Mitochondrial Pathway of Cadmium-induced Apoptosis in Primary Rat Cerebral Cortical Neurons Culture
|Clinical Veterinary Medicine
|cadmium Rat Cerebral cortical neurons apoptosis mitochondrial pathway Z-VAD-fmk BAPTA-AM
To study the mitochondrial pathway of cadmium-induced apoptosis in rat’s cerebral cortical neurons and the effect of calcium overload on mitochondrial pathway in cadmium-induced apoptosis, the model of cortical neurons cultured in vitro was established successfully, which were obtained from the foetal Sprague-Dawley rats at18-19days of gestation. Cortical neurons were incubated with cadmium acetate of different concentrations (0,5,10,20μmol/L), in the absence or the presence of100μmol/L Z-VAD-fmk or10μmol/L BTPTA-AM. Changes in ultrastructure of mitochondria were detected by transmission electron-microscope. The cleaved-caspase-3, cleaved-caspase-9, cleaved-PARP, Bax and Bcl-2were detected by immunoblot. The viabilities of cortical neurons were determined by MTT and the morphology of the nuclei of rat’s cerebral cortical neurons was observed by hoechst33258staining.The results were as follows:①In comparison with the control group, the results showed that the changes of ultrastructure in the cadmium-treated groups is obvious, the most ultrastructural modifications involved mitochondrial swelling and disappearance of mitochondrial cristae. The above-mentioned changes were time-and dose-dependent.②the increased cleaved-caspase-3, cleaved-caspase-9, cleaved-PARP, Bax and decreased Bcl-2protein expression levels were obtained after exposure to cadmium. Z-VAD-fmk and BAPTA-AM can restrain the activation of caspase-3, caspase-9and PARR.③After exposure to Cd, the viability of cortical neurons decreased, and the above-mentioned changes caused by Cd are time-and dose-dependent. In contrast, the cell vialility increased when co-treated with Z-VAD-fmk or BAPTA-AM.④The neuronal cells exposed to Cd resulted in morphology changes of nucleus, which including nucleus crimpled and chromatin condensedation, even nucleus disintegratation. These changes were dose-dependent. Compared to the poisoning groups, the damage to nucleus induced by cadmium can be efficiently prevented by Z-VAD-fmk and BAPTA-AM.Conclusion:①Cadmium of different concentrations can decrease the cell viability, change the morphology of nucleus and mitochondrial.②Caspase-3, caspase-9and PARP were activated after exposure to cadmium, bax and Bcl-2protein expression levels was adjusted by cadmium.③Z-VAD-fmk can inhibit the aboved changes induced by cadmium:decrease of viability, morphology changes of nucleus, increased cleaved-caspase-3, cleaved-caspase-9, cleaved-PARP protein expression, which showed that caspase played a vital role in the apoptosis of cortical neuron caused by cadmium.④Compared to the poisoning groups, apoptosis induced by cadmium can be efficiently prevented by BAPTA-AM, furthermore, the viability increased, the cleaved-caspase-3, cleaved-caspase-9, cleaved-PARP protein expression decreased. It showed that calcium overloading played a vital role in the apoptosis of cortical neuron caused by cadmium and is related to mitochondrial pathway in cadmium-induced apoptosis of cerebral cortical neurons intimately.