The Role Of Calcium In Cadmium-induced Apoptosis On Rat’s Cerebral Cortical Neuron Cultured In Vitro

To study the role of calcium in cadmium-induced apoptosis on rat’s cerebral cortical neurons, the model of cortical neurons cultured in vitro was established successfully, which were obtained from the foetal Sprague-Dawley rats at18-19days of gestation. The normal neuronal morphology was observed at different culturing time, and the cell purity was identified with the NSE immunohistochemical method. Cortical neurons were incubated with cadmium acetate of different concentrations (0,5,10,20μmol/L), in the absence or the presence of10μmol/L BAPTA-AM for12hours. The damage of Cd to the morphology of cortical neurons was observed. The viabilities of cortical neurons treated with cadmium acetate or BAPTA-AM were determined by MTT. The effects of Cd and BAPTA-AM on apoptosis rate, intracellular [Ca2+]i, ROS level, mitochondrial membrane potential were measured by flow cytometry (FCM). The mRNA expression of CaM was measured by FQ-PCR. The activities of ATPase were measured by spectrophotometer method. The results were as follows:①At day6post culture, the cell showed the best morphology and activity, which was best for further experiments, meanwhile, the cell purity is more than95%at this time.②The neuronal cells exposed to Cd resulted in morphology changes, which including the axon disappearance, loss of neuronal integrity and the appearance of apoptotic bodies. In addition, the above-mentioned changes caused by Cd are dose-dependent.③After exposure to Cd, the viability of cortical neurons decreased, in contrast, the cell vialility increased when co-treated with BAPTA-AM.④The apoptosis rate increased with the increase of Cd concertration, on the contrary, the apoptosis rate decreased when the cells co-inoculated with Cd and BAPTA-AM compared with the corresponding controls.⑤With the increased concertration of Cd, the intracellular [Ca2+]i increased significantly or very significantly (P<0.05or P<0.01) compared with the control group. However, the intracellular [Ca2+], decreased when co-treated with BAPTA-AM, and 10μmol/L groups decreased very significantly (P<0.01),20μmol/L groups significantly (P<0.05).⑥Cadmium induced a significant increase in ROS level compared with the control group(P<0.05), in contrast, the ROS level decreased significantly in the presence of BAPTA-AM(P<0.05or P<0.01).⑦Neuron’s mitochondrial membrane potential decreased with the increase of cadmium concdentrations, in which10μmol/L groups decreased very significantly (P<0.01) and20μmol/L groups significantly (P<0.05); in the presence of BAPTA-AM, mitochondrial membrane potential increased compared with corresponding Cd-treated group, and20μmol/L groups increased very significantly (P<0.01).⑧The activities of neuron’s Na+-K+-ATPase and Ca2+-Mg2+ATPase decreased significantly or very significantly (P<0.05or P<0.01); in the presence of BAPTA-AM, compared with20μmol/L group, the activities of Na+-K+-ATPase increased significantly (P<0.05) while Ca2+-Mg2+-ATPase very significantly (P<0.01).⑨The mRNA expression of CaM in20μmol/L group decreased very significantly (P<0.01) and in the presence of BAPTA-AM increased significantly (P<0.01), and the other group had no obvious change.Conclusion:①Exposure to different cadmium concentration can decrease the cell viability and change the neuronal morphology.②Exposure to different cadmium concentration can increase the cell apoptosis rate, intracellular [Ca2+],, ROS level, and decrease mitochondrial membrane potential, the activities of ATPase, the mRNA expression of CaM.③BAPTA-AM can inhibit the above change induced by cadmium, which showed that calcium overloading played a vital role in the apoptosis of cortical neuron caused by cadmium.