目的 探讨三叶青Tetrastigma Hemsleyani Radix黄酮（THRF）对肝癌细胞增殖、侵袭和细胞焦亡的影响，以及其对腺苷酸活化蛋白激酶（AMPK）/哺乳动物雷帕霉素靶蛋白（mTOR）信号介导的线粒体自噬的调控机制。方法 以低、高质量浓度（50、100 μg·mL^-1）的THRF处理SK-HEP-1细胞；同时以AMPK信号抑制剂化合物C（CC）进行功能挽救实验，即设置THRF（100 μg·mL^-1） ＋CC（50 μmol·L^-1）组；以自噬抑制剂3-甲基腺嘌呤（3-MA）进行功能回复实验，即设置THRF（100 μg·mL^-1）＋ CC（50 μmol·L^-1） ＋3-MA（5 μmol·L^-1）组；对照组不加药，THRF、CC、3-MA同时加药，加药后培养24 h。5-乙炔基-2'-脱氧尿苷（EdU）试剂盒检测细胞的增殖； Transwell小室检测细胞的侵袭； JC-1检测线粒体的膜电位； DCFH-DA染色实验检测活性氧（ROS）的含量； MitoTracker Red-Lysotracker green双染色检测线粒体自噬；¹²⁵IPCR（qRT-PCR）检测线粒体自噬、细胞焦亡相关的mRNA的表达； Western blotting检测细胞中AMPK、mTOR、磷酸化-mTOR（p-mTOR）、酵母自噬相关基因6同源物（Beclin1）、自噬相关基因5（Atg5）、核苷酸结合寡聚化结构域样受体家族含热蛋白结构域蛋白3（NLRP3）和消皮素D（GSDMD）的表达水平。结果 与对照组比较，THRF明显降低SK-HEP-1细胞的增殖、侵袭以及线粒体膜电位（P＜0.05），明显升高细胞中ROS含量（P＜0.05），明显升高自噬体的表达；明显升高BECN1、Atg5、NLRP3和GSDMD mRNA相对表达量（P＜0.05）；升高细胞中AMPK、BECN1、Atg5、NLRP3和GSDMD的蛋白表达（P＜0.05），降低p-mTOR的蛋白表达（P＜0.05）。CC可部分逆转THRF的上述效应，而3-MA可部分恢复THRF的抑癌效用，差异均具有统计意义（P＜0.05）。结论 THRF能明显下调细胞的线粒体膜电位，促进细胞的线粒体自噬过度激活，抑制肝癌细胞的增殖与侵袭活性，上调细胞焦亡相关基因的表达，这可能与THRF对APMK/mTOR信号介导的线粒体自噬的调控有关。

Objective To investigate the effcts of Tetrastigma Hemsleyani Radix flavone (THRF) on the proliferation, invasion, and the pyroptosis of hepatocellular carcinoma cells, as well as its regulatory mechanism on AMP-activated protein kinase(AMPK)/ mammalian target of rapamycin (mTOR) signaling-mediated mitochondrial autophagy. Methods SK-HEP-1 cells were treated with low and high concentrations (50, 100 μg·mL^-1) of THRF. Meanwhile, a functional rescue experiment was conducted using the AMPK signaling inhibitor compound C (CC), setting up a THRF (100 μg·mL^-1) + CC (50 μmol·L^-1) group. A functional recovery experiment was also performed using the autophagy inhibitor 3-methyladenine (3-MA), setting up a THRF (100 μg·mL^-1) + CC (50 μmol·L^-1) + 3-MA (5 μmol·L^-1) group. The control group was not treated with any drugs. All groups were treated for 24 hours. Cell proliferation was detected using the 5-ethynyl-2'-deoxyuridine (EdU) kit; cell invasion was detected using Transwell chambers; mitochondrial membrane potential was detected using JC-1; reactive oxygen species (ROS) content was detected using DCFH-DA staining; mitochondrial autophagy was detected using MitoTracker Red-Lysotracker green double staining; the expression of mRNAs related to mitochondrial autophagy and pyroptosis was detected using real-time fluorescence quantitative PCR (qRT-PCR); and the expression levels of AMPK, mTOR, phosphorylated-mTOR (p-mTOR), Beclin1, Atg5, NLRP3, and GSDMD in cells were detected using Western blotting. Results Compared with the control group, THRF significantly reduced the proliferation, invasion, and mitochondrial membrane potential of SK-HEP-1 cells (P < 0.05), significantly increased the ROS content in cells (P < 0.05), significantly increased the expression of autophagosomes; significantly increased the relative expression levels of BECN1, Atg5, NLRP3, and GSDMD mRNAs (P < 0.05); increased the protein expression of AMPK, BECN1, Atg5, NLRP3, and GSDMD in cells (P < 0.05), and decreased the protein expression of p-mTOR (P < 0.05). CC could partially reverse the above effects of THRF, while 3-MA could partially restore the anti-cancer effect of THRF, and the differences were statistically significant (P < 0.05). Conclusion THRF could significantly down-regulate the mitochondrial membrane potential, promote the excessive activation of mitochondrial autophagy, inhibit the proliferation and invasive activity of hepatocellular carcinoma cells, and up-regulate the expression of cell pyrogenesis-related genes, which may be related to the regulation of APMK/mTOR signal-mediated mitochondrial autophagy.

R285.5

浙江省自然科学基金项目（LY17C020003）