摘要
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.
作者
杨根梦
李娟
洪仕君
薛凤麟
何永旺
周一卿
曾晓锋
李利华
YANG Gen-meng;LI Juan;HONG Shi-jun;XUE Feng-lin;HE Yong-wang;ZHOU Yi-qing;ZENG Xiao-feng;LI Li-hua(School of Forensic Medicine,Kunming Medical University,Kunming 650500,China;Chinese Academy of Medical Sciences/Peking Union Medical College,Institute of Medical Biology,Yunnan Key Lab of Major Infectious Diseases Vaccine Development,Kunming 650118,China)
出处
《中国药理学通报》
CAS
CSCD
北大核心
2018年第4期577-583,共7页
Chinese Pharmacological Bulletin
基金
国家自然科学基金资助项目(No81560303,81660310);云南省科技厅-昆明医科大学应用基础研究联合专项(No2016FE467);云南省重大传染病疫苗研发重点实验室开放研究基金资助项目(NoKF2015-01);昆明医科大学毒品滥用法医病理学科技创新团队(NoCXTD201604);云南省教育厅研究生科技创新项目(No2017YJS077)