Relationship Between Autophagy And Apoptosis In PC12 Cells Under Hypoxia Injury

Part I Hypoxia injury induces the activation of autophagy in PC12 cellsObjective To discover the level of autophagy in neuron-like adrenal pheochromocytoma tumor cells(PC12 cells) after hypoxia injury.Methods The hypoxia incubator was used to establish the hypoxia injury model; GFP-LC3 plasmid was constructed as the method introduced before. In the case of giving or not giving lysosomal proton pump inhibitors Baf A1, western blot assay was used to detect the expression of autophagy-related protein; RT-PCR was used to detect the m RNA levels of autophagy microtubule-associated protein 3 light chain 1(LC3); confocal microscopy assay was applied to detect changes in the expression and distribution of LC3. Statistical analysis was used to evaluate the relationship between hypoxia injury and autophagy.Results Western blot results showed that after hypoxia injury, the expression of autophagy-related proteins in PC12 cells significantly increased with prolonged hypoxia time, which declined 24 later. RT-PCR results showed that after hypoxia injury, m RNA levels of LC3 significantly increased. Confocal microscopy examination showed that after hypoxia injury, cells containing GFP-LC3 particles autophagic bodies in PC12 cels increased significantly. Since there is a correlation between the occurrence of autophagy and hypoxia damage.Conclusion Hypoxia injury can induce autophagy in PC12 cells and there exist a correlation between the two events.Part II The role and mechanism of autophagy in hypoxia injuryObjective We use the hypoxia-sensitive neuron-like PC12 cells as hypoxia injury model to discover the role of autophagy in hypoxia injury and its possible mechanism.Methods The hypoxia incubator was used to establish the hypoxia injury model. PC12 cells were treated by autophagy inhibitor, 3-MA, simultaneously as hypoxia treatment. MTT assay was used to detect cell viability, and LDH assay was used to measure cell toxicity. PI staining assay was applied to estimate cell death. Caspase-3 activity assay kit was used to detect the activity of caspase-3, and western bolt was used to check the change of cleaved-caspase-3, which is the active form of caspase-3. Statistical analysis was used to analyze the changes of cell viability and apoptosis after autophagy was inhibited by 3-MA, and its correlation.Results MTT results showed that the viability of autophagy inhibitor 3-MA treated PC12 cells decreased further. LDH results showed that cell toxicity of 3-MA treated PC12 cells increased correspondingly. PI staining assay showed that more cells dead after autophagy inhibitor 3-MA treatment. Caspase-3 activity assay also showed that the activity of caspase-3 enhanced after 3-MA treatment. Finally, western blot results showed the increase of the expression of active form of caspase-3, cleaved-caspase-3.Conclusion Autophagy protects PC12 cell from hypoxia injury, which is related to the inhibition of apoptosis.Part III Apoptosis inhibits autophagyObjective Autophagy deceased gradually 24 hours after hypoxia treatment, with the increased apoptosis. The role of apoptosis and its possible mechanism on autophagy is not fully understood. The purpose of this study is to explore whether the decreased autophagy is caused by apoptosis enhancement induced by hypoxia, and whether there is a correlation between this two events.Methods The hypoxia incubator was used to establish the hypoxia injury model. Flag-Bax WT, Flag-Bax S184 Vand Flag-Bax ?C overexpression plasmids were constructed. After cells were co-transfected with EGFP-LC3 and Flag-Bax WT(or Flag-Bax S184 V, Flag-Bax ?C), western blot was used to detect the changes of autophagy-related protein. Fluorescence microscope was applied to show the change of green fluorescent protein EGFP-LC3. Apoptosis inhibitor z-VAD-FMK, proteasome inhibitor MG132 and lysosomal inhibitors chloroquine were given to cells respectively, then western blot was used to detect the changes of LC3 expression in cells co-transfected with the two plasmids mentioned above. Western blot was used to detect the expression of LC3 after cells were treated with hypoxia and apoptosis inhibitor z-VAD-FMK.Results Western blot results showed that when EGFP-LC3 was co-transfected with the wild-type Flag-Bax WT plasmids or active Flag-Bax S184 V, the expression of LC3 was significantly reduced, while being co-transfected with the mutant Flag-Bax ?C plasmids do not have this phenomenon. Apoptosis inhibitor z-VAD-FMK partially reversed the reduction of LC3 expression caused by overexpression of wild-type Flag-Bax WT or Flag-Bax S184 V plasmid. Apoptosis inhibitor z VAD also can inhibit the reduction of LC3 B expression after hypoxia injury.Conclusion 24 hours after hypoxia injury, increased apoptosis can inhibit the activation of autophagy. Overexpression of Bax induced apoptosis can inhibit autophagy, which may be related to the degradation of LC3 B protein executed by caspase. There exist a correlation between apoptosis and autophagy.