The Relationship Between Resveratrol And Cd-induced Autophagy And Apoptosis In HEK 293T Cells

Our laboratory study had shown that low concentration of cadmium (< 10 microns) could induce cell autophagy, and under the high concentration of cadmium cell apoptosis would happen. This study used cell culture, transmission electron microscopy (TEM), Western blot, inverted phase contrast microscope, confocal technology and flow cytometry and cell biology to explore the relationship between resveratrol and cadmium-induced autophagy in HEK 293T cell, to explore the relationship between resveratrol and autophagosome-lysosome fusion(basised on the cell autophagy induced by cadmium), to explore the relationship between resistance to oxidation of resveratrol and Cd-induced autophagy, to explore the relationship between resveratrol and Cd-induced apoptosis. The following are our study results:1. Relationship can strengthen the Cd-induced autophagy in HEK 293T cells.HEK 293T cells (5×105) were seeded in 6-well plates and randomly divided into control group and experimental group. Experimental group: ①The cells were exposed to 8 μM Cd for 12 h;②cells pretreated with resveratrol (40 microns) for 1 h, and then exposed to (0～12 μM) Cd for 24 h;③ Resveratrol (40 μM) and 3-MA (5 mM) were chosen and co-treated with cadmium (8 and 12 μM) for 24 h in HEK 293T cells. The changes of autophagosomes and autophagic vacuoles were analyzed by transmission electron microscopy (TEM), the change of LC3B-Ⅱ/Ⅰ protein were measured by western blotting and the gather of GFP-LC3 was analyzyed by confocal microscope. After HEK 293T cells were exposed to cadmium, we found that the number of autophagosome were significantly increased. Resveratrol can strengthen the autophagy induced by cadmium, and changes obviously when under 8 and 12 μM, after joined 3-MA (an autophagy inhibitors) this kind of phenomenon was suppressed. These findings suggest that resveratrol can strengthen the autophagy induced by cadmium.2. Resveratrol maybe play a role of autophagy before the stage of autophagosome-lysosome fusion.HEK 293T cells was seeded in 6-well plates and resveratrol (40 μM) and/3-MA (5mM) were chosen and co-treated with cadmium (8 and 10 μM) for 24h, or chose resveratrol (40 μM) and/TG (3μM) to co-treated with cadmium (8 and 10 μM) for 24h. The expression of cathepsin L and LC3B-Ⅱ/Ⅰ protein were measured by western blotting and the colocalization of LAMP2 and GFP-LC3 was analyzyed by confocal microscope. We observed that resveratrol can enhance the activity of lysosomes(under the cell autophagy induced by cadmium). When autophagy was inhibited the activity of lysosomal is also reduced, TG increased cell autophagy ability but decreased the activity of lysosomes, and resveratrol has failed to change the results. These findings suggest that resveratrol maybe play a role of autophagy before the stage of autophagosome-lysosome fusion.3. Resveratrol can enhance Cd-induced autophagy related to its oxidation resistance, and the SIRT1 plays an important role in it.HEK 293T cells (5×105) were seeded in 6-well plates. Experimental group:cells pretreated with resveratrol (40 microns) for 1 h, and then exposed to(0～12 μM)Cd for 24 h, resveratrol (40 μM) and/EX-527 (1μM) were chosen and co-treated with cadmium (10 μM) for 24h, or chose resveratrol (40 μM) and/EX-527 (1μM) to co-treated with H2O2 (1mM) for 24h. The expression of ROS was analyzyed by microplate reader, the expression of SIRT1 and LC3B-Ⅱ/Ⅰ protein were measured by Western blottingting and the colocalization of LAMP2 and GFP-LC3 was analyzyed by confocal microscope. We observed that with the increase of cadmium concentrations ROS also increases, and after joining resveratrol ROS was reduced; Resveratrol can activate SIRT1, and when the amount of SIRT1 were inhibited, resveratrol on autophagy role also weakened. These findings suggest that resveratrol can enhance Cd-induced autophagy may be related to its oxidation resistance, and the SIRT1 plays an important role in it.4. Resveratrol can cause the inhibition of Cd-induced apoptosis.HEK 293T cells were seeded in 6-well plates. After adaptation training, with 40 μM RSV,10 μM Cd,20 μM Cd,10 μM Cd+RSV and 20 μM Cd+RSV processing HEK 293T cells, respectively. The cellular morphology was observed by phase-contrast microscopy, the expression of caspase-3 was measured by western blotting and the cell apoptosis was analyzyed by Flow cytometry. We observed that HEK 293T cells shrinkage and distortioneven death, after treated with 8 μM Cd for 24h, but RSV changed this situation; Resveratrol reduces the caspase-3, which increasesed by Cd; Apoptosis induced by cadmium can be blocked by resveratrol. These findings suggest that resveratrol can suppress the apoptosis induced by cadmium.The present study has shown that the low concentrations (<10 μM) of Cd induces cells autophagy. Resveratrol can strengthen the autophagy induced by cadmium and its effect is likely to be before the autophagy lysosome fused. Resveratrol can enhance Cd-induced autophagy may be related to its oxidation resistance, and the SIRT1 plays an important role in it, what’s more resveratrol can suppress the apoptosis induced by cadmium.