自噬参与低氧预处理抗PC12细胞糖氧剥夺损伤过程

Hypoxic preconditioning alleviates oxygen-glucose deprivation in PC12cells: the involvement of autophagy

目的:观察低氧预处理(HPC)对氧糖剥夺(OGD)损伤的PC12细胞的作用,同时探讨自噬在其中的作用。方法:培养的PC12细胞按照处理因素分为6组:对照组、HPC模型组、3-甲基腺嘌呤(3-MA)组、低氧预处理后氧糖剥夺(HPC+OGD)组、3-甲基腺嘌呤预处理后行低氧预处理及氧糖剥夺(3-MA+HPC+OGD)组和OGD组。CCK-8检测细胞存活率;caspase-3活性检测试剂盒检测酶活性;TUNEL染色和流式细胞术检测细胞凋亡的情况;Western blot法检测自噬相关蛋白LC3、beclin-1和凋亡相关蛋白caspase-3的表达。结果:与对照组相比,OGD组细胞存活率明显降低;与3-MA+HPC+OGD组及OGD组相比,HPC+OGD组细胞存活率明显升高(P<0.05)。与对照组相比,OGD组细胞的caspase-3酶活性明显升高;与3-MA+HPC+OGD组及OGD组相比,HPC+OGD组的caspase-3酶活性明显降低(P<0.05)。与对照组相比OGD组细胞凋亡明显增多;HPC+OGD组与OGD组相比凋亡明显减少(P<0.05)。此外,与对照组相比,OGD组的激活型caspase-3蛋白水平明显升高(P<0.05);且HPC+OGD组与OGD组相比激活型caspase-3蛋白水平明显减少而LC3、beclin-1的蛋白水平明显升高(P<0.05)。结论:HPC抗OGD损伤的机制可能与其激活细胞自噬有关。

AIM： To examined the effects of hypoxic preconditioning （ HPC） on oxygen-glucose deprivation （ OGD）-induced PC12 cells, and to investigate its possible mechanisms of autophagy .METHODS： Cultured PC12 cells were randomly divided into control group , HPC group, 3-methyladenine （3-MA） group, HPC＋OGD group, 3-MA＋HPC＋OGD group and OGD group .CCK-8 assay was used to detect the cell viability .The caspase-3 activity was also tested . TUNEL staining and flow cytometry were used to detect the cell apoptosis .The protein levels of apoptosis-related protein caspase-3 and autophagy-marked protein LC3-2 and beclin-1 were determined by Western blot .RESULTS：Compared with control group, the viability of PC12 cells was significantly reduced , and the activity of caspase-3 was significantly increased in OGD group.Compared with 3-MA＋HPC＋OGD group and OGD group , the viability of PC12 cells was significantly in-creased, and the activity of caspase-3 was significantly reduced in HPC ＋OGD group （P〈0.05）.The PC12 cell injury was apparent after OGD with a great increase in the apoptotic rate （P〈0.05）.Compared with OGD group, the apoptotic rate significantly decreased in HPC ＋OGD group （ P〈0.05 ） .Compared with control group , the protein level of cleaved caspase-3 was significantly increased in OGD group （ P〈0.05） .Compared with OGD group , the protein level of cleaved caspase-3 was significantly decreased , and the levels of LC3-2 and beclin-1 were significantly increased in HPC ＋OGD group （P〈0.05）.CONCLUSION：OGD decreases cell survival and induces apoptosis .Activation of cell autophagy may be the mechanism by which hypoxic preconditioning protects the PC 12 cells from OGD induced injury .