Effect Of Cannabinoid Receptor â…¡ AM1241 On Rat Hepatic Stellate Cell HSC-T6 And Its Mechanism

Objective: To investigate the effect of cannabinoid CB2 receptor agonist AM1241 on rat hepatic stellate cell line HSC-T6 and its mechanism. Methods:1, MTT method were used to detect the HSC-T6 survival rate under the intervention of(0, 20, 50, 80umol/L)AM1241 and(0, 10, 20, 30, 40umol/L) AM630 for 24 hours respectively. According to the experimental cell survival rate choose the best concentration of the drug. 2,The HSC-T6 was divided into control group, oxidative stress group, AM1241(20, 80 umol/L) intervention group. The control group was treated with low glucose DMEM medium for 2 hours, the oxidative stress group was treated with 100mU/L glucose oxidase enzymelow low glucose DMEM medium for 2hours.AM1241 intervention group were treated with 20, 80 umol/L AM1241 low glucose DMEM medium irespectively for 3 hours, then cultured with 100 mU/L glucose oxidase for 2 hours. Immunocytochemical staining method was used to observe the expression of α-SMA in each group of HSC-T6.ELISA was used to detect the content of Col I in the supernatant in each group of HSC-T6.Detecting the content of MDA and SOD.Western blot was used to detect the total and nuclear content of Nrf2 protein in each group of HSC-T6.3, The HSC-T6 was divided into control group,oxidative stress group, AM1241 intervention group and AM1241+AM630 antagonist group. The control group was treated with low glucose DMEM medium for 12 hours,the oxidative stress group was treated with 100mU/L glucose oxidase enzymelow low glucose DMEM medium for 12 hours.AM1241 intervention group was treated with50umol/L AM1241 low glucose DMEM medium for 12 hours.AM1241+ AM630 antagonist group was treated with 20umol/L AM630 for 2 hours,then treated with50umol/L AM1241 low glucose DMEM medium for 12 h. The content of Col Ι and Col III were detected by ELISA. Western blot method was used to detect the expression of Nrf2 and HO-1 protein.Results:Compared with the 0umol/L AM1241 intervention group, the HSC-T6 survival rate in(0, 20, 50, 80umol/L) AM1241 intervention group decreased,which had statistical significance(p<0.05) and the HSC-T6 survival rate in(0, 10, 20, 30, 40umol/L) AM630 intervention group had no significant effect(p>0.05). 2, HSC-T6 cytoplasmic in control group had no obvious expression of α-SMA.Compared with oxidative stress group,the expression of α-SMA in AM1241(20,80umol/L) intervention group decreased significantly(p<0.05).Compared with the control group, the content of Col I in the oxidative stress group increased significantly(p<0.05), and compared to oxidative stress group,content of Col in AM1241(20, 80 umol/L) intervention group decreased significantly(p <0.05).Compared to the control group,the content of MDA decreased, and SOD activity decreased significantly(p<0.05)while content of MDA decreased,and SOD activity was significantly increased in AM1241(20, 80umol/L) intervention group,(p<0.05).The total expression of Nrf2 protein in Control group, oxidative stress group,AM1241(20,80umol/L) intervention group had no significant difference(p >0.05).Compared with AM1241(20,80umol/L) intervention group,expression of nuclear Nrf2 protein in oxidative stress group increased significantly(p<0.05).3.Compared with the control group, the expression of Col I and Col III in oxidative stress group increased significantly(p<0.05). the expression of Col I and Col III in the AM1241 intervention group were significantly reduced as compared with the oxidative stress group(p<0.05).The difference of the content of Col I and Col III between AM1241+AM630 antagonist group and oxidative stress group showed no significace(p>0.05).Compared with the control group, the content of GSH in oxidative stress group increased(p<0.05),the content of GSH in AM1241+AM630antagonist group was significantly higher than the AM1241 intervention group(p<0.05),while with no significant difference with oxidative stress group. The CB2 protein expressed in Control group, oxidative stress group, AM1241 intervention group and AM1241+AM630 antagonist group.Compared with the control group,the expression of CB2 protein in the AM1241 intervention group was increased(p<0.05).Compared with the oxidative stress group,the capacity of Nrf2 and HO-1protein in the AM1241 intervention group were significantly higher.The capacity ofNrf2 and HO-1 protein in AM1241+AM630 antagonist group were was significantly lower than that in AM1241 intervention group(p<0.05),with no significant difference compared with the oxidative stress group. Conclusion: The cannabinoid CB2 receptor excited agent AM1241 inhibit the proliferation and activation of HSC-T6 and the mechanism may invole with that AM1241 activate Nrf2 in HSC-T6,lead the Nrf2 protein transfer into the nucleus,and increase the expression of Nrf2 and HO-1 protein,meanwhile increase the antioxidant effect of HSC-T6 by the cannabinoid CB2 receptor channels.