The Mechanism Of Resveratrol Inhibitting Hepatocellular Carcinoma HepG2 Cells Proliferation By Inducing Autophagy

Objective1.To study the effects of Resveratrol(Res)on the proliferation and apoptosis of Hep G2 cells,and to explore its possible mechanism.2.After pretreatment with autophagy inhibitor 3-MA,the effects of Res on apoptosis and autophagy of hepatocellular carcinoma Hep G2 cells were further observed to clarify the effect of autophagy on the anti-hepatocellular carcinoma effect of Res.3.According to the experimental results,we analyzed the specific mechanism of Res as the extract of Polygonum cuspidatum and its Anti-hepatocarcinoma effect,in order to further explore the role and feasibility of Polygonum cuspidatum in the treatment of hepatocarcinoma.Methods1.CCK-8 assay was used to detect the changes of cell proliferation inhibition rate of Hep G2 cells treated with different concentrations of Res for 24,48 and 72 hours.At the same time,after pretreatment with autophagy inhibitor 3-MA,the inhibition effect of Res on the proliferation of Hep G2 cells at different concentrations and at different time was further tested.2.Flow cytometry was used to detect the effects of different concentrations of Res on cell cycle and apoptosis,and the effects of pretreatment with autophagy inhibitor 3-MA on cell apoptosis at 48 h.3.The effect of Res on autophagy was observed under fluorescence microscope after staining with MDC(Dansylcadaverine).4.Real-time fluorescence quantitative PCR(RT-PCR)was used to detect the effects of different concentrations of Res and pretreatment with autophagy inhibitor 3-MA on the m MRN expression of Beclin1,Bcl-2 and BTG2 in hepatocellular carcinoma Hep G2 cells.5.Western blot(WB)was used to detect the effects of different concentrations of Res and pretreatment with autophagy inhibitor 3-MA on the protein expression of Beclin1,Bcl-2 and BTG2 in hepatocellular carcinoma Hep G2 cells.Results1.The results of CCK-8 experiment showed that with the increase of Res concentration and the prolongation of Res action time,the inhibition rate of proliferation of Hep G2 cells increased gradually,and the difference was statistically significant compared with the control group(P < 0.01).Flow cytometry showed that the proportion of G2/M phase and the total apoptosis rate increased with the increase of Res concentration 48 hours after the intervention of Hep G2 cells,compared with the control group.The difference was statistically significant(P < 0.01).RT-PCR and WB experiments showed that the expression of Beclin1,BTG2 m MRN and protein increased with the increase of Res concentration,while the expression of Bcl-2 m MRN and protein decreased with the increase of Res concentration.The difference was statistically significant compared with the control group(P < 0.01).2.After pretreatment with autophagy inhibitor 3-MA,the inhibition rate of Res on the proliferation of Hep G2 cells increased in a time-and concentration-dependent manner,but compared with the corresponding concentration of Res experimental group,the inhibition rate of cell proliferation decreased in different degrees,and the difference was statistically significant(P < 0.05).At the same time,the role of Res in promoting apoptosis was also weaker than before,with statistical significance(P < 0.01).The results of MDC staining showed that the number of green fluorescent groups of MDC increased after Res intervention,and the number of green fluorescent groups decreased significantly after 3-MA treatment,while the fluorescence intensity of control group and 3-MA group was weak and the dot structure was very few.RT-PCR and WB experiments showed that after pretreatment with 3-MA,the expression of Beclin1,BTG2 m MRN and protein decreased,while the expression of Bcl-2 m MRN and protein increased(P < 0.05).Conclusion1.Res can inhibit the proliferation of Hep G2 cells,promote their apoptosis and induce cell cycle arrest in a concentration-dependent manner.2.After pretreatment with autophagy inhibitor 3-MA,the effect of Res on the proliferation of Hep G2 cells was partially reversed,suggesting that this effect may be autophagy-dependent,and its mechanism may be related to the effect on the autophagic signaling pathway of BTG2-Bcl-2/Beclin1.3.It is confirmed that Res can play an Anti-hepatocarcinoma role at the molecular level,which provides a theoretical basis for the clinical application of Polygonum cuspidatum in the treatment of hepatocarcinoma.