| OBJECTIVE:P2X7 receptor is an ATP-mediated gated ion channel.Activition of P2X7 receptor leads to the assembly of NLRP3 inflammasome,promoting the release of IL-1β.Our previous study confirmed that activation of the P2X7R-NLRP3 signaling pathway leads to deposition of extracellular matrix in hepatic stellate cells and promotes the progression of liver fibrosis.However,the role of P2X7R-NLRP3 signaling pathway in alcoholic steatohepatitis is unclear.Therefore,the aim of this study is to investigate the effect and mechanism of P2X7R-NLRP3 signaling pathway on liver fat accumulation and inflammation in alcoholic hepatic steatohepatitis via silencing or inhibiting P2X7 receptor.Methods:1.In vivo,firstly,C57BL/6 mice were used to establish acute and chronic alcoholic liver injury models,and then we chose acute alcoholic liver injury model to observe the role of P2X7R in alcoholic steatohepatitis via A438079 or SiRNA of P2X7R.And lastly,macrophages in mice were depleted by clodronate liposome to determine the effect of macrophages on liver fat accumulation.1)Acute alcoholic liver injury model.The mice were randomly divided into 5 groups(n=5),the normal group and the 4,12,24,and 48-hour sacrifice groups after the last alcoholic administration.Alcohol-administered mice were given 5 g/kg alcohol once every 12 hours for a total of 3 doses.After the last dose,alcohol-administered mice were sacrificed at the corresponding time and the normal group was sacrificed together with the 4 hours group.The levels of serum ALT,TG and IL-1β and TG contant in liver tissue were detected.Liver fat accumulation and macrophage recruitment were determined via HE staining and F4/80 immunofluorescence staining.The mRNA levels of Fasny Scd1,CXCL1 and CXCL2 of mouse liver tissue were detected by Q-PCR.2)Chronic alcoholic liver injury model.All mice were randomly divided into 2 groups(n=6);a pair-fed group,an ethanol-fed plus a single dose of ethanol group.Briefly,after a 5-day period of acclimation to the control Lieber-DeCarli liquid diets ad libitum,mice were fed Lieber-DeCarli liquid diets containing 5%(v/v)ethanol or isocaloric dextrin maltose for 10 days.On the 11th day.mice were gavaged with a single dose of ethanol(5 g/kg body weight)or isocaloric maltose dextrin solution in the early morning.The mRNA levels of the target genes of SREBP1.Fasn,Scd1,Acly and PPARa target genes Cd36,Cpt2 as well as P2x7r,Nlrp3,CxcI1,Cxcl2,Prkaal,Stkll in mouse liver tissues were detected by Q-PCR.3)Acute alcoholic liver injury plus A438079 model.mice were randomly divided into 3 groups(n=5):normal group,alcohol group and alcohol plus A438079 group.Alcohol plus A438079 group was subcutaneously injected with A438079(30 mg/kg)30 minutes after alcoholic administration.All mice were sacrificed 4 hours after the last alcoholic administration.Then,serum ALT,TG and mRNA levels of IL-1β,Fasn,Scdl,CXCL1 and CXCL2 were detected.Fat accumulation in liver tissue was determined via HE staining,Nile red staining and oil red O staining.Recruitment of macrophage was observed via F4/80 immunofluorescence and expression of SREBP1 and HMGB1 in liver tissue were observed seperatly via immunohistochemical staining.4)Acute alcoholic liver injury plus P2X7R SiRNA model.The mice were randomly divided into 7 groups,normal group,control group,P2X7R SiRNA64 or 65 group,alcohol group and alcohol plus P2X7R SiRNA64 or 65 groups.In SiRNA groups,SiRNA(7.5 nmol)was injected into the mouse via the tail vein,and the second dose was performed 48 hours later.Then,after 1 hour,aclcohol groups were treated with 3 doses alcohol gavage to establish acute alcoholic liver injury models as above.Mice were sacrificed after 4 hours of the last alcohol gavage and serum ALT,TG and liver TG content were detected;Fat accumulation in liver tissue was determined via HE staining,Nile red staining and oil red O staining;The expression of SREBPI and HMGB1 in liver tissue were determined via immunohistochemistry;Gene levels of IL-1β,Fasn,Scdl,Acly and Cpt2 in liver tissue were detected by QPCR5)Macrophage depletion