甲基苯丙胺诱导心肌细胞自噬作用的发生

Methamphetamine induces autophagy in myocardial cells

目的探讨甲基苯丙胺(methamphetamine,METH)引起心肌细胞发生自噬现象的过程。方法体内实验:将60只约6周龄的雄性C57Bl/6J小鼠随机(随机数字法)分为3组(对照组、3 d染毒组、7 d染毒组),每组20只,建立METH染毒模型。染毒组小鼠给予腹腔注射单次剂量15 mg/kg METH,2次/d,分别染毒3 d或7 d。对照组小鼠相同时间点给予腹腔注射0.9%的生理盐水共7 d。最后一次腹腔注射24 h后收获小鼠心脏。Western blot法检测心肌自噬相关蛋白的表达。体外实验:心肌细胞系(H9C2)分为两组,对照组(正常培养基),METH组(用900 mmol/mL的培养基干预24 h)。Western blot、免疫荧光检测细胞中自噬相关蛋白表达;透射电子显微镜观察细胞内自噬现象。结果在体内实验和体外实验中,甲基苯丙胺干预后,心脏自噬相关蛋白p62,Beclin-1和LC3表达量与对照组相比均明显上调,差异有统计学意义(P<0.05);激光共聚焦检测免疫荧光结果示,METH组心肌细胞LC3-Ⅱ较对照组明显增多;电镜结果显示,与对照组相比,METH组细胞的自噬体数量明显增多。结论甲基苯丙胺可激活心肌细胞的自噬程序。

Objective To investigate the process of autophagy in myocardial cells induced by methamphetamine (METH). Methods In vivo study: sixty 6-week-old male C57Bl/6 J mice were randomly(random number) divided into three groups evenly, control group, three-day METH treated group and seven-day METH treated group. Mice in control group was given physiological saline through intraperitoneal injection 2 times per day and lasted 7 days. Mice in three days group and seven days group intake methamphetamine at a dose of 15 mg/kg every time through intraperitoneal injection 2 times a day, lasted 3 days and 7 days respectively. The hearts of the mice were then obtained by anatomical method 24 hours after the last intraperitoneal injection of METH, then autophagy related proteins were detected by western blotting. In vitro study: the model was established by H9C2 cells. The cells were divided into two groups, control group (cells were cultured by normal medium) and METH group (cells were cultured by medium includes 900 mmol/mL METH for 24 hours). The expressions change of autophagy related proteins in cells were tested by Western blotting. Additionally, LC3-Ⅱ was tagged by red fluorescent and then the stained cells were visualized under a Zeiss LSM710 confocal microscope. Furthermore, the numbers of autophagosomes in cells were visualized by transmission electron microscopy. Results The expression of p62,Beclin-1and LC3 were significantly increased in METH group when compared with control group (P<0.05). The level of LC3 was significantly increased in METH treated group compared with control group visualized under a Zeiss LSM710 confocal microscope. The numbers of autophagosomes in METH group are more than control group visualized by transmission electron microscopy. Conclusions Autophagy can be induced by METH in myocardial cells.