The Mitochondrial Mechanism Of Resveratrol Inhibits HeLa Cell Proliferation And Autophagy

Objection:To explore the mitochondrial mechanism of inhibition of cell proliferation and autophagy after treatment of HeLa cells with different concentrations of Resveratrol（RSV）for 30 min.Methods:1.Mitochondrial mechanism of RSV inhibition of HeLa cell proliferationHeLa cells were cultured by cell culture method.The effect of RSV on cellproliferation was detected by MTT assay,and the change of intracellular ROS levels was detected using the active oxygen kit,and then passed through Oxygraph-2k Oroboros.The mitochondrial respiration apparatus measures changes in the OCR of intact cells.The kit was used to detect changes in mitochondrial membrane potential（MMP）;mitochondrial DNA Copy Number assay;mitochondrial electron transport chain complexes and complexes were detected;and changes in lactic acid levels before and after incubation were measured.2.To investigate the occurrence of autophagy in HeLa cells induced by RSVAfter treatment of HeLa cells with different concentrations of RSV,the expressionlevel of autophagy gene LC3 was detected by fluorescence quantitative PCR;the occurrence of intracellular autophagy was observed by confocal fluorescence microscopy;the effect of RSV on cell proliferation was detected by MTT assay.Result:1.After RSV treatment of HeLa cells for 30 min and 24 h,there was a dose-and time-dependent effect of RSV on cell proliferation inhibition.With the increase of RSV concentration and prolongation of the action time,apoptosis increased significantly（P<0.01）.Subsequent experiments were performed on cells treated with RSV for 30 minutes.After basal respiration of cells incubated with 6.25μM RSV State4,CI,ADP/O,and RCR were significantly increased,indicating that RSV has an effect on cellular mitochondrial respiration.However,after treatment with high concentration RSV HeLa cells,the basal respiration,State3,Leak,SRC,ETS and CI and CII values of the cells were significantly decreased compared with the control group（P<0.01）.The results of detection of lactic acid content showed that there was no significant change in intracellular lactate content during the experimental period.When the RSV concentration was 50μM and 100μM,the mitochondrial membrane potential of HeLa cells was significantly decreased（P<0.05）.The results of quantitative real-time PCR showed that When the concentration of RSV was greater than or equal to 50μM,the copy number of mtDNA was significantly decreased（P<0.05）.The results of flow cytometry and kit detection of membrane potential showed that high concentration of RSV significantly damaged the mitochondrial membrane.2.After treatment with RSV for 30 min,the results of fluorescence quantitative PCR showed that the expression of LC3 in the cells was significantly decreased at concentrations of 6.25,12.5,and 25μM（P<0.05）,and the expression of LC3 in cells was significant at the concentration of 100μM.Increased sex（P<0.05）,which shows that high concentrations of RSV will induce autophagy in HeLa cells.Fluorescence confocal microscopy was used to observe the expression of autophagic protein in the cells.It was found that the expression of LC3 protein in HeLa cytoplasm increased with the increase of RSV drug concentration,and there was no significant change in LC3 protein expression after 30 min.LC3 was detected after 24 h.There was a significant change in protein expression.Conclusion:1.Resveratrol treatment of cells can cause mitochondrial damage.2.Resveratrol inhibited HeLa cells in a concentration-dependent manner.Low concentrations of resveratrol promoted cell respiration and increased mitochondrial coupling.ADP was used to generate ATP,and O₂ produced less free radical ROS.3.High concentrations of resveratrol can inhibit mitochondrial activity caused by changes in the expression of autophagy LC3.4.Resveratrol induces autophagy and inhibits cell proliferation in a dose-and time-dependent manner.