Cadmium Induces Apoptosis Through The Mitochondrial Pathway And The Protection Of NAC In Rat’s Neuronal Cells

Cadmium (Cd) is recognized as one of the most toxic environmental and industrial pollutants due to its ability to damage the functions of several organs and tissues following either acute or chronic exposure. Cd can destroy the blood-brain barrier and enter the brain, leading apoptosis in neuronal cells. In addition, clinical studies showed that Cd exposure was considered to be an essential etiological factor in the learning disabilities of children. Cd of micro level could activated the Mitogen-activated protein kinase(MAPK) signal pathway, resulting in mitochondrial dysfunction, the decreases of mitochondrial membrane potential, the change of the mitochondrial membrane permeability and the release of the mitochondrial proteins into the cytosol. However, the mechanisms of Cd induces apoptosis through the mitochondrial pathway have not been well elucidated. In the current experiment, the model of primary neurons and PC12cells cultured in vitro were used to explore the mechanism of apoptosis induced by Cd in neuronal cells via mitochondrial pathway, the role of MAPK activation in mitochondrial pathway and the protection of NAC in the process of the above.1. Apoptosis induced by Cd in neuronal cellsTo explore the apoptosis induced by Cd in neuronal cells, the model of primary neurons and PC12cells cultured in vitro were used. The cells were treated with Cd (0,5,10,20μmol/L) for different time periods (12and24h). The cell viabilities were measured by MTT assay, the cell apoptosis rates were measured by Annexin V-FITC fluorescence staining assay. The results showed that the cell viabilities of primary neurons and PC12cells after12h or24h treatment decreased significantly with the increasing dosage of exposure compared with control group (P<0.05or P<0.01). After24h treatment, in comparison with the control group, the cells in Cd-treated groups showed morphological changes, such as crimpled and inadhesion of cell, disappearance of axon, dendrites and neural network, and the apoptosis rates of primary neurons and PC12cells increased significantly with the increasing dosage of exposure compared with control group (P<0.05or P<0.01). These data suggested that Cd could induce apoptosis in primary neurons and PC12cells in a dose-dependent manner.2. Apoptosis induced by Cd through mitochondrial apoptotic pathway in neuronal cellsTo investigate the role of mitochondrial pathway in apoptosis induced by Cd in neuronal cells, the model of primary neurons and PC12cells cultured in vitro were used. The cells were exposed to Cd of different concentrations (0,5,10,20μmol/L) for24h. Then, the immunoblot was used to measure the release of cytochrome c, caspase-3, the cleavage of poly (ADP-ribose) polymerase, the protein expressions of Bcl-2and Bax and the role of Z-VAD-fmk in the caspase-dependent pathway. The immunofluorescence was used to measure the nuclear translocation of apoptosis-inducing factor (AIF) and endonuclease G (Endo G). The result showed that Cd significantly decreased the Bcl-2/Bax ratio and increased release of cytochrome c, the cleavage of caspase-3and PARP, and the nuclear translocation of apoptosis-inducing factor and endonuclease G depend on the concentration of Cd, compared to the control group. In addition, Z-VAD-fmk(100μmol/L) could inhibit the cleavage of caspase-9, caspase-3and PARP, the decrease of the cell viabilities, the morphological changes of nuclear induced by Cd. These data suggested that mitochondrial pathway was involved in apoptosis induced by Cd in primary neurons and PC12cells.3. Cd activated MAPK singal pathway in neuronal cellsTo investigate the role of MAPK activation in Cd induced neuronal apoptosis, the model of primary neurons and PC12cells were cultured in vitro. The cells were exposed to Cd of different concentrations (0,5,10,20μmol/L) for3h or12h. Then, the activation of ERK, JNK and p38MAPK were measured by immunoblot and immumohistochemical staining. In addition, primary neurons