Effect Of Cannabinoid CB2 Agonist On Liver Fibrosis And Its Mechanism

Objective:To investigate the mechanism of prevention and treatment of canine receptor 2?CB2?agonists on CCl₄-induced liver fibrosis in mice.Methods:1.CCl₄-induced hepatic fibrosis in WT and CB2^-/-mice.WT control group,CB2^-/-control group,WT model group,and CB2^-/-model group were injected intraperitoneally with CCl₄?5 mL/kg?,The first week dose is 10%,adjust dose from the second week to 30%,3 times a week,a total of 16 weeks replication of liver fibrosis model.Enzyme assay was used to detect the expression of ALT and AST in mouse serum.H&E staining and Sirius red collagen staining were used to observe the inflammatory reaction,hepatocyte necrosis and extracellular collagen deposition area in mouse liver tissue.2.Intraperitoneal injection of CCl₄ induced liver fibrosis in WT mice.The experiments were divided into control group,model group,AM1241intervention group?3 mg/kg and 9 mg/kg?and AM630+AM1241 antagonist group?3mg/kg?.The same method replicates the hepatic fibrosis model with CCl₄.From the date of model establishment,AM1241 intervention group?3 mg/kg and 9 mg/kg?and AM630+AM1241 antagonist group?3 mg/kg?were given intraperitoneal injection of CCl₄ and CB2 receptor agonist AM1241 was injected intraperitoneally?3 mg/kg and9 mg/kg?and CB2 receptor antagonist AM630+AM1241?3 mg/kg?.Enzyme assay was used to detect the expression of ALT and AST in mouse serum.H&E staining and Sirius red collagen staining were used to observe the inflammatory reaction,necrosis and the area of extracellular collagen deposition in the liver of mice.Real-time PCR was used to detect CB2,TGF-?1,PDGF-B,?-SMA,and Col-I/III mRNA expression levels;?-SMA and collagen-I/III protein expression of liver tissue was detected by immunohistochemistry;Western blot analysis CB2,p-ERK1/2,ERK1/2,p-CREB,CREB,Bcl-2,Bax and cleaved caspase-3 protein expression levels.Results:1.Serum ALT,AST levels and H&E and Sirius red collagen staining experiments showed that there was no significant change in serum ALT and AST levels in CB2^-/-control group compared with WT control group?P>0.05?.No inflammation and necrosis were observed in the liver tissue.Only a small amount of collagen deposition was observed.The expression area was not statistically significant?P>0.05?.Compared with the WT model group,the ALT in the serum of the CB2^-/-model group mice was not found.The level of AST expression was significantly increased?P<0.05?,liver tissue inflammation,necrosis,and collagen deposition area were increased?P<0.05?;2.Serum ALT,AST expression levels and H&E,Sirius red collagen staining experimental results showed that the model group compared with the control group,serum ALT and AST levels increased significantly?P<0.05?,liver inflammation and necrosis were severe,and collagen deposition area increased?P<0.05?.Compared with the model group,serum ALT and AST levels in the AM1241 intervention group?3 mg/kg and 9 mg/kg?decreased?P<0.05?,liver inflammation,necrosis relief,and collagen deposition area decreased?P<0.05?.Compared with AM1241 intervention group?3 mg/kg and 9 mg/kg?,AM630+AM1241 antagonist group?3 mg/kg?reversed the above results?P<0.05?;3.Real-time PCR and western blot method shown,compared with the control group,the mRNA and protein levels of CB2 in the model group increased significantly?P<0.05?.4.Real-time PCR results showed that the model group compared with the control group,the mouse liver TGF-?1 and PDGF-BB mRNA was up-regulated?P<0.05?.Compared with the model group,the expression levels of TGF-?1 and PDGF-BB mRNA in liver tissue of mice treated with AM1241?3 mg/kg and 9 mg/kg?were down-regulated?P<0.05?.Compared with the AM1241 intervention group?3 mg/kg and 9 mg/kg?,AM630+AM1241 antagonist group?3 mg/kg?reversed the above results?P<0.05?;5.Real-time PCR and immunohistochemistry results showed that compared with the control group,the mRNA and protein levels of?-SMA and Col-I/III in the liver of the model group were significantly higher?P<0.05?.Compared with the model group,the mRNA and protein levels of?-SMA and Col-I/III in the hepatic tissue of the hepatic fibrosis mice treated with AM1241?3 mg/kg and 9 mg/kg?decreased significantly?P<0.05?.Compared with the AM1241 intervention group?3 mg/kg and 9 mg/kg?,the AM630+AM1241 antagonist group?3 mg/kg?reversed the above-mentioned results?P<0.05?;6.Western blot showed that the compared with control group.the expression of p-ERK1/2,p-CREB,and Bcl-2 in liver tissue of model group were increased,and the expression of Bax and cleaved caspased-3 protein decreased?P<0.05?.Compared with the model group,the expression levels of p-ERK1/2,p-CREB,and Bcl-2 protein were down-regulated in liver tissue of mice treated with AM1241?3 mg/kg and 9 mg/kg?.Bax and cleaved caspase-3 protein The level of expression increased?P<0.05?.Compared with the AM1241 intervention group?3mg/kg and 9 mg/kg?,the AM630+AM1241 antagonist group?3 mg/kg?reversed the above results?P<0.05?.Conclusion:1.CCl₄-induced more severe liver fibrosis in CB2^-/-mice indicates that CB2 receptor has anti-hepatic fibrosis effect in CCl₄-induced liver fibrosis in mice;2.CB2 receptor agonist can alleviate liver injury and hepatic fibrosis development;3.CB2 receptor agonist can alleviate CCl₄-induced liverfibrosisinmicemayberelatedtoitsdown-regulationof p-ERK1/2-p-CREB-Bcl-2 signaling pathway and promote activation of HSC apoptosis.