| Cadmium (Cd) is an extremely toxic metal commonly found in industrial workplaces, food contaminants and cigarette smoke. It is toxic even at low doses since the metal accumulates and has a long biological half-life in humans (10-30years). Cd has been shown to cause severe damage to a variety of organs, including the lung, liver, kidney, testis, brain and even to the placenta. Cd can enter into the brain parenchyma and neurons causing neurological alterations in humans and animal models, leading to lower attention, hypeociception, olfactory dysfunction and memory deficits. Moreover, there are studies showing the neurotoxicity of Cd at μM range on cell culture models like neurons and glial cells, and the Cd-mediated toxicity is thought to involve, at least in part, the induction of apoptosis. Furthermore, previous study demonstrated that apoptosis of cortical neurons caused by Cd is the main reason of abnormality in central nervous system, leading to memory deficits and mental retardation. However, the mechanisms of Cd-induced apoptosis have not been well elucidated. In the current experiment, the model of cerebral cortical neurons cultured in vitro was used, which were obtained from foetal Sprague-Dawley rats at18-19days of gestation, and the potential mechanism of Cd-induced cytotoxicity and apoptosis in rat’s cerebral cortical neurons was investigated, which will offer theoretic evidences for future exploring the mechanism in neurotoxicity of Cd.1. Mitochondrial pathway in cadmium-induced apoptosis of rat’s primary cultured cerebral cortical neuronsTo explore the mechamism of Cd-induced apoptosis in rat’s cerebral cortical neurons from mitochondrial pathway, the model of rat’s primary cerebral cortical neurons in vitro was established successfully. The neurons were exposured to cadmium acetate of different concentrations (0,5,10,20μmol/L) for12h. Then, the morphological changes of apoptosis in neurons with Cd treatment was observed by Hoechst33258staining, and the level of reactive oxygen species (ROS), mitochondrial membrane potential (AΨm) were detected by flow cytometry, meanwhile, the transcriptional level of Caspase-3mRNA was detected by real-time fluorescent quantitative PCR. In addition, the neurons were exposured to10μmol/L cadmium acetate for0,12,24,48h. Then, the Cleaved Caspase-9and Cleaved PARP were detected by immunoblot at each time-point. With immunocytochemical detection, the cultured cells were affirmed as neurons, which are positive to neurons specific enolase (NSE) antibody and represent classical features of neurons. After Cd exposure, in comparison with the control group, the cells in Cd-treated groups showed typical morphological changes of apoptosis with nucleus crimpled and chromatin condensedation, even nucleus disintegratation, on the other hand, ROS level were increased significantly (P<0.05or P<0.01), ΔΨm were decreased significantly (P<0.05or P<0.01), and the transcriptional level of Caspase-3mRNA increased significantly (P<0.01). Caspase-9was activated early at12h after treatment of10μmol/L Cd and PARP was cleaved subsequently. It was suggested that Cd-induced apoptosis involves mitochondrial pathway in rat’s cerebral cortical neurons.2. Effect of the disequilibrium of calcium homeostasis on the apoptosis in rat’s primary cultured cerebral cortical neurons induced by cadmiumTo investigate the effect of the disequilibrium of calcium homeostasis on the apoptosis in rat’s cerebral cortical neurons induced by Cd and ER-released calcium on the disequilibrium of calcium homeostasis induced by Cd, the neurons were exposured to cadmium acetate of different concentrations (0,5,10,20μmol/L), in the absence or the presence of10μmol/L BAPTA-AM [1,2-bis(2-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid-tetraacetoxymethyl ester], a specific intracellular Ca2+chelator and the inositol1,4,5tri-sphosphate receptor (IP3R) inhibitor,50μmol/L2-APB (2-aminoethoxydiphenyl borate) for12h. Then, the morphological changes of apoptosis in neurons with Cd treatment were observed by Hoechst33258staining and the intracellular [Ca+]i was detected by flow cytometry. Meanwhile, the activities of Na+-K+-ATPase as well as Ca2+-Mg2+-ATPase were measured by ATPase test kits, and the transcriptional level of CaM mRNA was detected by real-time fluorescent quantitative PCR. In comparison with the control group, the results showed that typical morphological changes of apoptosis were observed in cerebral cortical neurons with Hoechst33258staining after Cd treatment, intracellular [Ca2+]iwas increased significantly (P<0.01), in contrast, the activities of Na+-K+-ATPase and Ca2+-Mg2+-ATPase were decreased significantly (P<0.05or P<0.01) in the Cd-treated groups, and the transcriptional level of CaM mRNA was decreased significantly (P<0.01) in20μmol/L Cd group. Compared to the poisoning groups, apoptosis induced by cadmium can be efficiently prevented by BAPTA-AM, furthermore, the intracelluar [Ca2+]i were decreased significantly (P<0.05or P<0.01) in the presence of BAPTA-AM and2-APB groups. It was suggested that Cd