Study On The Apoptosis In Rats H9c2 Cells Induced By Cadmium Through Mitochondria Pathway

To study the apoptosis in rats H9c2 cell line induced by cadmium and its mechanism. Myocardial cell in rats, H9c2 cell line, was chosen as the model and toxicological and cytobiocological methods, such as MTT, Giemsa staining, FCM and immunohistochemical method were used to detect to the process of apoptosis and related mechanisms induced by cadmium chloride. The results showed cadmium chloride could depress the growth of H9c2 cells, induce apoptosis, decrease mitochondrial membrane potential and change the expression levels of related apoptosis proteins. So the apoptosis of H9c2 cells induced by cadmium may be through mitochondrial pathway.1. The effects of cadmium chloride on survival rate of H9c2 cell The H9c2 cells were exposed to cadmium chloride at different concentrations (5, 10, 30, 50and 80μmol/L) for different times (6, 12, and 24h). MTT assay was applied to observe the toxic effects of cadmium chloride. The result showed the inhibitory rate induced by cadmium chloride increased gradually with the increase of dosage at each time-spot, and existed a time-dose-effect relationship. It indicated that 5~80μmol/L cadmium chloride had toxic effects on H9c2 cells and could depress the growth of H9c2 cells.2. The effects o f cadmium chloride on apoptosis of H9c2 cells Giemsa staining was applied to observe the morphological changes of H9c2 cells induced by cadmium chloride and calculate the apoptotic rate. The resultshowed the typical morphological changes of apoptosis could be observed under microscope. When the H9c2 cells were exposed to cadmium chloride for 6h, no obvious morphological changes could be observed. The percentage of apoptotic cells increased significantly from 8% to 15.2% compared with NC group (P<0.05). When H9c2 cells were exposed to cadmium chloride for 12 h, the percentage of apoptotic cells in 30μmol·L-1 group had increased to 28.8% and that in 80μmol·L-1 group was 38%. There was significant difference in the percentage of apoptotic cells compared with NC group (P<0.05). When the H9c2 cells were exposed to cadmium chloride for 24 h, the apoptotic rate in 5, 50 and 80μmol·L-1 groups were 26.2%, 76.6% and 88.6% respectively. The apoptotic rate increased significantly compared with NC group (P<0.001). AnnexinV-FITC/PI double fluorescent staining FCM was adopted to determine the changes of apoptosis in H9c2 cells. The result showed cadmium chloride with different concentration could all induce apoptosis in H9c2 cells and the apoptotic rate increased significantly compared with NC group(P<0.001). When the H9c2 cells were exposed to cadmium chloride for 6 h, apoptotic rate in 5μmol·L-1 cadmium chloride had increased significantly to 46.95% compared with NC group. When the H9c2 cells were exposed to cadmium chloride for 24 h, apoptotic rate increased slightly. So cadmium chloride could induce apoptosis in H9c2 cells.3. The effects of cadmium chloride on mitochondrial membrane potential (MMP) in H9c2 cellsRhodamine123 as a fluorescent probe combined with FCM was applied to determine mitochondrial membrane potential in H9c2 cells. The result suggested cadmium chloride with different concentration could affect MMP of H9c2 cells. MMP decreased gradually with the increase of the doses of cadmium chloride. When the H9c2 cells were exposed to 5, 30, 50 and 80μmol·L-1 cadmiumchloride for 6 h respectively, MMP decreased significantly compared with NC group (P<0.05). When the H9c2 cells were exposed to cadmium chloride for 12 h, although there were no obvious differences between exposure groups, the MMP decreased significantly compared with the same doses in 6 h (P<0.001). When the H9c2 cells were exposed to cadmium chloride for 24 h, the MMP decreased quickly from 50μmol·L-1 cadmium chloride. The MMP in 80μmol·L-1 cadmium chloride decreased to the lowest (P<0.001). Besides, the MMP decreased gradually with the increase of time. Therefore, cadmium chloride could decrease the MMP in H9c2 cells.4. The effects of cadmium chloride on the levels of proteins related to apoptosis in H9c2 cells.Immunohistochemical method was applied to detect the expression levels of Cyt-c, Bax, Bcl-2 and Caspase-9. The result showed cadmium chloride could induce the up-regulation of Bax not Bcl-2; increased Bax/ Bcl-2 ratio; furthermore, cadmium chloride could enhance the expression of Cyt-c and activate the cascade reaction to lead to the occurance of apoptosis.