The Study Of Inhibitory Effect Of Ecliptasaponin A On HepG-2 Cells

Objective:With the development of bioscience,the cognition of the pathogenesis of tumor is gradually deepened.In order to improve the efficiency of cancer treatment and explore the best therapeutic effect,multifunctional therapeutic drugs have aroused widespread attention.Traditional chinese medicine for its low toxicity and pharmacological activities have been recognized,the pharmacological effects of ecliptasaponin A have been found.Currently,but limited to the study of extraction and simple cell model,the specific antitumor effect and mechanism is not clear,so still worth exploring.Ecliptasaponin A as potential anticancer drugs,the extraction and separation technology has matured,but its antitumor activity and is involved in cell signaling pathway mechanism is not clear,therefore,the specific antitumor effect we investigated ecliptasaponin A.Methods:The antitumor activity,apoptosis and autophagy mechanism of ecliptasaponin A was studied.In vitro antitumor activities of ecliptasaponin A test by MTT for liver cancer;The AO/EB double staining and Hoechst 33258 staining experiments were used to verify whether the drug can induce apoptosis and the changes of apoptotic from cell morphology;MDC staining to detect whether ecliptasaponin A could induce cell autophagy was detected;The mitochondrial membrane potential and reactive oxygen detection and flow ratio observation and cytometry assay of apoptosis or cell cycle arrest;Single cell gel electrophoresis was used to observe DNA damage in single cells;Western blot of ecliptasaponin A acting on cells,the expression level of apoptosis and autophagy related protein in cells.Results:After treatment with ecliptasaponin A for 48 h,the ecliptasaponin A significantly inhibited the growth of HepG-2 cells in a concentration-dependent manner.The IC50 was(29.8±1.6)μmol/L(Compared with the control group.P<0.05).Cell morphology assay showed that apoptotic cells significantly increased in AO/EB group with the concentration of 12.5 and 25 μmol/L,showing obvious apoptotic morphological characteristics.Flow cytometry showed that the cells exposure to ecliptasaponin A for 48 h,the apoptosis ratio increased by 4.06% and 11.82%.With use to the concentration of 12.5,25 μmol/L of ecliptasaponin A for 48 h,the effect can significantly block the cell cycle,the S arrest rates were 24.03%,43.19%.The result of single cell gel electrophoresis(comet assay)showed that ecliptasaponin A could induce DNA damage in cells for 48 hours,and cell scratch test could effectively inhibit tumor cell migration and proliferation.The results of MDC staining showed that cells after exposure to ecliptasaponin A for 48 h,ecliptasaponin A could induce autophagy obviously.Western blot showed that the hepatic cancer cells after treatment with ecliptasaponin A for 48 h,the levels of Bcl-2 and PARP proteins were down regulated,and the caspase-3 and Bax proteins were increased.Ecliptasaponin A induces apoptosis through interference with the mitochondrial signaling pathway.Western blot results showed that ecliptasaponin A can down-regulate the ratio of p62 and LC3I/II,increase the proportion of Beclin1,and indicate that ecliptasaponin A can reduce cell proliferation through autophagy.In summary,it can be presumably presumed that a certain concentration of ecliptasaponin A can promote the activation of mitochondria-dependent apoptosis pathway and play a role in apoptosis after HepG-2 cells are treated;It can increase Beclin1 and down-regulate LC3I/II.This ratio promotes autophagy in liver HepG-2 cells.