天麻素在甲基苯丙胺诱导SH-SY5Y细胞自噬中的作用及机制研究

Effects of gastrodin against SH-SY5Y cell autophagy induced by methamphetamine and underlying mechanisms

目的研究天麻素(gastrodin)在甲基苯丙胺(methamphetamine,METH)诱导SH-SY5Y细胞自噬中的作用,并探讨其作用机制。方法体外培养人神经母细胞瘤SH-SY5Y细胞,不同浓度的METH(0.5、1.0、1.5、2.0、2.5、3.0 mmol·L^(-1))处理SY5Y细胞,并在6、12、24、48 h观察SY5Y细胞自噬的情况,确定自噬的最佳浓度和时间点。提前1 h给予1mmol·L^(-1)天麻素进行干预,用显微镜观察细胞形态变化,采用激光共聚焦显微镜观察LC3-Ⅱ的改变,Western blot检测天麻素干预前后LC3-Ⅱ、Beclin-1以及Akt、p-Akt、mTOR、p-mTOR的表达情况。结果随着METH浓度(0~3.0 mmol·L^(-1))的增加,SY5Y细胞逐渐变圆、突起逐渐变短、消失,同时部分细胞质内可见大小不等的空泡结构形成,细胞间隙逐渐增宽,最终细胞呈漂浮状态。随着METH浓度的增加,SY5Y细胞LC3-Ⅱ表达水平逐渐增强。激光共聚焦显微镜结果显示,天麻素+METH组LC3-Ⅱ的表达水平比METH组降低。与对照组相比,METH组LC3-Ⅱ和Beclin-1表达水平明显增强(P<0.01),mTOR和Akt差异无显著性,p-mTOR、p-Akt表达水平明显降低(P<0.01);给予天麻素干预后,与METH组相比,LC3-Ⅱ和Beclin-1表达水平降低,而mTOR、p-mTOR、Akt、p-Akt表达水平升高。结论 METH可诱导SY5Y细胞自噬,天麻素可减弱METH诱导的SY5Y细胞自噬,这一作用与天麻素调控Akt和mTOR信号通路密切相关。

Aim To investigate the effects of gastrodin on SH-SY5Y cell autophagy induced by methamphetamine(METH)and the underlying mechanisms.Methods SY5Y cells were treated by METH with the concentration of 0.5,1.0,1.5,2.0,2.5,3.0 mmol·L-1 for 24 h.The morphological changes were observed by microscopy,the expression of LC3-Ⅱ,Beclin-1,Akt,p-Akt,mTOR and p-mTOR were detected by Western blot.Gastrodin was added to the medium 1 h before METH treatment.Results The SY5Y cells were morphologically featured by shrinkage and dendrite disruption after exposed to METH(0~3 mmol·L-1),and autophagic vacuoles occurred in cytoplasm.The expression of LC3-Ⅱincreased over METH dose.Confocal results showed that LC3-Ⅱsignificantly increased in METH group as compared with control,while decreased in METH+Gastrodin group.The expression levels of LC3-Ⅱand Beclin-1 significantly increased(P<0.01)in METH group,p-mTOR and p-Akt decreased,and mTOR and Akt showed no significant difference as compared with control.However,the gastrodin could decrease the expression of LC3-Ⅱand Beclin-1 and increase the expression of mTOR,p-mTOR,Akt and p-Akt as compared with METH-treated groups.Conclusions METH can induce SY5Y cells autophagy.The protective effect of gastrodin against METH-induced autophag may be related to gastrodin regulation mTOR and Akt signaling pathway.