Prevention Of Cannabinoid Receptor-2 Agonists In ConA-induced Liver Injury

Objective:To study the protective effect and mechanism of cannabinoid receptor-2（CB2）agonist AM1241 on acute liver injury induced by concanavalin-A（ConA）in mice.Methods:1.The model of acute liver injury induced by wild type mice and CB2^-/-knockout mice was induced by type IV ConA to verify the protective effect of CB2 in liver injury.They were divided into four groups:wild type WT control group,CB2^-/-gene knockout control group,wild type WT model group and CB2^-/-gene knockout model group,which were replicated by tail vein injection of ConA（20 mg/kg）.Acute immune liver injury model.Serum alanine aminotransferase（ALT）and aspartate aminotransferase（AST）levels were measured in mice,and H&E sections were stained to observe inflammatory cell infiltration and hepatocyte injury in mouse liver tissue.2.C57BL/6J mice were randomly divided into control group,ConA model group,AM1241 intervention group（3 mg/kg and 12mg/kg）and AM630（12 mg/kg）antagonist group.The AM1241 group was given a strong dose 1 h before the ConA injection,and the inhibitor group was administered intraperitoneally before the AM1241 injection.Mice were sacrificed 9 hours after ConA tail vein injection.The expression levels of ALT and AST in the serum of mice were detected.The infiltration,inflammation,necrosis and liver damage area of liver tissue were observed by H&E section staining.IL-1β,TNF-αand IL were detected by Elisa in liver tissue homogenate.-6,IFN-γexpression;real-time PCR detection of TNF-α,IL-6,IFN-γmRNA relative expression;immunohistochemistry（IHC）detection of cleaved caspase-3,Bax,Expression levels of Bcl-2 and F4/80 proteins;Western blot analysis of MAPK,p-CREB,CREB,p-p65,p65,Bax,Bcl-2,cleaved caspase-3,and inflammatory NLRP3 Related protein expression levels.3.Mouse AML-12 hepatocytes and RAW264.7 macrophages were cultured,and the expression of CB2 was detected by Western blot.Results:1.Compared with the wild-type WT model group,the expression levels of ALT and AST in the CB2^-/-knockout model group increased significantly（P<0.05）,the liver tissue inflammation was more serious,and the necrotic area increased.Compared with the control group,serum ALT and AST levels in the model group were significantly increased（P<0.05）,liver tissue inflammation was obvious,inflammatory cell infiltration,and hepatocyte necrosis was serious.Compared with the model group,the serum levels of ALT and AST in the AM1241 intervention group（3 mg/kg and 12 mg/kg）decreased（P<0.05）,and the liver tissue inflammation and necrosis were significantly improved.The results of Elisa and Real-time PCR showed that the expression levels of TNF-α,IL-6 and IFN-γin the model group were significantly increased（P<0.05）,while AM241 intervention decreased the expression levels of these inflammatory factors（P<0.05）.Compared with the control group,the phosphorylation levels of p38,ERK1/2,and p65 and CREB proteins in the MAPK family of the model group increased,and the NLRP3-related proteins of the inflammatory corpuscles also increased significantly,while the AM1241 intervention group decreased significantly（P<0.05）.Compared with the control group,the expression levels of cleaved caspase-3,Bax,Bcl-2 and F4/80 in the liver of the model group were significantly increased（P<0.05）.Compared with the model group,the AM1241 intervention group decreased significantly（P<0.05）.Compared with the AM1241 intervention group（3 mg/kg and12 mg/kg）,the CB2 antagonist AM630（12 mg/kg）inhibited the results of AM1241（P<0.05）.3.Liver tissue and macrophage CB2 is abundantly expressed and CB2expression on hepatocytes is extremely weak.Conclusion:1.CB2 receptor may have protective effect in ConA-induced acute liver injury in mice;2.CB2 agonist may reduce ConA-induced liver injury;3.CB2 agonist alleviates ConA-induced acute immunity in mice Hepatic injury may be related to the regulation of MAPK-related signaling pathways,inhibition of inflammatory NFRP3 activation,and promotion of hepatocyte macrophage apoptosis.