摘要目的 探讨大麻素CB2受体激动剂AM1241对大鼠肝星状细胞系(HSC-T6)的作用及其对抗氧化酶的影响.方法 用噻唑蓝法分别检测0、20、50、80μmol/L) AM1241和0、10、20、30、40 μ mol/L AM630干预24h下HSC-T6的存活率,根据实验所测得的细胞存活率选择最佳的用药浓度.将HSC-T6随机分成对照组、氧化应激组、AM1241干预组、AM12;41+AM630拮抗组共4组.除对照组,其余各组用含100 mU/L的葡萄糖氧化酶(GO)的低糖DMEM完全培养液培养12h制备细胞氧化应激模型,然后AM1241干预组用含50 μ mol/L AM1241的低糖DMEM完全培养液培养12 h,AM1241+AM630拮抗组用大麻素CB2受体拮抗剂AM630 (20 μmol/L)干预2h后,再用含50 μ mol/L AM1241的依氏低糖完全培养基培养12 h;酶联免疫吸附法检测各组HSC-T6培养液上清液中Ⅲ型胶原(ColⅢ)的含量;分光光度法检测各组HSC-T6中谷胱甘肽(GSH)的含量;Western blot法分别检测HSC-T6中CB2受体与血红素加氧酶1(HO-1)蛋白的表达量.多组间比较用单因素方差分析. 结果 AM1241各浓度组明显抑制HSC-T6的增殖且呈现浓度依赖性(P＜0.05),80 μ mol/L时抑制作用最大,细胞存活率为41.61％士3.13％(P＜0.05).AM630各浓度组对HSC-T6的增殖无明显抑制作用(P＞0.05);各组中的HSC-T6可以表达CB2受体,与对照组相比,AM1241干预组CB2的表达量有所增高(P＜0.05);与对照组相比,氧化应激组ColⅢ的表达量明显升高(P＜0.05),AM1241干预组Col Ⅲ的表达较氧化应激组明显减低(P＜0.05),而AM1241+AM630拮抗组Col Ⅲ的表达与AM1241干预组相比明显增高(P＜0.05),与氧化应激组相比无明显差异;与对照组相比,氧化应激组HO-1、GSH含量有所升高(P＜0.05),AM1241干预组GSH、HO-1含量较氧化应激组相比有所升高,而AM1241 +AM630拮抗组HSC-T6中HO-1 、GSH含量较AM1241干预组明显下降(.P＜0.05),与氧化应激组差异无统计学意义.结论 大麻素CB2受体激动剂AM1241可抑制HSC-T6的增殖与活化,其机制可能与AM1241结合HSC-T6大麻素CB2受体,激活HSC-T6 Nff2转录到细胞核内,启动了抗氧化酶HO-1、GSH蛋白表达量上调,增加HSC-T6的抗氧化作用有关.

abstractsObjective 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/LAM-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/LAM-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 supematant 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 80μtmol/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.