[Study of antioxidant effect of cannabinoid receptor type 2 agonist on rat hepatic stellate cell line]

Objective: To investigate the action and antioxidant effects of CB2 agonist AM-1241 on rat hepatic stellate cell line (HSC-T6). Methods: HSC-T6 was randomly divided into four groups: control group, oxidative stress group, AM-1241 intervention group and AM-1241+AM-630 antagonist group. Survival rate of HSC-T6 was detected by thiazolyl blue assay under 24 h interventions with 0, 20, 50, 80 μmol/L AM-1241 and 0, 10, 20, 30, 40 μmol/L AM-630, respectively. Besides control group, the remaining groups were well cultured in low-glucose DMEM containing 100 mU/L glucose oxidase (GO) for 12 h to prepare the oxidative stress model. Then, AM-1241 intervention group was treated with 50 μmol/L low-glucose DMEM medium. After incubation for 12 h, the AM-1241+AM-630 antagonist group was treated with CB2 antagonist AM-630 (20 μmol/L) for 2 h, and cultured with 50 μmol/L AM-1241 in complete low-glucose medium for 12 h. The optimal drug concentration was selected according to the cell viability considered by the experiment results. Type III collagen (C III) content in the HSC-T6 supernatant was detected by enzyme-linked immunosorbent assay. Glutathione (GSH) content in HSC-T6 was detected by spectrophotometry. CB2 and heme oxygenase-1(HO-1) in each group of HSC-T6 were detected by western blotting. Results: HSC-T6 proliferation was inhibited in each group of AM-1241 in a concentration-dependent manner (P < 0.05). The inhibition was highest at 80μmol/L, and the cell survival rate was (41.61% ± 3.13%) (P < 0.05). AM-630 concentration group had no significant inhibitory effect on the proliferation of HSC-T6 (P > 0.05). HSC-T6 expressed CB2 receptor in each group. The expression level of CB2 in the AM-1241 intervention group was higher compare with control group (P < 0.05).The expression of Col III were significantly higher in oxidative stress group (P < 0.05) than in control group, and the expression of Col III of AM-1241 intervention group was significantly lower than that in oxidative stress group (P < 0.05). Col III level in AM-1241+AM-630 antagonistic group was significantly higher than that in AM-1241 intervention group (P < 0.05). There was no significant difference between AM-1241+AM-630 antagonistic group and oxidative stress group (P > 0.05). The content of GSH and HO-1 in oxidative stress group was higher (P < 0.05) than control group. The content of GSH and HO-1 in the AM-1241 intervention group was higher compared with oxidative stress group, while content of AM-1241 + AM-630 antagonist group was lower compared to AM-1241 intervention group (P < 0.05), and the differences were not statistically significant for oxidative stress group. Conclusion: CB2 agonist AM-1241 can inhibit the proliferation and activation of HSC-T6 and its mechanism may activate the nuclear translocation of Nrf2 binding to HSC-T6, initiating the up-regulation of antioxidant enzymes HO-1 and GSH protein expression, and thus increase the antioxidant effect of HSC-T6.