P2X4 Purinergic Receptor Regulates Activation Of Hepatic Stellate Cells In Alcohol-induced Liver Fibrosis

Backgrounds:Alcoholic liver disease（ALD）is very common,within the range of liver diseases,and its main causes are long-term alcohol addiction and excessive drinking.Alcoholic liver fibrosis is one of the important factors in the development of alcoholic chronic liver disease.Liver fibrosis is a reaction of scar tissue formation due to the imbalance between the deposition and absorption of extracellular matrix（ECM）.In this process,the activation of HSC（hepatic stellate cells）as a key step has attracted much attention.P2X4 purinoceptor（P2X4）,as one of the purinoceptor families,has been reported to play an important role in a variety of liver diseases.It has been reported that inhibition of P2X4 receptor can alleviate liver fibrosis by reducing the activation of myofibroblasts,and hepatic stellate cells are the main source of myofibroblasts.Phosphorylation of PI3K/AKT pathway,as a classical pathway affecting liver fibrosis,has been reported to be closely related to the activation of hepatic stellate cells.Previous studies have also shown that TGF-βsignaling pathway affects the activation of hepatic stellate cells,and this signaling pathway is inextricably linked to the P2 receptor family in other diseases.Therefore,we aimed to investigate whether inhibition of P2X4receptor attenuates alcohol-induced liver fibrosis directly by inhibiting phosphorylation of the PI3K/AKT pathway and reducing hepatic stellate cell activation,and whether inhibition of P2X4 receptor attenuates alcohol-induced liver fibrosis indirectly by reducing TGF-βrelease.Objective:（1）To explore the relationship between P2X4 and PI3K/AKT pathway and its role in alcohol-related liver fibrosis;（2）To explore the relationship between P2X4and TGF-βsignaling pathway and its role in alcohol-related liver fibrosis.Methods:In the animal model experiment in vivo,compared with the Et OH-fed group,the serum indexes（AST,ALT）,collagen deposition in the pathological section andα-SMA,Col1 in the tissue were increased in the CCL₄group.Compared with Et OH-fed+CCL₄group,the liver pathological sections were significantly improved after intraperitoneal injection of 5-BDBD,the HE staining showed that the number of fat vacuoles was reduced,the deposition of collagen was improved,and the expression of tissue fibrosis was significantly reduced.Therefore,the results of in vivo experiments show that inhibiting the expression of P2X4 can alleviate alcohol-related liver fibrosis.In vitro,the expressions ofα-SMA,Col1 and P2X4 in T6 cells stimulated by acetaldehyde were increased in varying degrees,while the expressions ofα-SMA,Col1and P2X4 in T6 cells pretreated with 5-BDBD when compared with the model group were decreased in varying degrees.And the phosphorylation of PI3K/AKT signaling pathway was inhibited in the 5-BDBD group.By cell transfection,P2X4 was silenced in T6 cells,and compared with the model group,fibrosis indicators were also significantly down-regulated and PI3K/AKT phosphorylation was reduced.In vitro experiments showed that after administration of P2X4 inhibitor 5-BDBD or gene silencing,alcoholic liver fibrosis was alleviated by reducing PI3K/AKT pathway phosphorylation.The expression of TGF-βin the supernatant of the remaining RAW264.7cells was increased in the model group（acetaldehyde 200μm stimulation for 48 h）compared with the normal group,while it was decreased in the 5-BDBD group.The expressions ofα-SMA and Col1 were increased in the supernatant treated with acetaldehyde,but decreased in the 5-BDBD treated group.This in vitro study suggests that inhibition of P2X4expression may attenuate alcoholic liver fibrosis by reducing TGF-βsecretion.Results:Compared With Et OH-fed group,the serum AST,ALT,α-SMA and Col1 in Et OH-fed+CCL₄group were significantly increased,while compared with Et OH-fed+CCL₄group,the serum AST,ALT,α-SMA and Col1 were significantly decreased after intraperitoneal injection of 5-BDBD.Liver pathological sections were significantly improved,HE staining showed that fat vacuoles were reduced,collagen deposition was improved,and the expression of tissue fibrosis indicators was significantly reduced.Therefore,the results of in vivo experiments show that inhibiting the expression of P2X4 can alleviate alcohol-related liver fibrosis.In vitro,the expressions ofα-SMA,Col1 and P2X4 in T6 cells stimulated by acetaldehyde were increased in varying degrees,while the expressions ofα-SMA,Col1 and P2X4 in T6 cells pretreated with 5-BDBD were decreased in varying degrees compared with the model group,and the phosphorylation of PI3K/AKT signaling pathway was inhibited in the 5-BDBD group.By cell transfection,P2X4 was silenced in T6 cells,and compared with the model group,fibrosis indicators were also significantly down-regulated and PI3K/AKT phosphorylation was reduced.In vitro experiments showed that after administration of P2X4 inhibitor 5-BDBD or gene silencing,alcoholic liver fibrosis was alleviated by reducing PI3K/AKT pathway phosphorylation.The expression of TGF-βin the supernatant of the remaining RAW264.7 cells was increased in the model group（acetaldehyde 200μm stimulation for 48h）compared with the normal group,while it was decreased in the 5-BDBD group.The expressions ofα-SMA and Col1 were increased in the supernatant treated with acetaldehyde,but decreased in the 5-BDBD treated group.This in vitro study suggests that inhibition of P2X4 expression may attenuate alcoholic liver fibrosis by reducing TGF-βsecretion.Conclusion:（1）Inhibiting the expression of P2X4 receptor can reduce the alcohol-related liver fibrosis;（2）Inhibiting the expression of P2X4 receptor can reduce the phosphorylation of PI3K/AKT pathway,thereby reducing alcohol-related liver fibrosis;（3）Inhibiting P2X4 receptor expression,reducing the release of TGF-β,and inhibiting the activation of hepatic stellate cells,thereby reducing alcohol-related hepatic fibrosis.