Effects Of Maslinic Acid On Autophagy And Proliferation Of Gastric Cancer SGC-7901 Cells

Objective To observe the effect of maslinic acid(MA)on autophagy and proliferation of gastric cancer SGC-7901 cells,and to explore the role of AMPK/mTOR pathway in the process of autophagy.Methods 1.The inhibitory effect of maslinic acid on the proliferation of gastric cancer SGC-7901 cells.Taking gastric cancer SGC-7901 cells as the research object,maslinic acid was treated with different concentrations(5μM,10μM,20μM,40μM,80μM)for 24 h and48h,respectively.The CCK-8 method was used to detect the effect of maslinic acid on cell proliferation,to calculate the IC50 concentration,screening the concentration and action time of maslinic acid;2.The immunofluorescence method was used to detect the effect of maslinic acid on autophagy of gastric cancer SGC-7901 cells.Experimental groups: Solvent control group,different concentrations(10μM,20μM,40μM)maslinic acid group,40μM maslinic acid + AMPK inhibitor group.After 24 hours of treatment of cells in each group,the immunofluorescence method was used to detect LC3 B to observe the autophagy of gastric cancer SGC-7901 cells in each group;3.Western Blot was used to detect the expression changes of AMPK/mTOR signaling pathway related proteins(p-AMPK,AMPK,p-mTOR,mTOR)and LC3-I and LC3-II proteins after maslinic acid treatment of gastric cancer SGC-7901 cells.Results 1.CCK-8 results showed that:maslinic acid can inhibit the proliferation of SGC-7901 cells.As the concentration of maslinic acid increases,the cell proliferation inhibition rate increases,which is concentration-dependent under the experimental conditions(P<0.05);under the experimental conditions When the same concentration of maslinic acid acted on SGC-7901 cells for 24 h and 48 h,there was no significant difference in the cell proliferation inhibition rate between the two time points(P>0.05).Therefore,the maslinic acid treatment time was taken for 24 hours for follow-up experiments;according to the IC50(33.75μmol/L)of maslinic acid acting on SGC-7901 cells for 24 hours,the concentration of maslinic acid(10μM,20μM,40μM)was selected for follow-up experiments.2.Immunofluorescence results showed that: the relative fluorescence intensity of cells in the 10μM maslinic acid group(1.73±0.34)was not statistically different from that of the Solvent control group(P>0.05);the relative fluorescence intensity of cells in the 20μM maslinic acid group(2.97±0.30)and the relative fluorescence intensity of cells in the 40μM maslinic acid group(3.89±0.57)were higher than that of the Solvent control group(P<0.05);the relative fluorescence intensity of cells in the 40μM maslinic acid+AMPK inhibitor group(1.09±0.22)was lower than that in the 40μM maslinic acid group(3.89±0.57)(P<0.05).3.Western Blot results showed that: the protein expression levels of p-AMPK,LC3-Ⅰ,and LC3-Ⅱ in the maslinic acid(10μM,20μM,40μM)group gradually increased with the increase of maslinic acid concentration,and the upward trend of LC3-Ⅱ protein expression level was greater than LC3-I protein expression level,and the expression level of p-mTOR protein gradually decreases with the increase of maslinic acid concentration;compared with the 40μM maslinic acid group,the expression levels of p-AMPK,LC3-Ⅰ,and LC3-Ⅱ proteins decreased in the 40μM maslinic acid+AMPK inhibitor group,and the decline trend of the LC3-Ⅱ protein expression level was greater than that of the LC3-I protein expression level,while p-mTOR protein expression level increased.Conclusions 1.Maslinic acid can inhibit the proliferation of gastric cancer SGC-7901 cells in a concentration-dependent manner within the experimental range;2.Maslinic acid may induce autophagy by activating AMPK/mTOR signaling pathway to inhibiting the proliferation of gastric cancer SGC-7901 cells.