α-硫辛酸和NAC在镉致神经细胞氧化损伤和线粒体凋亡通路中的保护效应

发布时间：2018-07-29 16:18

【摘要】：为研究α-硫辛酸(α-lipoid acid, α-LA)和N-乙酰半胱氨酸(NAC)在镉导致的神经细胞氧化损伤及线粒体凋亡通路中的保护作用,本文以妊娠18-19日龄SD大鼠胎鼠原代大脑皮质神经元和PC12细胞系为模型,用10μmol/L镉处理细胞不同时间(0、6、12、24、48 h),不同浓度镉(0、5、10、20 μmol/L)及10或20μmol/L镉与100 μmol/L a-LA或NAC联合作用于细胞24 h后,用比色法测定神经细胞内超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GSH-Px)、谷胱甘肽还原酶(GR)、谷胱甘肽转移酶(GST)活力、谷胱甘肽(GSH)、丙二醛(MDA)含量及神经细胞线粒体内的SOD、CAT、GSH-Px活力和MDA含量,免疫荧光法观察细胞内活性氧(ROS)的含量变化,流式细胞术及Annexin V-FITC染色法测定细胞凋亡率,免疫印迹法检测线粒体通路中相关蛋白VDAC, cleaved caspase-3、9,Bcl-2和Bax蛋白的表达量和Cyt C的释放情况。结果表明：①与对照组相比,10 μmol/L镉暴露不同时间或不同浓度的镉暴露24 h能引起SOD、CAT活力显著或极显著升高(PCI2细胞)或降低(大鼠大脑皮质神经元),GSH-Px、GST、GR活力及GSH含量显著或极显著下降,MDA、ROS含量显著或极显著升高(P0.05或P0.01),且呈现一定的剂量、时间-效应关系。与染毒组相比,镉联合a-LA或NAC后,ROS含量极显著降低(P0.01),MDA含量显著或极显著降低(P0.05或P0.01),GSH-Px、GR、GST活力及GSH含量都显著升高(P0.05)；②与对照组相比,不同浓度的镉暴露24 h能引起神经细胞线粒体内SOD、CAT活力显著或极显著升高(PC 12细胞)或降低(大鼠大脑皮质神经元),GSH-Px活力显著或极显著下降及MDA含量显著或极显著升高(P0.05或P0.01),且呈现一定的剂量-效应关系。与染毒组相比,镉联合a-LA或NAC后,MDA含量显著或极显著降低(P0.05或P0.01),GSH-Px活力显著升高(P0.05)；③与对照组相比,20 μmol/L镉暴露极显著增加细胞凋亡率,促进线粒体内CytC的释放,降低VDAC,Bcl-2蛋白表达,提高cleaved caspase-3、cleaved caspase-9、Bax蛋白表达,差异都极显著(P0.01)。与染毒组相比,镉联合a-LA或NAC后,极显著抑制细胞凋亡,cleaved caspase-3、9,Bax蛋白表达及CytC的释放,极显著提高Bcl-2、VDAC蛋白表达(P0.01)。结论,镉能够导致神经细胞的氧化损伤并且激活线粒体凋亡通路,a-LA和NAC作为抗氧化剂,能够提高神经细胞的抗氧化能力,对镉导致的神经细胞氧化损伤及线粒体氧化损伤具有一定的保护作用,并可抑制线粒体凋亡通路的激活。
[Abstract]:To investigate the protective effects of 伪 -lipoid acid (伪 -LA) and N-acetylcysteine (NAC) on the oxidative damage of neurons and mitochondrial apoptosis pathway induced by cadmium. The primary cerebral cortical neurons and PC12 cell lines of fetal SD rats aged 18-19 days were used as models. The cells were treated with 10 渭 mol/L cadmium for different time (0 ~ 612A 24U 48 h),) and 10 or 20 渭 mol/L cadmium in combination with 100 渭 mol/L a-LA or NAC for 24 h. The activities of superoxide dismutase (SOD),) catalase (CAT), glutathione peroxidase (GSH-Px) and glutathione reductase (GR), glutathione transferase (GST) were measured by colorimetry. The content of glutathione (GSH), malondialdehyde (MDA), the activity of GSH-Px and the content of MDA in mitochondria of nerve cell were measured. The changes of (ROS) in cells were observed by immunofluorescence, and the apoptosis rate was determined by flow cytometry and Annexin V-FITC staining. The expression of VDAC, cleaved caspase-39, Bcl-2 and Bax protein and the release of Cyt C in mitochondrial pathway were detected by Western blot. The results showed that 10 渭 mol/L cadmium exposure at different time or concentration for 24 h could significantly or extremely increase the activity of PCI2 cells or decrease the activity of GSH-PxX GSTGR and the content of GSH in rat cerebral cortex neurons. The Ros content of MDAA was significantly or extremely significantly increased (P0.05 or P0.01), and showed a certain dose. Time-effect relationship. Compared with the exposed group, the content of Ros was significantly decreased (P0.01) after cadmium combined with a-LA or NAC (P0.05 or P0.01). The activity of GSH-PxGST and the content of GSH were significantly increased (P0.05) compared with the control group. Exposure to different concentrations of cadmium for 24 h resulted in a significant or extremely significant increase in the activity or decrease of GSH-Px in mitochondria (PC12 cells) and a significant or extremely significant decrease in GSH-Px activity and MDA content in mitochondria (PC12 cells). It was elevated (P0.05 or P0.01) and showed a dose-effect relationship. Compared with the exposed group, the activity of GSH-Px increased significantly (P0.05 or P0.01) after cadmium combined with a-LA or NAC (P0.05 or P0.01). Compared with the control group, 20 渭 mol/L cadmium exposure significantly increased the apoptosis rate and promoted the release of CytC in mitochondria. The expression of Bcl-2 protein and the expression of cleaved caspase-3 and caspase-9 protein were significantly decreased (P0.01). Compared with the exposed group, cadmium combined with a-LA or NAC significantly inhibited the expression of Bax protein and the release of CytC, and significantly increased the expression of Bcl-2VDAC protein (P0.01). Conclusion cadmium can induce oxidative damage of nerve cells and activate mitochondrial apoptotic pathways such as a-LA and NAC as antioxidants, which can enhance the antioxidant ability of neurons. It has a protective effect on oxidative injury and mitochondrial oxidative damage induced by cadmium, and can inhibit the activation of mitochondrial apoptosis pathway.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.3

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