A Preliminary Study On The Anti-cholangiocarcinoma Effect And Mechanism Of Ginsenoside Rh2 In Vitro

Currently the treatment for cholangiocarcinoma is limited and lack of effective medicines.In recent years,the anti-cancer effects of ginsenoside Rh2 have been shown to be superior in a variety of cancers,which can be summarized as inhibition of tumor cell proliferation,migration,induction of apoptosis,reversal of drug-resistant cells,enhancement of chemotherapeutic drug sensitivity,reduction of toxic side effects,and impact on tumor signaling pathways,while the study of the effects of ginsenoside Rh2 on cholangiocarcinoma and its mechanisms have not been clarified yet.In this research,we investigated the anti-cholangiocarcinoma effects and mechanisms of ginsenoside Rh2 in cholangiocarcinoma cells by in vitro experiments combined with network pharmacological methods.Methods: Study of the inhibitory effect of ginsenoside Rh2 on cholangiocarcinoma cells.Using CCK-8 assay the activity of ginsenoside Rh2 on different tumor cells as well as different sources of cholangiocarcinoma cells in relation to time and dose;and ordinary light microscopy to observe the changes on cell morphology from the morphological perspective,PI single-stain flow cytometry to detect the changes on the cycle,as well as the use of cell colony formation and wound healing to observe the effect on the clonogenic and migration ability of cholangiocarcinoma cells.2.Preliminary study of the mechanism of action of ginsenoside Rh2 against cholangiocarcinoma.Based on the virtual screening of anti-cholangiocarcinoma action targets by network pharmacology and molecular docking technology and preliminary validation,by collecting the targets of ginsenoside Rh2 and cholangiocarcinoma,obtaining the intersecting targets and analyzing to get the protein PPI network,using the core targets for enrichment analysis,gene expression validation and survival analysis,and simulating the binding process of ginsenoside Rh2 and cholangiocarcinoma targets by molecular docking,using Western blot for preliminary validation of the top 3 targets in the score ranking.3.Study on the apoptosis-inducing effect of ginsenoside Rh2 in cholangiocarcinoma cells.The changes in apoptotic status were observed qualitatively using Hoechst 33258 staining and AO/EB staining,the incidence of apoptosis was quantified by Annexin V-FITC/PI double-stained flow cytometry,and the expression of apoptosis-related proteins were verified by Western blot.Results: 1.20(S)-Rh2 showed that the strongest inhibition on cholangiocarcinoma cells QBC939 compared with hepatocellular carcinoma Hep G2 and cervical carcinoma He La cells;20(S)-Rh2 demonstrated that the significant inhibition on four cholangiocarcinoma cells,showing time-and dose-dependent effects;20(S)-Rh2 caused significant changes in the morphology of cholangiocarcinoma cells,as it was observed by the ordinary light microscopy.The cells became rounded,smaller in size,and the cell gap increased,and almost all cells died when the concentration of 20(S)-Rh2 was greater than 80 μM;the results of PI single staining exhibited that 20(S)-Rh2 could change the cycle of QBC939 and Hu CCT1 cells,and a significant increase of G0/G1 phase and decrease of S phase appeared with increasing concentration;the results of both colony formation and cell scratching showed that 20(S)-Rh2 at a maximum concentration of 80 μM could significantly inhibit clone formation and migration of cholangiocarcinoma cells compared to the control group(**p<0.01).2.Using a network pharmacology approach to screen and obtain 287 targets of ginsenoside Rh2 action and 723 targets of cholangiocarcinoma action,a total of 84 targets of ginsenoside Rh2anti-cholangiocarcinoma action were analyzed to obtain 5 core targets.The results of molecular docking simulations showed that the core targets all stably bound to ginsenoside Rh2,and the Western blot validation results were consistent with the predictions of network pharmacology and molecular docking.3.Fluorescence staining qualitatively observed the apoptosis of cells,and the results indicated that the cholangiocarcinoma cells treated with 20(S)-Rh2 were in an apoptotic state,and Annexin V-FITC/PI double-stained flow cytometry was in an apoptotic state.Flow cytometry quantitatively detected a significant increase in the proportion of early and late apoptotic rates of cholangiocarcinoma cells with increasing 20(S)-Rh2 concentration(*p<0.05),and Western blot results further showed that 20(S)-Rh2 increased Bax expression,Cleaved-caspase-3 expression,and Bax/Bcl-2 ratio was elevated.Conclusion: 1.20(S)-Rh2 can achieve anti-cholangiocarcinoma effect by affecting cell cycle and thus inhibiting proliferation,clone formation and migration.2.20(S)-Rh2anti-cholangiocarcinoma effect is closely related to the targets TP53,AKT and EGFR.3.20(S)-Rh2 can induce apoptosis of cholangiocarcinoma cells in vitro.