Purinergic P2X7 Receptor Mediates Acetaldehyde-induced Hepatic Stellate Cells Activation Via PKC-dependent GSK3? Pathway

Backgrouds:The major spectrum of alcoholic liver disease(ALD),which is induced by long-term and heavy alcohol drinking,includes steatosis,steatohepatitis(alcoholic hepatitis),fibrosis,cirrhosis,and hepatocellular carcinoma(HCC).The role of hepatic stellate cells(HSCs)played in the patheogenesis of alcoholic liver fibrosis is critical.Acetaldehyde,the principal metabolite of ethanol,could promote Collagen I production and deposition of extracellular matrix(ECM)by activating HSCs,the critical cell type involving in the process of alcoholic liver fibrosis.Purine receptor P2X7 R subtype plays an important role in fibrotic diseases and has close association with liver diseases.P2X7 receptor is a unique member of extracellular ATP-activated purinergic signal family and activating P2X7 R contributes to influx of Ca2+as well as the release of pro-inflammatory cytokines.GSK-3?(glycogen synthase kinase-3?),an extensively expressed serine/threonine protein kinase,is previously regarded as a mediator of glycogen metabolism.Except its critical role played in glycogen metabolism,recent studies have reported that GSK-3? also plays its unique role in response to inflammation and the regulation of fibrotic disease.Recent reports have suggested that GSK-3? inhibition could significantly reduce renal fibrosis by suppressing Collagen I synthesis and inflammatory cytokines in renal fibrosis model.Bz ATP,the P2X7 R selective agonist provokes GSK3 phosphorylation and regulates neuronal survival via the PKC-dependent GSK3 pathway.Purposes:Our group established acetaldehyde-induced activation of HSC model to simulate alcoholic liver fibrosis in vitro model.To observe the m RNA and protein expression of purine capable P2X7 receptor in HSC.To explore the effect of P2X7 receptor on cell cycle in acetaldehyde-induced HSC activation model.And to explore in acetaldehyde-induced HSC activation process,whether the expression of various inflammatory cytokines have changes,while observing whether the effect is associated with P2X7 receptor.And to investigate the effect of P2X7 receptor on the expression of ?-SMA and Collagen I during the activation of HSC induced by acetaldehyde,and whether it is affected by PKC-GSK3? signaling pathway.And then further verified the PKC-GSK3? signaling pathway.To provide a new target for the prevention and treatment of alcoholic liver fibrosis.Methods:In this study,stimulating HSC with 200?M acetaldehyde for 48 h was used to simulate the isolated model of alcoholic liver fibrosis.The expression of P2X7 R was observed by Western blot and QPCR.While treating HSC with P2X7 R agonist /inhibitor and RNA interference technique,using flow cytometry to observe the influences of P2X7 R on cell cycle,using Western blot and QPCR to explore the effects of P2X7 R on inflammatory cytokines(involving TNF-?,IL-6,IL-18 and IL-1?)and then using WB method to investigate the function of P2X7 R for ?-SMA and Collagen I levels,and also for PKC-GSK3? signaling pathway and further for the expression of p AKT and p ERK1/2.PKC agonist / PKC inhibitor and GSK3? selective inhibitor were used to explore the effects of PKC and GSK3? on the expression of?-SMA and Collagen I during the activation of HSC induced by acetaldehyde,and to further confirm whether P2X7 R mediates acetaldehyde-induced HSC activation through PKC-GSK3? signal pathwayResults:The Western blot and QPCR results have shown that P2X7 R expression was significantly increased in the activation of HSCs after acetaldehyde treatment.Obviously,activation of P2X7 R by stimulating with P2X7 R agonist Bz ATP significantly promoted acetaldehyde-induced Cyclin D1 expression,cell proportion in S phase,inflammatory response,and also protein and m RNA levels for ?-SMA and Collagen I.In contrast,blockage of P2X7 R or stimulating with the inhibitor A438079 dramatically suppressed acetaldehyde-induced HSCs activation.Furthermore,PKC activation treated with PMA could obviously up-regulate the expression of ?-SMA and Collagen I and the phosphorylation of GSK3?,while PKC inhibition could significantly reduce GSK3? activation.Moreover,GSK3? inhibition harvested a dramatic dropping of the m RNA and protein levels of ?-SMA and Collagen I by suppressing GSK3? phosphorylation.Interestingly,Bz ATP could also significantly increase the phosphorylation of AKT and ERK1/2,while P2X7 R blockage or A438079 could also dramatically suppress the levels of p AKT and p ERK1/2.Taken together,these results suggested that purinergic P2X7 R mediated acetaldehyde-induced activation of HSCs via PKC-dependent GSK3? pathway.