P2x7R-mediated NLRP3 Inflammasome Regulates Alcoholic Fatty Liver And Liver Fibrosis

OBJECTIVE:Fibrosis is the progressive pathological process in which the body's wound healing and tissue remodeling mechanisms respond to liver injury in all chronic hepatopath.Purinergic receptor P2x7(P2x7R)is a key modulator of fatty liver and fibrosis.The present study aimed to investigate the role of P2x7R in hepatic stellate cells activation.METHODS:(1)Lipopolysaccharide(LPS)was supplemented to human hepatic stellate cells,LX-2 for 24 h and was detected the expression of Interleukin-1?(IL-1?),Interleukin-1?(IL-1?)and tumor necrosis factor alpha(TNFa)by ELISA.When lipopolysaccharide(LPS)was supplemented to human hepatic stellate cells(LX-2)for 4 h,we added adenosine-triphosphate(ATP)for 30min,and P2x7R selective antagonist A438079(10?M),A438079 was supplemented to LX-2 cells 10 min prior to ATP.Detect the expression of NOD-like receptor family,pyrin domain containing 3(NLRP3)and a-smooth muscle actin(a-SMA),type ? collagen(collagenI).(2)In TAA liver fibrosis model,we set 3 groups:normal,TAA group and TAA plus A438079 group.Daily administration of intraperitoneal injection of 1,for 6 weeks at a dose of 100 mg/kg.From the second week,the dose of TAA raised to 200 mg/kg.The mice were given subcutaneous injection 30mg/kg,3 times a week in the fifth and sixth week.At the end of the sixth week,all mice were executed.We used hematoxylin-eosin staining(HE),Immunohistochemistry(IHC),MASSON(Masson's Trichrome Staining),IF(Immunofluorescence Staining)and Western Blot to evaluate the expressions of.(3)Ethanol(50mM)was supplemented to AML12 cells for 24h,we added metformin(500?M),the inhibitor of AMP dependent kinase pathway Compound C(10?M)and P2x7R selective antagonist A438079(10?M).Then we detected the expression of metabolism related factor Sirtuin 1.(4)In vivo,the chronic ethanol model,we set 4 groups:Pair-fed,ethanol group,ethanol-fed plus A438079 group and A438079 group.The experiment lasted for 11 days.On the 11st day,the mice were alcohol administration and were executed 9h later.We analyzed the expressions of fibrosis marks of liver.RESULTS:(1)LPS Treatment can activate HSC to release great number of inflammatory factors.Pretreatment of A438079 on LX-2 cells can stimulate cytokines.These events were remarkably suppressed by A43 8079 pretreatment.siRNA against P2x7R reduced protein expression of NLRP3 and a-SMA and suppressed deposition and secretion of type I collagen.(2)In TAA-induced mice liver fibrosis,A438079 could suppress the expression of liver fibrosis markers and cytokines caspase-1?NLRP3?collagen I?a-SMA and TIMP 1.(3)Treated with ethanol(50mM)can suppress the expression of SIRT1 and P-AMPK in AML12 cells.A438079 could suppress the expression of PPAR??caspase-3.(4)In chronic binge model,A438079 could deduce the expression of ?-SMA,deposition of type I,LIPIN 1 and ALT/AST,regulated the expression of P-AMPK?SIRT 1 and pathological expression.CONCLUSION:The involvement of P2x7R-mediated NLRP3 inflammasome activation of HSC might stimulate alcoholic fatty liver and liver fibrosis.It suggests that blockade of the P2x7R-NLRP3 inflammasome axis represents a potential therapeutic target.