| [Objective] Through detecting the expression levels of the proteins related to cAMP-PKA-CREB signal pathway and apoptosis in the control group ,different PC 12 cell groups exposed to different concentrations of manganese and inhibitor-treated group, to explore the potential mechanism of cAMP-PKA-CREB signal pathway in manganese-induced neurotoxicity.[Methods] PC 12 cells were cultured in vitro and randomly exposed to different concentrations (0?100?200?300?400?500?600?700?800?900?M) of MnCl2·4H2O for 24 h, cell morphology was observed under inverted phase contrast microscope and cell viability was measured by MTT method,then the Mn-treated concentrations were confirmed as follow: the control group(0 ?M), the low dose of manganese-treated group (400 ?M), the intermediate dose of manganese-treated group (500 ?M), and the high dose of manganese-treated group (600 ?M). Additionally, to set an inhibitor-treated group: PC12 cells were pre-treated with 10.0 ?M Rolipram for 1 h, subsequently exposed to 500 ?M MnCl·4H2O for 24 h. Collected all PC 12 cell in the control group, different manganese-treated groups and inhibitor-treated group after 24 h manganese exposure, then used Annexin V-FITC apoptosis detection kit to determine the apoptosis rate of different groups of PC 12 cell, used ELISA kits to determine the expression levels of cAMP, pCREB and BDNF proteins related to cAMP-PKA-CREB signal pathway, and western blot assay was used to detect the expression levels of PDE4 and PKA proteins related to cAMP-PKA-CREB signal pathway as well as the contents of Bax and Bcl-2 proteins related to apoptosis in different groups.[Results]1. When manganese exposure concentration higher than 300 ?M, as manganese exposure concentration increased, cell viability decreased significantly. Cell viability in the low dose, intermediate dose and high dose of manganese-treated groups decreased significantly when compared with the control group (p <0.05). Different concentrations of manganese exposure can lead to changes in cell morphology and different degrees of damage.2. With increasing concentration of manganese exposure, the expression levels of proteins related to cAMP-PKA-CREB signal pathway were significantly changed. The expression levels of cAMP, PKA, pCREB and BDNF protein were significantly decreased along with manganese exposure levels increased; whereas PDE4 protein levels increased significantly along with manganese exposure levels increased (p<0.05).3. With manganese-treated concentration increased, the expression levels of apoptosis-related protein changed significantly. The content of pro-apoptotic protein Bax increased significantly along with manganese exposure concentrations increased; whereas the content of anti-apoptotic protein Bcl-2 was significantly decreased along with manganese exposure concentrations increased (p<0.05).4. Pre-treated with inhibitor can significantly alter the expression levels of cAMP-PKA-CREB signal pathway associated proteins and its downstream apoptosis-related proteins. In the cAMP-PKA-CREB signal pathway, as compared to the intermediate dose of manganese-treated group (500 ?M), the expression levels of cAMP,PKA, pCREB and BDNF protein in the inhibitor-treated group increased significantly, whereas the content of PDE4 was significantly reduced. Among apoptotic-related proteins, the expression level of pro-apoptotic protein Bax in the inhibitor-treated group were significantly lower than the intermediate dose of manganese-treated group, while the content of anti-apoptotic protein Bcl-2 in the inhibitor-treated group was significantly higher than the intermediate dose of manganese-treated group.5. With manganese exposure level increased, the apoptosis rate of PC 12cell was increased significantly. The apoptosis rate in different concentrations of manganese-treated groups were increased when compared with the control group (p<0.05). Furthermore, Compared with the intermediate dose of manganese-treated group, the apoptosisrate in the inhibitor-treated group was decreased (p<0.05).[Conclusion]1. Manganese exposure lead to change of morphology and different degrees of damage in PC 12 cell, and further reduced the cell viability. PC 12 cell can be induced apoptosis by maganese exposure. Inhibitor can reduce the apoptosis rate of PC 12 cell.2. Manganese can affect the contents of cAMP-PKA-CREB signal pathway associated proteins, and alter the expression levels of the downstream apoptotic-related proteins.3. Inhibitor may reduce the toxic effects induced by manganese in PC 12 cell, through improving the expression levels of cAMP-PKA-CREB signal pathway associated proteins and changing the contents of apoptosis-related proteins.4. cAMP-PKA-CREB signal pathway may play an important role in Mn-induced apoptosis of PC 12 cell. |
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