Effect Of Cannabinoid Receptor 2 Agonist AM1241 On ERK1/2-RSK1 Signaling Pathway In Liver Fibrosis Mice

Objective:To investigate the impact and mechanism of cannabinoid receptor 2（CB2）agonist AM1241 on carbon tetrachloride（CCl₄）-induced liver fibrosis in mice through the ERK1/2-RSK1 signaling pathway.Methods:In vivo:Replicate liver fibrosis model in mice（the model has been successfully replicated in previous experiments of our research group）:1.wild type（WT）C57BL/6J mice were randomly divided into WT control group and WT model group,CB2 receptor gene knockout(CB2^-/-)C57BL/6J mice were randomly divided into CB2^-/-control group and CB2^-/-model group.Two model groups mice were injected with CCl₄（5mg/kg,diluted with edible olive oil）,3 injections per week for 16 weeks,two control groups were injected with edible olive oil.Enzymatic method was used to detect ALT and AST contents of mice in each group,HE staining and Masson staining were performed on liver tissue sections of each group,Western blot was used to detect expression levels ofα-SMA,TGF-β1,A20,NF-κB,p-NF-κB,TNF-αand IL-6proteins in liver tissue,RT-PCR was used to test gene transcription levels of TNF-αand IL-6.2.WT C57BL/6J mice were randomly set into control group,model group,AM1241（3mg/kg）group,AM1241（9mg/kg）group,AM630+AM1241（3mg/kg）group,and liver fibrosis model was replicated in the same way.AM1241（3mg/kg）group,AM1241（9mg/kg）group and AM630+AM1241（3mg/kg）group all were injected with AM1241 3mg/kg,AM1241 9mg/kg and AM630+AM1241 3mg/kg respectively before injecting CCl₄.Enzymatic method was used to detect the ALT and AST contents of mice in each group,HE staining and Masson staining were performed on liver tissue sections of each group,expression levels of VEGF,c TGF,b FGF,ERK1/2,p-ERK1/2,RSK1,p-RSK1,PPAR-γand C/EBPβproteins in liver tissue were tested by Western blot.ELISA was used to analyze the contents of GSH-px,IL-6 and IL-1βin liver tissue homogenates,RT-PCR was used to detect m RNA levels of PPAR-γ,C/EBPβ,GSH-px,IL-6 and IL-1β.In vitro experiments:1.Cultured rat hepatic stellate cells（HSC-T6）were stimulated with 0,2.5,5,10,and 15μg/L TGF-β1 for 24 hours.Western blot was used to analyze expression level ofα-SMA protein in each group;2.HSC-T6 cells stimulated with 5μg/L TGF-β1 were treated with 0,20,40,80,and 160μmol/L AM1241 for 24 hours and 48 hours respectively,control group（medium containing HSC-T6 cells）and blank group（only medium）were set.The influence of AM1241 on the HSC-T6 cells proliferation was examined by CCK-8,and the 50%suppression ratio concentration（IC50）of AM1241on HSC-T6 cells for 24 hours was calculated.Western blot was used to detect expression levels ofα-SMA,b FGF,caspase-3,cleaved caspase-3 and BAX proteins on HSC-T6 cells treated with AM1241 for 24 hours.3.HSC-T6 cells stimulated with5μg/L TGF-β1 were treated with 0,20,30,40,60,80,and 160μmol/L AM1241 for24 hours and 48hours respectively,the effect of AM1241 on HSC-T6 apoptosis was detected by flow cytometry;4.The control group,TGF-β1 stimulation group,34μmol/L AM1241 group（simultaneously was given 5μg/L TGF-β1）were set,HSC-T6cells of each group were cultured for 24 hours and 48 hours respectively,Western blot was used to detect expression levels ofα-SMA,cleaved caspase-3,BAX,ERK1/2,p-ERK1/2,RSK1,p-RSK1 proteins on HSC-T6;5.The control group,TGF-β1stimulation group,34μmol/L AM1241 group,AM630+AM1241 group,TBHQ group,TBHQ+AM1241 group were set,in addition to the control group,5μg/L TGF-β1 was given to the rest of groups concurrently,all groups were cultured for 48hours,the relative expression levels of ERK1/2 and p-ERK1/2 proteins were alanyzed by Western blot.Results:In vivo experiments:1.There were no statistical difference in serum ALT and AST content between the