摘要
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(m TOR)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、100μg·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(100μg·mL^(-1))+CC(50μmol·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(100μg·mL^(-1))+CC(50μmol·L^(-1))+3-MA(5μmol·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;实时荧光定量PCR(qRT-PCR)检测线粒体自噬、细胞焦亡相关的m RNA的表达;Western blotting检测细胞中AMPK、m TOR、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 m RNA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(50,100μg·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.
作者
张云飞
叶春林
陈信佳
ZHANG Yunfei;YE Chunlin;CHEN Xinjia(Zhejiang Key Laboratory of Agricultural Chemistry and Bioprocessing Technology,College of Biological and Chemical Engineering,Zhejiang University of Science and Technology,Hangzhou 310023,China;Traditional Chinese Medicine Department,Qianhai Life Insurance Nanning Hospital,Nanning 530000,China)
出处
《药物评价研究》
北大核心
2025年第8期2135-2144,共10页
Drug Evaluation Research
基金
浙江省自然科学基金项目(LY17C020003)。
关键词
三叶青黄酮
肝癌
增殖
侵袭
细胞焦亡
腺苷酸活化蛋白激酶/哺乳动物雷帕霉素靶蛋白信号
线粒体自噬
Tetrastigma Hemsleyani Radix flavone
hepatocellular carcinoma cell
proliferation
invasion
pyroptosis
AMP-activated protein kinase/mammalian target of rapamycin signaling pathway
mitochondrial autophagy