model.mice were randomly divided into 4 groups(n=6),PBS/liposome group,clodronate/liposome group,PBS/liposome plus ethanol group and clodronate/liposome plus ethanol group.150μL of PBS/liposomes or clodronate/liposomes were injected into the tail vein of the mouse.48 hours after injection,acute alcoholic liver injury model was established as above.The macrophage depletion was detected via F4/80 fluorescence staining.HE and Nile red staining were used to observe if macrophage depletion can influence fat accumulation caused by alcohol gavaged2.In vitro,mouse primary hepatocytes,HepG2 or HepG2-ADH1 cells were cultured in presence of alcohol or LPS/ATP to observe the effect of A438079 or P2X7R SiRNA on hepatic steatosis or inflammatory response caused by alcohol exposure.1)Mouse primary hepatocytes were isolated by collagenase perfusion and cultured in vitro with alcohol or LPS/ATP.The expression of SREBPI in mouse primary hepatocytes was observed by immunofluorescence staining.Oil red O staining was used to observe fat deposition in primary hepatocytes and the expression of IL-1β and HMGB1 was detected through Western blotting.2)Alcohol or LPS/ATP was used to stimulate mouse primary hepatocytes,and P2X7R selective inhibitor A438079,TLR4 inhibitor CLI-095 and Caspasel inhibitor VI were used to observe the expression of Caspase1,ASC,IL-1β and HMGB1 through Western blotting.3)HepG2 and HepG2-ADH1 cells were stimulated with different concentrations of alcohol,and the expression and location of HMGB1 was observed via immunofluorescence staining Total protein,plasma protein and nuclear protein of HepG2-ADH1 cells were extracted and cultured medium was concentrated to determine expression of HMGB1 in each component via Western blotting.4)HepG2 cells were exposed to alcohol in presence of A438079 or Caspasel inhibitor VI,to observe the expression of SREBP1 via immunofluorescence stainning,and the protein was extracted to detect the expression of LKBI,AMPK and P-LKBI via Western blotting.Results:1.In vivo,liver fat accumulation and elevation of inflammation-related factors appeared both in acute and chronic alcoholic liver injury models.In acute alcoholic liver injury model,the liver fat accumulation was most obviously in 4 hours after the last alcohol administration.Obviously,inhibition or silencing of P2X7R by chronic P2X7R selective inhibitors A438079 and P2X7R SiRNA can reduce the levels of ALT,TG and TG content in liver tissue,down-regulate the expression of SREBPI,and reduce the accumulation of fat in liver tissue and down-regulated the expression of inflammation-related proteins NLRP3,ASC,HMGB1;In addition,A438079 reduced alcohol induced recruitment of macrophages in mouse liver and translocation of HMGB1 from nucleus to cytoplasm;P2X7R SiRNA reduced the expression of P2X7R in mouse liver and the levels of SREBP1-targeted gene Fasn,Scd1,Acly and up-regulated the expression of PPARa-targeted gene Cpt2;macrophage depletion can alleviate fat accumulation in mouse liver caused by alcohol exposure.2.In vitro,ETOH or LPS/ATP stimulation can cause fat deposition in hepatocytes and increase the expression SREBP1 and IL-1β in mouse primary hepatocytes;A438079,TLR4 inhibitors CLI-095 and Caspasel Inhibitor VI can reduce the expression of P2X7R-NLRP3 pathway-related protein and decrease the expression and release of IL-1β.Alcohol causes the translocation of HMGB1 from nucleus to cytoplasm in concentration-dependent way in HepG2 and HepG2-ADH1;LKB1-AMPK in HepG2 was restored after administration of A438079 and Caspasel inhibitor VI in HepG2.Conclusion:P2X7R inhibition or silencing can reduce liver fat accumulation and release of IL-1β as well as other inflammatory factors both in vivo and in vitro by blocking the P2X7R-NLRP3 signal axis to reduce steatosis and inflammatory response during ASH.IL-1β release in hepatocyte is part dependent on P2X7R-NLRP3 axis.A safe and effective inhibitor of P2X7R-NLRP3 inflammatory signaling pathway is a new option for the treatment of ASH. |
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