目的：探索中药单体20（ S）-人参皂苷Rg3（ Rg3）对肝癌细胞自噬介导的索拉非尼药物敏感性的影响。方法以人肝癌细胞株Hep3B为观察对象，分别接受Rg3、自噬抑制剂3-甲基腺嘌呤（3-MA）、索拉非尼、Rg3＋索拉非尼和3-MA＋索拉非尼处理，采用MTT法检测Hep3B细胞对Rg3、索拉非尼及3-MA的敏感性，CCK-8法检测Rg3、3-MA分别联合索拉非尼后对Hep3B细胞的增殖抑制作用，q值判断联合用药效果；LC3 Conversion实验及LC3 Turnover实验检测Rg3和索拉非尼对Hep3B细胞自噬的影响，Western blotting检测Rg3和索拉非尼对自噬相关蛋白Beclin1、LC3-Ⅰ／LC3-Ⅱ、p62水平的影响。结果 MTT检测显示，3种药物对Hep3B细胞的增殖抑制作用呈剂量和时间依赖性。 CCK-8检测显示，低、中浓度（0．5、1μg／ml）索拉非尼联合Rg3或3-MA对Hep3B细胞抑制作用均增强（ P均＜0．01），表现为协同作用；高浓度（2μg／ml）索拉非尼联合Rg3亦呈抑制增强作用（ P＜0．05），但联合3-MA的差异无统计学意义（ P＞0．05），高浓度索拉非尼与两药联合后均表现为相加作用。 LC3 Conversion实验显示，随着药物作用时间的延长，Rg3及索拉非尼均使LC3-Ⅱ蛋白水平逐渐升高；LC3 Turnover实验显示，索拉非尼联合溶酶体抑制剂氯喹（ CQ）后，LC3-Ⅱ蛋白水平明显升高（ P＜0．01），而Rg3联合CQ前后则无明显差异（ P＞0．05）。 Western blotting检测显示，与对照组相比，Rg3、索拉非尼均可使Beclin1蛋白表达升高（ P＜0．01），索拉非尼联合3-MA后Beclin1水平明显降低（ P＜0．01），而联合Rg3后下降不明显（ P＞0．5）。三药均可使LC3-Ⅱ水平升高（ P＜0．05），索拉非尼联合Rg3后LC3-Ⅱ水平高于索拉非尼单药（ P＜0．01），而联合3-MA后LC3-Ⅱ水平无明显变化（ P＞0．05）。3-MA、索拉非尼作用后，p62蛋白水平明显低于对照组（ P＜0．05），索拉非尼联合Rg3后p62水平明显升高（ P＜0．01），联合3-MA后水平明显降低（ P＜0．01）。结论索拉非尼可通过增加Beclin1的表达诱导肝癌细胞自噬导致药物敏感性下降，Rg3可增加索拉非尼的敏感性，其机制可能是通过抑制自噬降解从而抑制肝癌自噬活性来实现的。%Objective To investigate the effect of 20( s)-Ginsenoside Rg3 ( Rg3) on regulating autophagy-mediated sorafenib sensitivity of hepatocellular carcinoma cells. Methods Hep3B cells were used in this study and treated by Rg3, autophagy inhibitor 3-Methyladenine ( 3-MA) , sorafenib, Rg3 plus sorafenib and 3-MA plus sorafenib. The sensitivity of Hep3B cells to Rg3, sorafenib and 3-MA was detected by MTT method. Hep3B cells proliferation inhibition of Rg3, 3-MA plus sorafenib were detected by CCK-8 method. The interaction of sorafenib with 3-MA and Rg3 was assessed by q value. LC3 conversion and LC3 turnover assay were used to detect the autophagic regulation effect of sorafenib and Rg3. Autophagy related proteins, including Bcelin1, LC3 and p62, were detected by Western blotting. Results MTT showed that Rg3, sorafenib and 3-MA inhibited Hep3B cell proliferation in a dose and time-dependent manner. CCK-8 test showed that the inhibition effects of low ( 0. 5 μg/ml) and medium ( 1 μg/ml) doses of sorafenib in combination with Rg3 or with 3-MA were significantly higher than sorafenib alone with synergistic effect ( P<0. 01) . High dose ( 2 μg/ml) of sor-afenib in combination with Rg3 showed a higher inhibitory rate to Hep3B cells( P<0. 01) , but no difference was observed on the inhibi-tory rate of high dose of sorafenib with or without 3-MA ( P>0. 05) . The interaction of high dose of sorafenib in combination with Rg3 or 3-MA appeared addictive effect. LC3 conversion assay showed that with the extension of the duration of drug action, Rg3 and sorafenib increased the LC3-II level gradually. LC3 turnover assay showed that LC3 II level was significantly higher in sorafenib combined with CQ than that of sorafenib alone (P<0. 01), while Rg3 combined with or without CQ had no obvious changes of LC3-II levels (P>0. 05) . Western blotting showed that compared with control group, Rg3 and sorafenib increased Beclin1 protein expression obviously ( P<0. 01) . Compared with sorafenib, Beclin1 decreased significantly when sorafenib combined with Rg3 instead of in combination with 3-MA. Rg3, 3-MA and sorafenib caused the rising levels of LC3-II ( P<0. 05) . LC3-II level in Rg3 combined with sorafenib was higher than that of sorafenib alone ( P<0. 01) , but there was no significant difference between 3-MA and 3-MA in combination with sorafenib ( P>0. 05) . The level of p62 in Hep3B cells treated by 3-MA or sorafenib alone was significantly lower than that of control group ( P<0. 05) . Rg3 combined with sorafenib increased p62 level significantly ( P<0. 01) , while sorafenib combined with 3-MA decreased p62 level significantly ( P<0. 01) . Conclusion Sorafenib induced autophagy in hepatocellular carcinoma cells leads to decreased drug sen-sitivity by increasing the expression of Beclin1. Rg3 can increase the sensitivity of hepatocellular carcinoma to sorafenib possibly through inhibiting degradation of sorafenib-induced autophagy.
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