could disturb intracellular Ca2+homeostasis by affecting the transcriptional level of CaM mRNA as well as the activities of Na+-K+-ATPase and Ca2+-Mg2+-ATPase and ER-released calcium, the disequilibrium of calcium homeostasis played a vital role in the Cd-induced apoptosis of rat’s cerebral cortical neurons.3. Influence of cadmium on MAPK signaling pathway in rat’s primary cultured cerebral cortical neurons.To observe the effect of Cd on the levels of the phosphorylation of extracellular signal regulated kinase1/2(ERK1/2), c-Jun NH2-terminal kinases(JNK), p38mitogen-activated protein kinases (p38MAPK) in rat’s primary cultured cerebral cortical neurons, the neurons were exposured to cadmium acetate of different concentrations (0,5,10,20μmol/L) for3h and10μmol/L cadmium acetate for different time points (0,1,2,3,4,5,6h). The total and phosphorylated protein of ERK1/2, JNK and p38MAPK were assayed by immunoblots. The result showed that after treatment of5,10,20μmol/L Cd for3h the phosphorylation of ERK1/2, JNK1and p38MAPK was increased in all other Cd-treated groups with the exception of ERK1/2in5μmol/L Cd-treated group, while the total protein had no obvious changes. Following the treatment of10μmol/L Cd, the phosphorylation of ERKl/2begin to increase from2h and keeps to6h, at the same time, the phosphorylation of JNK and p38MAPK begin to be activated from1h with a peak at3h and decreased gradually. However, the phosphorylation of JNK and p38MAPK was also higher than that in the control within6h. The total expression of ERK1/2, JNK and p38MAPK had no obvious changes with the Cd exposure. It was suggested that cadmium can activate the key proteins in the MAPK signaling pathway.4. Effect of cadmium on the oxidative damage of mitochondria in rat’s primary cultured cerebral cortical neurons To investigate the oxidative damage of mitochondria in rat’s cerebral cortical neurons caused by Cd. The neurons were exposured to cadmium acetate of different concentrations (0,5,10,20μmol/L) for6h. Then, changes in ultrastructure of mitochondria were detected by transmission electron-microscope and the contents of malon-dialdehyde (MDA) were detected by colorimetric method. In addition, the relative gene expression levels of cytochrome oxidase submits (COX Ⅰ/Ⅱ/Ⅲ) were quantified by real-time fluorescent quantitative PCR. In comparison with the control group, the results showed that the changes of ultrastructure in the Cd-treated groups are obvious, the most ultrastructural modifications involved mitochondrial swelling, fragmentation and disappearance of mitochondrial cristae. The contents of MDA were significantly increased in the poisoning groups (P<0.01). The relative expression levels of COX Ⅰ and COX Ⅱ in the three exposed groups were significantly decreased (P<0.05or P<0.01), the relative expression levels of COX Ⅲ were also significantly decreased (P<0.01) in10,20μmol/L groups. Therefore, the damage of mitochondria may be medicated by cadmium. Moreover, the decreased expression levels of COX Ⅰ, COX Ⅱ, COX Ⅲ may be related to the mitochondrial lipid peroxidation.5. Influence of cadmium on Bcl-2and Bax expression in rat’s primary cultured cerebral cortical neurons.To investigate the effects of Cd on the expressions of apoptosis-related genes Bcl-2, Bax in rat’s primary cerebral cortical neurons, the neurons were exposured to Cd of different concentrations (0,5,10,20μmol/L) for12h. Real-time quantitative PCR was used to detect the mRNA transcriptional levels of Bcl-2and Bax. Furthermore, the protein expressions of Bcl-2and Bax were also detected by immunoblots. In comparison with the control group, the results showed that transcriptional levels of Bax mRNA in Cd-treated groups increased significantly (P<0.01). On the contrary, the transcriptional levels of Bcl-2mRNA and the ratios of Bcl-2/Bax decreased significantly (P<0.05or P<0.01). Correspondingly, the decreased Bcl-2and increased Bax protein expression levels were obtained by immunoblots detection with the increasing concentration of Cd. These results together indicating that cadmium can promote apoptosis by regulating the transcription and protein expression of apoptosis-related genes, including Bcl-2and Bax.6. Effect of cadmium on mRNA transcription of nitric oxide synthase in rat’s primary cultured cerebral cortical neuronsTo study the impact of Cd on mRNA transcriptional levels of nitric oxide synthase in rat’s primary cultured cerebral cortical neurons, the neurons were exposured to Cd of different concentrations (0,5,10,20μmol/L) for12h. Then, transcriptional levels of neuronal nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS) mRNA were detected by real-time quantitative PCR. In comparison with the control group, the results showed that the transcriptional levels of nNOS mRNA increased significantly (P<0.05and P<0.01) in5,10μmol/L groups and transcriptional levels of iNOS mRNA increased significantly (P<0.01) in20μmol/L group. It was concluded that cadmium can regulate the transcription of nNOS and iNOS. |
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