线粒体功能障碍在青光眼视网膜神经节细胞凋亡中的作用

Effect of mitochondrial dysfunction on the apoptosis of retinal ganglion cells in glaucoma

目的探讨青光眼视神经损伤中线粒体功能改变及其在视网膜神经节细胞(RGCs)凋亡中的作用。方法在标准大气压环境(100 kPa;1 kPa=7.5 mmHg)下,利用压力装置额外予以70 mmHg的气体压力培养RGC-5细胞,按照不同培养时间将RGC-5细胞分为12 h组、24 h组、48 h组。并将标准大气压下培养的RGC-5细胞设为空白组。通过Annexin V/PI染色检测细胞凋亡,JC-1染色及DCFH-DA荧光探针染色分别检测RGC-5细胞线粒体膜电位(MMP)及细胞内活性氧(ROS)水平。Western blot检测细胞内B淋巴细胞瘤-2(BCL-2)蛋白、BCL-2相关X蛋白(BAX)、活化的含半胱氨酸的天冬氨酸蛋白水解酶-3(cleaved Caspase-3)及胞质内细胞色素C(Cyt C)蛋白的表达变化。结果与空白组相比,12 h组、24 h组、48 h组细胞内MMP均显著下降(均为P<0.05)。与空白组相比,24 h组、48 h组细胞凋亡率、ROS水平均显著增加(均为P<0.05),12 h组细胞凋亡率、ROS水平差异均无统计学意义(均为P>0.05)。与空白组相比,24 h组、48 h组细胞内cleaved Caspase-3及BAX蛋白相对表达量均显著增加,差异均有统计学意义(均为P<0.05),12 h组细胞内cleaved Caspase-3、BAX蛋白相对表达量差异均无统计学意义(均为P>0.05)。与空白组相比,12 h组、24 h组、48 h组细胞内BCL-2蛋白相对表达量均显著下降,差异均有统计学意义(均为P<0.05)。与空白组相比,48 h组胞质内Cyt C蛋白相表达量显著增加(P<0.05),12 h组、24 h组胞质内Cyt C蛋白相对表达量差异均无统计学意义(均为P>0.05)。结论RGC-5细胞在70 mmHg压力下培养后,表现出细胞凋亡增加及线粒体功能障碍。压力损伤早期线粒体损伤机制可能与外源性细胞凋亡途径密切相关。

Objective To explore the mitochondrial dysfunction in glaucoma-related optic nerve injury and its effect on the apoptosis of retinal ganglion cells(RGCs).Methods The RGC-5 cells were incubated under elevated pressure(70 mmHg,1 kPa=7.5 mmHg)on the basis of standard atmospheric pressure(100 kPa)for 12 h,24 h,and 48 h,respectively.The RGC-5 cells cultured under the standard atmospheric pressure were selected as the blank group.The apoptosis of RGC-5 cells was detected through Annexin V/PI staining.The mitochondrial membrane potential(MMP)and reactive oxygen species(ROS)level were measured using the JC-1 staining and DCFH-DA fluorescent probe.The expression levels of B cell lymphoma-2(BCL-2)protein,BCL-2-associated X(BAX)protein,cleaved Caspase-3,and cytochrome C(Cyt C)protein were detected using Western blot.Results Compared with the blank group,the MMP in the 12 h,24 h and 48 h groups significantly decreased(all P<0.05).Compared with the blank group,the cell apoptosis and ROS level in the 24 h and 48 h groups significantly increased(all P<0.05),while there was no statistically significant difference in the cell apoptosis and ROS level between the 12 h group and the blank group(both P>0.05).Compared with the blank group,the relative expression levels of cleaved Caspase-3 and BAX protein in the 24 h and 48 h groups significantly increased(all P<0.05),while the relative expression levels of cleaved Caspase-3 and BAX protein in the 12 h group showed no significant difference(both P>0.05).Compared with the blank group,the relative expression level of BCL-2 protein in the 12 h,24 h and 48 h groups decreased,and the differences were statistically significant(all P<0.05).Compared with the blank group,the relative expression level of Cyt C protein in the 48 h group significantly increased(P<0.05),while the relative expression level of Cyt C protein in the 12 h and 24 h groups showed no significant difference(both P>0.05).Conclusion After culture at 70 mmHg pressure,RGC-5 cells show increased apoptosis and mitochondrial dysfunction.The mitochondrial damage at the early stage of stress injury may be closely related to exogenous apoptosis pathways.