The Mechanism By Which ATP Regulates Alcoholic Steatohepatitis Through P2X4 And CD39

Backgrounds: Alcoholic liver disease（ALD）caused by chronic excessive drinking has become one of the most common liver disease types.The inflammatory immune response caused by alcoholic liver injury plays an important role in the development of ALD.Kupffer cells are liver-dwelling macrophages that influence inflammatory immune responses by secreting cytokines and chemokines.As a newly discovered "danger signal",extracellular adenine nucleoside triphosphate（ATP）plays an important role in the pathophysiological process of chronic liver disease,including ALD,by acting on p2-purine receptors on cell surface.P2X4 purine receptor（P2X4）is a typical ligand-gated ion channel belonging to the P2 X family with unique structure and high sensitivity.P2X4 is reported to be the most sensitive of all the purinergic receptors to ATP,and can be activated by ATP to open cationic channels that mediate calcium influx,thereby affecting a range of signaling pathways.Extracellular ATP can be hydrolyzed to phosphate and adenosine monophosphate by adenosine triphosphate diphosphate hydrolase 1（CD39）.It has been reported that P2X4 receptor-mediated ATP-based intercellular communication is a feature of pro-inflammatory macrophages,and CD39 hydrolyzed ATP may be a regulatory target of macrophages.Therefore,this study aims to investigate whether ATPmediated P2X4 and CD39 can be used as therapeutic targets for alcohol-associated steatohepatitis and the interaction between ATP-mediated P2X4 and CD39 in alcoholassociated steatohepatitis.Objective:（1）to investigate the role of ATP-mediated P2X4 and CD39 in alcohol-related steatohepatitis;（2）Explore the-mediated interaction between P2X4 and CD39;Methods: Mice model of alcohol-associated steatohepatitis was established by feeding alcoholic liquid diet in vivo,and the mice were randomly divided into control liquid diet group（Pair-fed）,alcoholic liquid diet group（Et OH-fed）,control liquid diet + Ivermectin group（P2X4 specific agonist,10mg/kg）,alcohol liquid diet group + 5-BDBD（P2X4specific inhibitor,1mg/kg）,alcohol liquid diet group + Ivermectin group（10mg/kg）,alcohol liquid diet group + 5-BDBD（1mg/kg）.Liver injury and lipid accumulation were detected by HE staining and BODIPY staining.The expressions of CD39,P2X4,CD39,IL-1β,IL-6 and TNF-ɑ were detected by Western blot and/or RT-QPCR and/or ELISA.In vitro,RAW264.7 cells were stimulated with alcohol（75m M）for 12 h to establish cell inflammation model.Alcohol stimulated RAW264.7 cells were treated with ATP,POM-1（CD39 specific inhibitor）and 5-BDBD respectively.The expressions of CD39,P2X4,TNF-ɑ,IL-1β and IL-6 were detected by Western blot and/or RT-QPCR and/or ELISA.Results: Compared with Et OH-fed group,intraperitoneal injection of 5-BDBD（P2X4specific inhibitor）reduced serum ALT,AST,TC,TG,IL-1β,IL-6,TNF-α.Histopathological examination of liver showed that alcohol-induced fat vacuole and inflammatory cell infiltration were significantly reduced in the Et OH-fed + 5-BDBD group.In vivo studies have shown that reducing P2X4 expression alleviates alcoholinduced liver injury and inflammation.However,in vitro experiments,exogenous ATP treatment of alcohol-stimulated RAW264.7 cells up-regulated the expression of P2X4 and CD39,and also up-regulated the contents of IL-1β,IL-6 and TNF-α in cell supernatant.Exogenous ATP aggravated alcohol-induced inflammatory response.After treatment with POM-1（CD39 specific inhibitor）,the contents of IL-1β,IL-6 and TNF-α in the supernatant of alcohol-stimulated RAW264.7 cells were up-regulated.Pom-1 decreased the expression of CD39 but up-regulated the expression of P2X4,which also aggravated alcohol-induced inflammatory response.The contents of IL-1β,IL-6 and TNF-α in the supernatant of ALCOHOL-stimulated RAW264.7 cells were decreased by 5-BDBD（P2X4 specific inhibitor）.The experimental results showed that 5-BDBD inhibited the expression of P2X4 and the influx of calcium ions.Reduces NLRP3 activation and thus alcohol-induced inflammation.Conclusion:（1）inhibition of P2X4 expression can reduce alcohol-related steatohepatitis;（2）Exogenous ATP can aggravate alcohol-induced inflammation by activating P2X4;（3）Inhibition of P2X4 expression can reduce the expression of CD39,and inhibition of CD39 expression can hinder ATP hydrolysis and aggravate p2X4-mediated inflammatory response;（4）ATP-P2X4 induces inflammation by mediating calcium influx and activating NLRP3 inflammasome.