and PC12cells were exposed to10μmol/L Cd for3h after30-min pretreatment with the specific ERK inhibitor U0126(10μmol/L), JNK inhibitor SP600125(20μmol/L) and p38MAPK inhibitor SB202190(20μmol/L). Then, the cell viabilities were measured by MTT assay, the cell apoptosis rates were measured by Annexin V-FITC fluorescence staining assay. The results showed that the phosphorylation level of ERK begin to increase from1h and keeps it untill12h in PC12cells treated with Cd(10μmol/L). Simultaneously, the phosphorylation of JNK and p38MAPK increased from4h and keeps it untill12h. The phosphorylation level of ERK, JNK and p38MAPK in primary neurons and PC12cells treated with Cd(5,10,20μmol/L) increased significantly in a dose-dependent manner compared to the control group (P<0.05or P <0.01). U0126, SP600125and SB202190blocked Cd-induced ERK, JNK and p38MAPK phosphorylation, respectively. U0126and SP600125, but not SB202190, inhibited Cd-induced cell death in primary neurons and PC12cells, and each inhibitor alone did not affect cell viability in an obvious manner. U0126(inhibitors of the phosphorylation of ERK) and SP600125(inhibitors of the phosphorylation of JNK), but not SB202190(inhibitors of the phosphorylation of p38), could partially rescued cells from the apoptosis induced by Cd. Furthermore, U0126and SP600125could inhibit the drop of cell viabilities and the rise of apoptosis rate induced by Cd in primary neurons and PC12cells significantly or extremely significantly (P<0.05or P<0.01). These findings suggest that ERK and JNK activation might be involved in apoptosis induced by Cd in primary neurons and PC12cells.4. Cd induced apoptosis via an ERK-and JNK-mediated mitochondrial apoptotic pathwayTo investigate the role of ERK, JNK activation in cadmium induced neuronal apoptosis via a mitochondrial pathway, the model of primary neurons and PC12cells cultured in vitro were used. Primary neurons and PC12cells were exposed to10μmol/L Cd for24h after30min pretreatment with the specific ERK inhibitor U0126(10μmol/L), JNK inhibitor SP600125(20μmol/L) and p38MAPK inhibitor SB202190(20μmol/L). Then, the release of cytochrome c, caspase-3, the cleavage of PARP, and the protein expressions of Bel-2and Bax were measured by immunoblot. The nuclear translocation of AIF and Endo G were observed by immunofluorescence. The results showed that the inhibition of the phosphorylation of ERK or JNK suppressed the release of cytochrome c, the cleavage of Caspase-3and PARP, the decrease of the ratio of Bcl-2/Bax and the nuclear translocation of AIF and Endo G induced by Cd significantly (P<0.05or P<0.01). These findings indicate that primary neurons and PC12cells apoptosis induced by Cd via a mitochondrial apoptotic pathway (involving caspase-dependent and caspase-independent pathways) is mediated by ERK and JNK activation.5. The protection of NAC in Cd-induced neuronal apoptosisTo investigate the protection of NAC in Cd induced neuronal apoptosis, the model of primary neurons and PC12cells cultured in vitro were used. Primary neurons and PC12cells were exposed to Cd (10μmol/L) for24h after30min pretreatment with100μmol/L NAC. Then, cell viabilities were measured by MTT assay, cell apoptosis rates were measured by flow cytometry, SOD, GSH-Px, CAT activity and MDA content were measured by colorimetric method, the release of cytochrome c, caspase-3and PARP cleavage, the protein expressions of Bcl-2and Bax were measured by immunoblot, and nuclear translocation of AIF and Endo G were observed by immunofluorescence. The results showed that NAC inhibited Cd-induced cell death, partially rescued cells from Cd-induced apoptosis; and suppressed the increased of SOD, CAT activity, MDA content, the decreased of GSH-Px activity; the release of cytochrome c, the cleavage of Caspase-3and PARP, the decrease of the ratio of Bcl-2/Bax and the nuclear translocation of AIF and Endo G induced by Cd significantly (P<0.05or P<0.01). These findings suggest that NAC can relieve oxidative damage to suppress the Cd-induced apoptosis via a mitochondrial apoptotic pathway.