CB2^-/-control group and the WT control group（P>0.05）.HE staining of liver tissue sections showed that the liver cells were intact,the liver tissue had no inflammatory changes,Masson staining showed almost no collagen production,expression levels ofα-SMA,TGF-β1,A20,p-NF-κB,TNF-αand IL-6 proteins were not obviously different in liver tissues between the CB2^-/-control group and the WT control group,there were no remarkable changes in TNF-αand IL-6 m RNA levels.Compared with the WT model group,the levels of serum ALT and AST in the CB2^-/-model group were obviously increased（P<0.05）,HE staining showed increased hepatocytes damage and inflammatory response,Masson staining showed more collagen fibers produced,the expression levels ofα-SMA,TGF-β1,A20,p-NF-κB,TNF-αand IL-6 proteins in liver tissues all were up-regulated（P<0.05）,TNF-αand IL-6 m RNA levels were consistent with levels of protein（P<0.05）.2.Compared with the mice of the wild type control group,levels of ALT and AST in the mice of the wild type model group were significantly increased（P<0.05）,HE staining showed severe hepatocyte necrosis and inflammation in liver tissues,Masson staining showed a significant increase in collagen fibers secreted by liver tissue,the expression levels of VEGF,c TGF,b FGF,p-ERK1/2,p-RSK1,and C/EBPβin liver tissues were all increased（P<0.05）,and the expression levels of PPAR-γprotein and m RNA were decreased（P<0.05）,the content and m RNA level of GSH-px were decreased（P<0.05）,the contents and m RNA levels of IL-6 and IL-1βwere increased（P<0.05）.Compared with the model group,AM1241 intervention group（3mg/kg,9mg/kg）had significantly decreased serum ALT and AST levels（P<0.05）,HE staining showed less hepatocyte necrosis,fewer inflammatory cells,and Masson staining showed a decrease in collagen fibers secreted by the liver tissue,the expression levels of VEGF,c TGF,b FGF,p-ERK1/2,p-RSK1,and C/EBPβin liver tissues were all decreased（P<0.05）,and the expression levels of PPAR-γprotein and m RNA were increased（P<0.05）,the content and m RNA level of GSH-px were increased（P<0.05）,the contents and m RNA levels of IL-6 and IL-1βwere decreased（P<0.05）.Compared with AM1241（3mg/kg）group,AM630+AM1241 group（3mg/kg）could reverse the above results（P<0.05）.In vitro experiments:1.The expression level ofα-SMA protein was up-regulated with the increase of TGF-β1concentrations;2.The cells absorbance values of each group were negatively correlated with the concentration and treatment time of AM1241（P<0.05）,IC50=34μmol/L;The expression levels ofα-SMA and b FGF proteins were gradually decreased when the concentrations of AM1241 went up,and cleaved caspase-3 and BAX proteins were gradually increased（P<0.05）;3.The apoptosis rate of HSC-T6cells was increased when the concentrations of AM1241 went up（P<0.05）,moreover,the apoptosis rate of 48 hours was higher than that of 24 hours.4.Compared with the control group,the expression level ofα-SMA protein was increased in TGF-β1stimulation group（P<0.05）,compared with TGF-β1 stimulation group,the expression levels ofα-SMA,p-ERK1/2,and p-RSK1 proteins were decreased with the increase of cultured time in 34μmol/L AM1241 group（P<0.05）,expression levels of cleaved caspase-3 and BAX were increased;5.Compared with TGF-β1 stimulation group,the relative expression level of p-ERK1/2 protein in 34μmol/L AM1241 group was decreased（P<0.05）,while relative level of p-ERK1/2 protein in the AM630+AM1241group and TBHQ+AM1241 group were reversed（P<0.05）.Conclusion:Cannabinoid receptor 2 agonist AM1241 can reduce hepatocytes damage,inhibit liver inflammatory response,block the activation of hepatic stellate cells,and promote their apoptosis to play an anti-fibrotic role.The mechanism may be related to ERK1/2-RSK1 signaling pathway.