Relation Between Autophagy And Apoptosis In EA.hy926 Cell Line Under Hypoxic Microenvironment

Partâ… Hypoxic micro-environment induce autophagy in endothelial cellsObjective:Hypoxia micro-environment play an important role in the tumor growth, metabolism and resistance.However, the mechanism of the tumor cells and endothelial cells to avoid damage to them in the tumor hypoxia micro-environment is not fully understood yet. Our study was to test hypoxic microenvironment the autophagy in endothelial cells.Methods:The hypoxia incubator was used to establish the hypoxic microenvironment; transmission electron microscopy was used to detect the autophagic bodies in endothelial cells; Real time PCR was used to test the expression level of ATG; laser confocal microscopy was used to detected the expression of microtubule-associated protein 1 light chain 3 (LC3) in autophagic body surface; Monodansylcadaverine (MDC) staining and acridine orange staining assay were used to quantify the number of autophagic bodies by different time of hypoxia; statistical analysis the relationship between hypoxic micro-environment and the autophagy in endothelial cells.Results:The TEM analysis showed that, autophagic bodies can be observed in endothelial cell sunder hypoxic microenvironment, showing vacuolar membrane structure, high electron density, suggesting that hypoxia can induce autophagy in endothelial cell by micro-environment. The result of RT-PCR shown the the expression of ATG were up-regulate by the hypoxic micrornvironment. Confocal laser microscopy showed that LC3-GFP particles in autophagic bodies in endothelial cells under hypoxic conditions increased significantly, MDC and acridine orange staining showed that the autophagic bodies increased significantly with different hypoxia time.Conclusion:Hypoxia can induce autophagy in endothelial cell.and this phenomenon is related with the hypoxia time.Partâ…¡The regulatory pathway of autophagy in endothelial cells induced by hypoxic micro-environmentObjective:The molecular mechanism regulate autophagy is divided into dependent and non-dependent mTOR molecular pathways. In this study, through the application of classâ…¢PI3K inhibitor 3-MA, mTOR inhibitor rapamycin and MPKAp38 inhibitor SB202190, explore the molecular mechanism of regulate autophagy in endothelial cells under hypoxic microenvironment.Methods:The hypoxia incubator was used to establish the hypoxic micro-environment;classâ…¢PI3K specific inhibitor 3-MA, mTOR specific inhibitor rapamycin, and SB202190, a specific inhibitor MPKAp38 were used to treated the endothelial cells in vitro; laser scanning confocal microscopeautophagic was used to detect the expression of microtubule-associated protein 1 light chain 3 (LC3); Monoda-nsylcadaverine(MDC) staining and acridine orange staining assay were used to quantify the number of autophagic bodies by different time of hypoxia; statistical analysis the relationship of three different types of inhibitors and the autophagy in endothelial cells.Results:Western blotting showed rapamycin can reduce the phosphorylation of mTOR and Akt, SB202190 can inhibit MAPKp38 phosphorylationin in endothelial cells; the laser confocal microscope analysis showed that, the number of autophagic bodies containing LC3 in endothelial cells treated by 3-MA was significantly reduced, while the autophagy body was increased treated by rapamycin, and SB202190 didn't have such effect;.MDC staining and acridine orange staining method also prompted 3-MA can inhibit endothelial cell production of autophagic bodies, and the rapamycin have the opposite effect, while SB202190 did not have such a role.Conclusion:mTOR/PI3K/Akt pathway involved in mediating the autophagy in endothelial cells under hypoxic micro-environment, the MAPKp38 pathway did not have such a role.Partâ…¢Autophagy induced by hypoxia on endothelial cell apoptosisObjective:Apoptosis and autophagy, two different type of programmed cell death were both exist in endothelial cells under the hypoxic micro-environment.In this study, we aim to test the relationship of autophagy and apoptosis by using the specific autophagy inhibitor 3-MA.Methods:TThe hypoxia incubator was used to establish the hypoxic micro-environment;autophagy-specific inhibitors were used in endothelial cells in vitro;flow cytometry were used to test the level of endothelial cell apoptosis, caspase-8,caspase-9 activity kit were used to detect cell regulatory pathway of apoptosis; caspase inhibitor Z-DEVD-FMK were used to detected the role of apoptosis in autophagy; MDC staining were used to test different level of autophagy; Transwell chamber migration and phalloidin staining were used to test cell function changes in endothelial cell after treated by autophagy inhibiter,Results:Flow cytometry showed that, by using 3-MA, inhibitor of autophagy, the cell apoptosis rate was significantly increased; caspase-8, caspase-9 activity assay suggest that after inhibition by 3-MA, caspase-8 did not significant change in activity, while caspase-9 activity was significantly increased, suggesting that autophagy inhibit endothelial cell apoptosis mainly through mitochondrial regulatory pathway; by transwell chamber migration and phalloidin detection of endothelial cell function, the results showed that after using 3-MA inhibit autophagy, the migration of endothelial cells was significantly increased as well as the intracellular filaments. MDC staining showed that by using caspase activity inhibitor, Z-DEVD-FMK, the level of autophagy in endothelial cell did not make any change, suggesting that apoptosis is not on the regulation of autophagy.Conclusion:Autophagy can avoid the apoptotic pathway in endothelial cells under hypoxia micro-environment as well as the ell function.