积雪草酸对人肝癌SMMC-7721细胞增殖和自噬的影响

目的  探讨积雪草酸（asiatic acid，AA）对人肝癌SMMC-7721细胞增殖和自噬的影响。方法 不同浓度AA作用于人肝癌SMMC-7721细胞24 h后，MTT法检测细胞活性并观察自噬抑制剂3-MA对40 μmol/L AA的干预作用；MDC染色检测自噬泡的形成，Western blot检测自噬相关蛋白LC3-Ⅰ、LC3-Ⅱ、p62、mTOR、p-mTOR及p53的表达。结果 MTT检测结果显示，不同AA浓度均可抑制人肝癌SMMC-7721细胞增殖，并呈浓度依赖性（F＝46.790，P＝0.006），IC₅₀ =37.313 μmol/L。与单独使用40 μmol/L AA相比，自噬抑制剂3-MA可部分逆转40 μmol/L AA对SMMC-7721细胞增殖的抑制作用［（46.400±9.099）% vs （22.000±3.391）%，P＜0.001］。MDC染色实验表明40 μmol/L AA干预可增加自噬泡形成。Western blot检测发现，与对照组比较，40 μmol/L AA可明显降低LC3-Ⅰ的蛋白表达，而提高LC3-Ⅱ表达（1.744±0.108 vs 1.529±0.065，t=2.928，P=0.043；0.113±0.031 vs 0.380±0.036，t=-9.754，P<0.001），降低p62蛋白表达（0.522±0.024 vs 0.123±0.019，t=22.565，P<0.001）和p-mTOR蛋白表达（1.252±0.039 vs 0.353±0.028，t=30.775，P<0.001），但对mTOR和p53的蛋白表达无影响（1.713±0.111 vs 1.556±0.076，t=1.555，P=0.190；0.671±0.040 vs 0.726±0.055，t=-1.555，P=0.210）。结论 AA能抑制人肝癌SMMC-7721细胞增殖，可能与其通过非p53依赖方式负调控mTOR通路诱发自噬有关。

Effects of asiatic acid on proliferation and autophagy of human hepatoma SMMC-7721 cells

Objective To investigate the effects of asiatic acid（AA） on cell proliferation and autophagy of human hepatoma SMMC-7721 cells. Methods The human hepatoma SMMC-7721 cells were exposed to different concentrations of AA alone or combined with autophagy inhibitor 3-MA for 24 h，after that，the cell viability was examined by MTT method，the formation of autophagic vacuoles were detected by MDC staining，the expression of the autophagy-related proteins including LC3-Ⅰ，LC3-Ⅱ，p62，mTOR，p-mTOR，and p53 were detected by Western-blot. Results  MTT assay results showed that different concentrations of AA could inhibit the proliferation of human hepatocarcinoma SMMC-7721 cells in a concentration-dependent manner（F＝46.790，P＝0.006） with IC₅₀= 37.313 μmon/L.When combined with autophagy inhibitor 3-MA，the cell proliferation inhibition rate （%） of AA to SMMC-7721 cells was significantly reduced compared with the group treated with 40 μmol/L AA alone［（22.000±3.391）% vs （46.400±9.099） %，P<0.001）］. AA increased the amount of autophagic vacuoles detected by MDC staining. Compared with the control group，40 μmol/L AA significantly down-regulated the protein expression level of LC3-I while up-regulated the protein expression of LC3-Ⅱ（1.744±0.108 vs 1.529±0.065，t=2.928，P=0.043；0.113±0.031  vs 0.380±0.036，t=-9.754，P<0.001），but down-regulated the p62 protein expression（0.522±0.024 vs 0.123±0.019，t=22.565，P<0.001）.　Besides，AA significantly reduced the protein expression level of p-mTOR（1.252±0.039 vs 0.353±0.028，t=30.775，P<0.001），but had no influence on the protein expression of mTOR and p53（1.713±0.111 vs 1.556±0.076，t=1.555，P=0.190；0.671±0.040 vs 0.726±0.055，t=-1.555，P=0.210）. Conclusions AA can inhibit the proliferation of human hepatoma SMMC-7721 cells，which may be related to the AA-inducted autophagy via the suppression of mTOR signaling pathway in a p53-indepandent manner.