues of cellular proteins and is mediated by OGT and OGA.O-GlcNAcylation regulates various important cellular processes such as signal transduction,gene expression,protein homeostasis,cellular stress response and cellular metabolism.Disruption of O-GlcNAcylation can lead to various diseases.However,the role of O-GlcNAcylation in NASH is still unclear,especially its role in the inflammatory response of NASH.Aims:(1)To explore the role of O-GlcNAcylation in the occurrence and development of NASH by constructing animal models and cell models of NASH.(2)To identify target proteins of O-GlcNAcylation by mass spectrometry,furthermore,to screen and verify O-GlcNAcylated proteins that may play a role in the occurrence and development of NASH.Methods:(1)The OGA hepatocyte-specific knockout(OGA-LKO)mice and their wild-type(WT)littermates were fed with methionine-choline deficient(MCD)diet or control diet.The degree of hepatic steatosis and fibrosis was evaluated by HE staining,Oil Red O(ORO)staining,and Sirius red staining.Hepatic inflammation was revealed by serum alanine aminotransferase(ALT),serum aspartate aminotransferase(AST),and hepatic F4/80 IHC staining.(2)HepG2 cells were treated with palmitic acid(PA)to establish a cell model of NASH.OGA inhibitor TMG was used to increase the level of O-GlcNAcylation.ORO staining was used to observe intracellular lipid droplets.The content of supernatant ALT/AST as well as the expression levels of cellular inflammatory factor were detected to evaluate hepatocyte inflammatory injury.(3)SK-Hep-1 cells were treated with TMG to increase the level of O-GlcNAcylation,and subsequently subjected to mass spectrometry,which identified differentially expressed proteins between TMG group and control group.At the same time,the O-GlcNAcylation antibody RL2 was used for immunoprecipitation to enrich O-GlcNAcylated proteins,which were identified by mass spectrometry as well.Results:(1)OGA hepatocyte-specific knockout significantly increased the level of OGlcNAcylation and aggravated hepatic steatosis and inflammation upon MCD challenge.F4/80 immunohistochemical staining showed that after feeding with MCD diet,the number of macrophages in the OGA-LKO group was significantly increased,and more inflammatory cell foci were also observed.The detection of ALT and AST showed that liver injury in the OGA knockout group was more severe.(2)Stimulation of HepG2 cells by PA can increase the level of O-GlcNAcylation.At the same time,PA can simulate the lipotoxic stimulus in the case of NASH,promoting hepatic steatosis and inflammatory injury.Inhibition of OGA can aggravate PA-induced lipid droplet formation and inflammatory injury in hepatocytes.(3)Treatment with TMG significantly increased O-GlcNAcylation in SK-Hep1 cell line.Label-free quantitative proteomics showed that there were 187 up-regulated proteins and 224 down-regulated proteins in the TMG-treated group compared with control group.A total of 221 glycosylated proteins were enriched by immunoprecipitation,and these proteins were mainly enriched in RNA metabolism,protein translation,transcription factors and other related pathways.Since O-GlcNAcylation can inhibit the ubiquitination of targeted proteins to improve the expression level of targeted proteins,we intersected the up-regulated protein after TMG treatment with the protein obtained by IP mass spectrometry to obtain 18 candidate O-GlcNAcylation proteins,in which HMGB1,an important factor mediating inflammatory response in NASH,was found.By Western blot and Co-IP,we confirmed that the protein expression level of HMGB1 was elevated as the level of O-GlcNAcylation increases.Co-IP confirmed that HMGB1 could be enriched by RL2 antibody,suggesting that HMGB1 is modified by O-GlcNAcylation.Conclusion:O-GlcNAcylation is of great importance in the development of NASH.OGA hepatocyte-specific knockout significantly increased the level of O-GlcNAcylation,and aggravated MCD diet-induced hepatic steatosis and inflammation.Inhibition of OGA can aggravate PA-induced lipid droplet formation and inflammatory injury in hepatocytes.HMGB1 can be modified by O-GlcNAcylation,which could increase its protein expression level.