目的 探討三葉青Tetrastigma Hemsleyani Radix黃酮（THRF）對肝癌細胞增殖、侵襲和細胞焦亡的影響，以及其對腺苷酸活化蛋白激酶（AMPK）/哺乳動物雷帕霉素靶蛋白（mTOR）信號介導的線粒體自噬的調(diào)控機制。方法 以低、高質(zhì)量濃度（50、100 μg·mL^-1）的THRF處理SK-HEP-1細胞；同時以AMPK信號抑制劑化合物C（CC）進行功能挽救實驗，即設(shè)置THRF（100 μg·mL^-1） ＋CC（50 μmol·L^-1）組；以自噬抑制劑3-甲基腺嘌呤（3-MA）進行功能回復(fù)實驗，即設(shè)置THRF（100 μg·mL^-1）＋ CC（50 μmol·L^-1） ＋3-MA（5 μmol·L^-1）組；對照組不加藥，THRF、CC、3-MA同時加藥，加藥后培養(yǎng)24 h。5-乙炔基-2'-脫氧尿苷（EdU）試劑盒檢測細胞的增殖； Transwell小室檢測細胞的侵襲； JC-1檢測線粒體的膜電位； DCFH-DA染色實驗檢測活性氧（ROS）的含量； MitoTracker Red-Lysotracker green雙染色檢測線粒體自噬；¹²⁵IPCR（qRT-PCR）檢測線粒體自噬、細胞焦亡相關(guān)的mRNA的表達； Western blotting檢測細胞中AMPK、mTOR、磷酸化-mTOR（p-mTOR）、酵母自噬相關(guān)基因6同源物（Beclin1）、自噬相關(guān)基因5（Atg5）、核苷酸結(jié)合寡聚化結(jié)構(gòu)域樣受體家族含熱蛋白結(jié)構(gòu)域蛋白3（NLRP3）和消皮素D（GSDMD）的表達水平。結(jié)果 與對照組比較，THRF明顯降低SK-HEP-1細胞的增殖、侵襲以及線粒體膜電位（P＜0.05），明顯升高細胞中ROS含量（P＜0.05），明顯升高自噬體的表達；明顯升高BECN1、Atg5、NLRP3和GSDMD mRNA相對表達量（P＜0.05）；升高細胞中AMPK、BECN1、Atg5、NLRP3和GSDMD的蛋白表達（P＜0.05），降低p-mTOR的蛋白表達（P＜0.05）。CC可部分逆轉(zhuǎn)THRF的上述效應(yīng)，而3-MA可部分恢復(fù)THRF的抑癌效用，差異均具有統(tǒng)計意義（P＜0.05）。結(jié)論 THRF能明顯下調(diào)細胞的線粒體膜電位，促進細胞的線粒體自噬過度激活，抑制肝癌細胞的增殖與侵襲活性，上調(diào)細胞焦亡相關(guān)基因的表達，這可能與THRF對APMK/mTOR信號介導的線粒體自噬的調(diào)控有關(guān)。

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

浙江省自然科學基金項目（LY17C020003）