P2Y2 Receptors Promote Inflammation In Alcoholic Liver Injury Via EGFR-ERK1/2 Pathway

Backgrouds: Alcoholic liver disease(ALD)is a serious human health problem that has a high morbidity and mortality in the world.The pathogenesis of alcoholic liver disease is very complex,including the role of ethanol and its toxic metabolites on various cells in the liver and intestine,inflammatory cascade response and reactive oxygen species production.The exact mechanism by which alcohol abuse leads to alcoholic liver injury is not currently clearly.Purposes: Vitro model of alcoholic liver injury was established by establishing ethanol-induced AML-12 cells injury model.We used this model to investigate the protein and m RNA expression of purinergic P2Y2 receptors in AML-12 cells,to explore the effect of P2Y2 receptors on inflammation in ethanol-induced AML-12 cells injury model,and to explore the role of EGFR and ERK1/2 in the pathway in ethanol-induced AML-12 cells injury model.At the same time,C57BL/6 male mice were intraperitoneally injected with ethanol to establish the vivo model of alcoholic liver injury,and we used this model to further verify the role of P2Y2 receptors in alcoholic liver injury.It may provide a new idea for the prevention and treatment of alcoholic liver injury.Methods: In this study,our group established alcohol-induced AML-12 cells injury model by simulating with ethanol.The expression of P2Y2 Rs was observed by WB and QPCR.P2Y2 Rs agonist or inhibitor,si RNA interference technique were used to study the effect of P2Y2 receptors on the inflammatory cytokines TNF-? and IL-1? as well as EGFR and ERK 1/2 phosphorylation.The effect of EGFR and ERK1/2 on the expression of TNF-? and IL-1? in ethanol-induced AML-12 cells injury were investigated by EGFR inhibitor and ERK 1/2 inhibitor.And to further confirmed whether P2Y2 receptors through EGFR-ERK 1/2 signaling pathway to affect alcoholic AML-12 cells injury,one group was given the P2Y2 receptors inhibitor suramin to verify the role of P2Y2 receptors in the in vivo model.Results: In ethanol-induced AML-12 cells injury model,P2Y2 Rs expression was significantly higher than the control group.The use of P2Y2 Rs agonist UTP significantly increased the expression of inflammatory cytokines(TNF-? and IL-1?).And the use of P2Y2 Rs inhibitor suramin and silence P2Y2 Rs also can significantly reduce the expression of inflammatory cytokines.In addition,UTP also increased the phosphorylation of EGFR and ERK1/2.Suramin and silencing P2Y2 Rs showed the opposite effect.In addition,the use of EGFR inhibitor AG1478 and ERK 1/2 inhibitor U0126 indicated that AG1478 could inhibit ERK 1/2 phosphorylation and the expression of TNF-? and IL-1?.U0126 also inhibited ERK 1/2 phosphorylation and reduced inflammation factor expression.Interestingly,suramin also significantly reduced alcohol-induced liver damage in vivo experiments.As HE staining shown,mice fed with suramin effectively alleviated the symptoms of the disorders of hepatic sinusoids and inflammatory cell infiltration.What's more,suramin not only reduced the levels of ALT and AST,but also reduced the protein and m RNA levels of inflammatory factors.Conclusion: P2Y2 receptors were existed in AML-12 cells and probably had a positive influence on inflammation via EGFR-ERK1/2 pathway in alcohol-induced AML-12 cells injury.And in vivo experiments showed that suramin ameliorated alcohol-induced liver injury.