Protective Effect Of α-lipoic Acid And N-acetylcysteine On Cadmium-induced Oxidative Damage And Apoptosis Via Mitochondria Pathway In Neuronal Cells

To investigate the protective effect of α-lipoic acid (α-LA) and N-acetylcysteine (NAC) treatment on Cadmium(Cd)-induced oxidative damage and apoptosis via mitochondria pathway in neuronal cells, PC 12 cells and primary cerebral cortical neurons of foetal Sprague-Dawley rats at 18-19 days of gestation were used. The primary cerebral cortical neurons and PC 12 cells were exposed to 10 μmol/L Cd for different times (0,6,12,24,48 h), Cd at different concentrations (0,5,10 and 20μmol/L),10 or 20μmol/L cadmium in the absence or the presence of a-LA (100μmol/L) or NAC (100 μmol/L) for 24 h. Then the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidation (GSH-Px), glutathione reductase (GR), glutathione-S-transferase (GST), the contents of glutathione (GSH) and malondialdehyde (MDA) in cells and the activities of SOD, CAT, GSH-Px, the contents of MDA in mitochondria were measured by analysis kits. ROS levels were detected by 2,7-dichloro-dihydrofluorescein diacetate (DCFH-DA). Cell apoptosis rates were measured by Annexin V-FITC fluorescence staining assay and flow cytometry (FCM). The expression of VDAC, Bcl-2, Bax, cytochrome c, cleaved caspase-9 and -3 were detected by immunoblots.The results were as follows:①In comparison with the control group,10 μmol/L Cd treated cells for different time or Cd(5,10,20 μmol/L) treated for 24 h significantly increased SOD and CAT activity in PC 12 cells and decreased SOD and CAT activity in primary neurons(P<0.05 or P<0.01); however, a-LA or NAC treatment normalized SOD and CAT activity compared with 10 μmol/L Cd alone. MDA contents were significantly increased after treatment with Cd (P<0.05 or P< 0.01), which was significantly reduced by treatment with a-LA or NAC (P<0.05). Similarly, treatment with 10 μmol/L Cd for 24 h was associated with a significant decrease in GSH content, as well as decreased GSH-Px, GST and GR activity (all P<0.01). Consistent with their effect on SOD and CAT activity, a-LA or NAC administration normalized all of these variables toward baseline in 10 μmol/L Cd-treated cells (P<0.05). ROS production was significantly increased after treatment with 10 μmol/L Cd for 24 h(P<0.01), which was significantly reduced by co-administration of a-LA or NAC (P<0.01). ②The neuronal cells exposed to Cd at different concentrations (0、5、10、20 μmol/L), in mitochondrion the activity of SOD and CAT increased(P<0.05 or P<0.01) in PC12 cells and decreased (P<0.05 or P<0.01) in primary neurons, the activity of GSH-Px decreased and the content of MDA increased (P<0.05 or P<0.01), the above-mentioned caused by Cd were dose-dependent, however, a-LA or NAC treatment normalized SOD and CAT activity, increased GSH-Px activity, decreased MDA content compared with 10 μmol/L Cd alone.③Cd decreased cell viability, released Cyt C to the cytosol, changed the level of VDAC, Bcl-2 and Bax and increased the expression of cleaved caspase-9 and -3. Treatment with a-LA or NAC inhibited Cd-induced apoptosis through preventing cytochrome c release, increasing the expression of VDAC and Bcl-2, decreasing the expression of Bax, and inhibiting caspase-9 and -3 activation (all P<0.01).Conclusion:These results suggest that both a-LA and NAC function as increasing antioxidant effect, reducing lipid peroxidation caused by cadmium and inhibiting the mitochondrial apoptotic pathway induced by Cd exposure in neuronal cells.