自噬参与氢溴酸樟柳碱对缺氧状态下PC12细胞损伤的保护作用

Protective Effect of Anisodine Hydromide on Autophagy-related Factors of PC12 Cell Induced by Hypoxia

目的:研究氢溴酸樟柳碱对缺氧状态下肾上腺嗜铬细胞瘤细胞(rats pheochromocytoma cells,PC12)自噬相关因子的影响。探讨氢溴酸樟柳碱对缺氧损伤神经细胞模型的保护作用及机制。方法:运用氯化钴(CoC l2)处理PC12细胞建立化学性缺氧模型,设空白组,模型组,阳性组(丁基苯酞),氢溴酸樟柳碱高、中、低浓度组(100,50,25μmol·L^(-1))。采用倒置显微镜观察细胞形态,噻唑蓝(MTT)比色法测定细胞相对存活率,单丹磺酰尸胺荧光染色(MDC)和透射电镜技术检测自噬小体的变化,蛋白免疫印迹法(Western blot)技术检测微管相关蛋白1轻链3(LC3),Beclin-1等蛋白的表达。结果:氢溴酸樟柳碱干预后细胞存活率显著提高,细胞形态恢复较好。MDC荧光染色和透射电镜下自噬小体的数量增多,LC3-Ⅱ及Beclin-1的表达显著提升。结论:自噬在缺氧状态下对细胞有保护作用,氢溴酸樟柳碱通过诱导低氧状态细胞自噬发挥了神经保护作用。

Objective： To observe the effect of anisodine hydromide on the expression of autophagy-related protein,in order to explore the protective effect and possible mechanism of anisodine hydromide on rats pheochromocytoma cells（ PC12） induced by hypoxia. Method： PC12 cells were divided into control group,model group,n-butylphthalide group,and high-dose,middle-dose and low-dose anisodine hydromide groups（ 100,50,25 μmol·L^（-1））. The relative viability of PC12 cells induced by hypoxia was measured by thiazole blue chromatomentry（ MTT）, and the morphology of PC12 cells was observed. The morphological changes inautophagosome were observed by transmission electron microscope and monodansylcadaverine（ MDC） fluorescence staining. The expressions of microtubule associated protein 1 light chain3（ LC3） and Beclin-1 protein were detected by Western blot method. Result： The viability rates of cells were significantly improved after intervention with anisodine hydromide,and the expressions of LC3-Ⅱ and Beclin-1 were significantly improved after intervention with anisodine hydromide. These results were consistent with the observation of transmission electronmicroscope and MDC fluorescence staining. Conclusion： Autophagy has a protective effect on PC12 cell induced by hypoxia,and anisodine hydromide could prevent the injury of PC12 cells induced by hypoxia through activating autophagy.