脂筏在CB2受体介导的内源性大麻素AEA抑制大鼠肝星状细胞增殖活性中的作用

摘要

目的 探讨脂筏在内源性大麻素受体2(CB2)介导的内源性大麻素(AEA)抑制大鼠肝星状细胞(HSC)增殖活性中的作用及作用机制.方法 构建大麻素受体2 shRNA(Cnr2-shRNA)转染HSC细胞,干扰CB2受体的表达,采用MTT法检测转染前后不同浓度的AEA和甲基-β-环糊精(MCD)对HSC的作用效应;采用Western blot检测不同浓度AEA及MCD作用后HSC中P38 丝裂原活化蛋白激酶(p-P38MAPK)和c-Jun氨基端激酶/应激活化蛋白激酶(p-JNK)的表达量;采用激光共聚焦法检测HSC上的脂筏(LRs)以及CB2受体的表达;蔗糖密度梯度离心法提取脂筏,Western blot鉴定脂筏并检测脂筏中CB2受体的表达.结果 成功构建Cnr2-shRNA转染筛选Cnr2-单克隆细胞株,MTT检测发现转染后CB2受体的减少能减弱AEA对HSC细胞增殖的抑制作用,然而用MCD预处理HSC细胞后CB2受体的减少对AEA的效应无明显影响.p-P38MAPK和p-JNK的表达与AEA浓度有依赖关系,且可以被MCD部分拮抗.CB2受体在HSC膜脂筏和胞质中均有表达,但用蔗糖密度梯度离心法提取AEA刺激前HSC细胞脂筏,发现未受AEA刺激时脂筏中含有的CB2受体量很少,CB2受体大部分存在于HSC细胞胞质中.结论 CB2受体参与AEA 抑制HSC细胞增殖的过程与脂筏相偶联,通过脂筏这个信号平台AEA的刺激可能使CB2受体聚集或增多从而发挥级联放大效应,且这一效应与细胞中p-P38MAPK和p-JNK信号途径的激活有关.脂筏和CB2受体介导的信号传导途径可能成为治疗肝纤维化有效的作用靶点.%Objective To investigate the roles of lipid rafts in cannabinoid receptor 2(CB2)-mediated inhibitory effects of endogenous anadamide(AEA)on proliferation of hepatic stellate cells in rats and the action mechanism. Methods Cell viability was measured by using MTT assay. CB2-shRNA(Cnr2-shRNA) was designed to decrease the amount of CB2 and methyl-β-cy-clodextrin(MCD)treatment designed to destroy the lipid rafts in AEA-treated hepatic stellate cells. Subcellular localization of lipid rafts and CB2 were examined by using laser confocal microscopy. The amount of CB2 in lipid-raft microdomains was detected after isolating lipid-rafts from hepatic stellate cells by Western blot. Results MTT assay showed either Cnr2-shRNA-trans-fected cells or MCD-treated cells had a noticeably higher ratio of cell proliferation than controls. No obvious difference in the ratio of cell proliferation was observed between Cnr2-shRNA-transfected cells and controls after MCD treatment. Meanwhile, the levels of P38 mitogen-activated protein kinases(p-P38MAPK)and c-Jun N-terminal kinases/stress-activated protein kinases(p-JNK)were significantly decreased after the hepatic stellate cells were treated with MCD. Laser confocal microscopy revealed the presence of lipid rafts and CB2 on the membranes of hepatic stellate cells. The lipid rafts isolated from hepatic stellate cells before AEA stimulation had little CB2. Conclusion Lipid rafts are required for the process of inhibition in the proliferation of HSC caused by AEA through CB2. This process was associated with the p-P38MAPK and p-JNK signaling pathway,suggesting that it is possible to be an effective treatment for liver fibrosis by controlling lipid rafts , CB2, and related signal.