Effect Of P2X7 Receptor Antagonist On Alcohol-induced Steatohepatitis And Intestinal Microecological Changes And Research Of Mechanism

Backgrouds: Alcoholic liver disease(ALD)is a series of pathological liver damage caused by long-term or excessive drinking,which has a high morbidity and mortality,and has become a public health problem in the world.ALD has a long course and complicated pathogenesis.A large number of studies have shown that the direct toxic effects of alcohol and its metabolites as well as the superposition of hepatitis virus play an important role in ALD.Alcohol consumption can also cause intestinal flora disorders,resulting in damage to the intestinal mucosal barrier and increased permeability.Then,bacteria and it's products will enter the liver through the damaged mucosal barrier and the hepatic portal vein,further aggravating liver damage by oxidative stress and activation of toll-like receptor 4(TLR4)on the surface of Kupffer cells.Thus,it can be seen that there is a close relationship between the liver and intestinal microecology.P2X7 receptor(P2X7R)is a adenosine triphosphate(ATP)-binding purinergic receptor,widely participating in a variety of inflammatory diseases.Studies have found that P2X7 R is abnormally highly expressed in the progress of carbon tetrachloride-caused fatty hepatitis,paracetamol-induced hepatotoxicity,and chemical-induced inflammatory bowel disease(IBD).Moreover,P2X7 R antagonist has certain improvement effect on the above tissue damage.Objective:(1)To investigate the expression of P2X7 R in alcohol-inducedsteatohepatitis and intestinal microecological changes.(2)To explore the effect of P2X7 R antagonist on alcohol-induced steatohepatitis and intestinal microecological changes and the possible mechanism.Methods: ? C57BL/6 mice aged 8-10 weeks(body weight > 20 g)were used to induce steatohepatitis and intestinal microecological changes by establishing a ALD murine model of chronic plus binge alcohol feeding(Liber-De Carli alcoholic liquid feed containing 5% alcohol for 10 days + a alcohol gavage in the morning on the 11 th day(31.5%,5 g/kg)).C57BL/6 mice were randomly divided into Pair-fed group(n = 10),Et OH-fed group(n = 15),P2X7 R antagonist Brilliant Blue G-low dosage group(BBG-L,25 mg/kg,n = 15),BBG-middle dosage group(BBG-M,50 mg/kg,n = 15),BBG-high dosage group(BBG-H,100 mg/kg,n = 15),and P2X7 R antagonist A438079group(100 mg/kg,n = 15).From day 4 to day 10,mice in each antagonist-administration group were intraperitoneally injected with corresponding doses of BBG and A438079 once a day,and mice in the Pair-fed group and the Et OH-fed group were given corresponding volumes of saline.On the 11 th day,the mice were intragastrically administrated with alcohol in the morning and sacrificed 9 hours later.Serum,liver,small intestine and cecum contents were collected.Serum levels of ALT,AST,T-CHO and TG were detected by colorimetric assay.Histopathological damage of liver and intestine was observed by HE staining.The expression of P2X7 R or Ly-6G in the liver and intestine was detected by immunohistochemical staining.Liver lipid infiltration was observed by Oil Red O staining.The expression of P2X7 R,TNF-?,IL-1?,IL-6,Ly-6G,MCP-1,ZO-1,claudin-1,p-MEK1/2,p-ERK1/2 and egr-1was measured by western blot or RT-q PCR.16 S r RNA sequencing was applied to evaluate and define taxonomic alterations of cecal microbiota.Results: Compared to the Pair-fed group,alcohol feeding significantly increased serum ALT,AST,T-CHO and TG levels,leading to liver fat accumulation,neutrophil infiltration,disorganized liver cord,disrupting intestinal mucosal barrier integrity,raising liver Ly-6G,MCP-1 expression,decreasing intestinal expression of ZO-1,claudin-1,promoting secretion of TNF-?,IL-1?,IL-6 in the liver and intestine.At the same time,the m RNA and protein expression levels of P2X7 R in the liver and intestine were significantly increased.In comparision with the Et OH-fed group,high-dose BBG and A438079 significantly improved alcohol-induced liver and intestinal damage.Interestingly,compared to the Pair-fed group,alcohol feeding increased the relative abundance of phylum Bacteroidetes while decreased the number of phylum Verrucomicrobia and genus Akkermansia in the cecal content.Relative to the alcohol group,high-dose BBG and A438079 significantly reversed alcohol-induced changes in cecal microflora taxonomy.In addition,alcohol feeding promoted MEK1/2-ERK1/2phosphorylation and up-regulated egr-1 expression in liver and intestinal tissues compared to mice fed with control diet.Relative to alcohol-fed mice,high-dose BBG and A438079 significantly inhibited alcohol-induced phosphorylation level of MEK1/2-ERK1/2 and high expression of egr-1.Conclusion:(1)P2X7R is abnormally highly expressed in alcoholic steatohepatitis,suggesting that P2X7 R may be one of the potential targets of alcoholic liver disease.(2)P2X7R antagonist effectively alleviates alcoholic steatohepatitis,which may be related to the inhibition of MEK1/2-ERK1/2 phosphorylation and egr-1 expression.(3)P2X7R antagonist can also indirectly alleviate alcoholic steatohepatitis by alleviating the secondary blow to the liver caused by alcohol-induced intestinal microecological changes.