Effect Of Ginsenoside On Apoptosis And Autophagy Of Hela Cells And Its Mechanism

Cervical cancer is the second most common cancer affecting women worldwide,especially in less developed areas.In the early stages of cervical cancer,women may have no symptoms and cervical cancer is characterized by high morbidity and mortality.Therefore,the development of drugs to treat cervical cancer,more and more attention.Natural drugs have the advantages of multi-target,low toxicity and wide source,so the selection of anticancer drugs from natural drugs has become a research hotspot.Ginseng is a famous traditional medicinal plant,which has been widely used in Asia for thousands of years and is becoming more and more popular in western countries recently.Ginsenoside is one of the most important active ingredients in ginseng,which has anti-tumor and immunomodulation effects.Therefore,in this study,ginsenosides Rh2,Rg3 and total ginsenosides with anti-cervical cancer activity were screened from various saponins,and their mechanism of action was studied.Objective: In this study,HeLa cells of cervical cancer were used as the model to screen out ginsenosides with regulatory effects on He La cells of cervical cancer,and to investigate the effects of ginsenosides on autophagy and apoptosis of cells under different cell culture conditions,as well as the basic research of pharmacodynamic substances.Methods: The cck-8 method was used in this study to investigate the effect of ginsenoside on cell viability,and determine the concentration and duration of ginsenoside administration.Hoechst 33342 staining and Annexin V/FITC-PI double staining were used to investigate the apoptotic effect of ginsenosides on He La cells and the cell cycle was analyzed by flow cytometry.The apoptosis pathway was detected by ROS and rhodamine 123.Western Blot and q RT-PCR were used to detect the expression levels of apoptotic and autophagy-related proteins and mrnas.The degree of autophagy was detected by AO staining and EGFP-LC3 transfection.Results:1.The serum starvation method was used to induce autophagy,and different monomers and total saponins of ginseng were screened.The results showed that under serum-free culture,the ginsenosides Rh2,Rg3 and total saponins could inhibit the proliferation of He La cells for 24 hours,with the concentration of 5 μM,20 μM and60 μg/m L,respectively.2.The total ginsenosides irreversibly enhanced the autophagy level of He La cells,including promoting the formation of AVO,the aggregation of EGFP-LC3,and the expression of autophagy related factors.With the participation of actinomycin,the degradation of p62 also increased significantly.Total ginsenoside(0-120 μg/m L)significantly inhibited the proliferation of He La cells under starvation,and autophagy inhibitor 3-ma partially restored this inhibition.At the same time,total ginsenosides down-regulated the transcription level of bst-2 in cells,indicating that the influence of total ginsenosides on He La cells is mediated by a variety of pathways.3.In serum-free starvation,ginsenoside Rh2 and Rg3 regulated the autophagy level of He La cells,causing changes in cell AO staining,increasing the transformation of LC3 I-LC3 II,the aggregation of EGFP-LC3 fluorescent spots,the red-green fluorescence ratio of m RFP-GFP-LC3,and inhibiting the degradation of autophagy substrate p62.It also leads to the increase of ROS,the decrease of mitochondrial membrane potential,the release of apoptosis inducer AIF from mitochondria,nuclear transfer,and a series of subsequent apoptotic events.Autophagy inducer rapamycin inhibited the apoptosis induced by ginsenoside Rh2 and Rg3,while autophagy inhibitor BA1 promoted the apoptosis induced by rapamycin.Therefore,Rh2 and Rg3 promote He La cell apoptosis by regulating autophagy.4.Under normal cell culture conditions,ginsenoside Rh2 can simultaneously improve the apoptosis rate and autophagy level of cervical cancer cells.Ginsenoside Rh2 significantly reduces mitochondrial membrane potential by affecting it,causing changes in expression of apoptosis-related factors and inducing apoptosis of cervical cancer cells.Meanwhile,ginsenoside Rh2 also increased the expression of autophagy initiating proteins,promoted the shear and aggregation of autophagy marker proteins,increased the degradation of autophagy substrate proteins,and induced autophagy by AMPK/m TOR pathway.Rapamycin,an autophagy inducer,reduced the apoptosis induced by ginsenoside Rh2,while the addition of autophagy inhibitor 3-MA increased the apoptosis induced by it.Meanwhile,the apoptosis inhibitor Z-VAD-FMK not only reduced the apoptosis rate,but also increased the level of autophagy.It indicates that apoptosis and autophagy are closely related and influence each other.Conclusion:1.Ginsenosides Rh2 and Rg3 play a role in the late stage of autophagy,inhibiting the autophagic flow of cells,making the cells unable to maintain their own state through autophagy,thus losing the balance between autophagy and apoptosis,and then causing cell apoptosis through the mitochondrial AIF pathway.2.Total ginsenosides can cause excessive autophagy and autophagic death of cells,and at the same time reduce the transcription level of bst-2,which is necessary for the survival of tumor cells,indicating that the influence of total ginsenosides on He La cells is mediated by a variety of pathways.3.Under normal culture conditions,ginsenoside Rh2 induced apoptosis and also induced protective autophagy through AMPK/m TOR pathway.It suggests that inhibiting autophagy may be a potential therapeutic strategy to improve Rh2 efficiency.