延胡索乙素通过线粒体活性氧调控能量代谢表型转换促进人肝细胞癌凋亡  

Levo-tetrahydropalmatine promotes apoptosis of human hepatocellular carcinoma through switching energy metabolism phenotype via upregulation of mitochondrial reactive oxygen species

作　　者：尹逊哲 刘作家[1] 汪劲[2] YIN Xun-zhe;LIU Zuo-jia;WANG Jin(State Key Laboratory of Electroanalytical Chemistry,Changchun Institute of Applied Chemistry,Chinese Academy of Sciences,Changchun 130022,China;Department of Chemistry and Physics,State University of New York at Stony Brook,Stony Brook,New York 11790-3400,USA)

机构地区：[1]中国科学院长春应用化学研究所电分析化学国家重点实验室,吉林长春130022 [2]纽约州立大学石溪分校化学与物理系,纽约11790-3400

出　　处：《中国中药杂志》2022年第23期6494-6504,共11页China Journal of Chinese Materia Medica

基　　金：吉林省科技发展计划项目(20200301009RQ)。

摘　　要：线粒体是维持细胞稳态的重要细胞器,在决定细胞命运中起着至关重要的作用。该文旨在探讨延胡索乙素(levo-tetrahydropalmatine, THP)上调线粒体活性氧(mitochondrial reactive oxygen species, mtROS)对人肝细胞癌自噬流和能量代谢表型的影响。选用SMMC-7721和BEL-7402细胞株作为模型,经THP(100μmol·L^(-1))和/或N-乙酰-L-半胱氨酸(N-acetyl-L-cysteine, NAC)(10μmol·L^(-1))处理SMMC-7721和BEL-7402细胞24 h后,利用MitoSOX荧光探针与流式细胞术检测mtROS变化水平,并同时通过荧光显微镜对mtROS进行可视化观察;利用CYTO-ID自噬荧光探针检测自噬流的变化情况,采用Western blot检测2种肝癌细胞中微管相关蛋白轻链3(microtubule-associated protein light chain 3,LC3)与磷酸化腺苷酸活化蛋白激酶(AMP-activated protein kinase, AMPK)蛋白表达;Seahorse XFp检测细胞线粒体能量代谢变化趋势,通过实时无标记RTCA检测对肝癌细胞增殖的影响;Annexin V-FITC和PI/RNase染色流式细胞术检测细胞凋亡率,MitoTracker Red CMXRos荧光探针可视化观察线粒体膜电位变化。结果表明,与对照组相比,THP可促进SMMC-7721和BEL-7402肝癌细胞内mtROS产生,发现联合NAC后削弱了由THP单独引发的自噬或自噬流的增加。添加自噬抑制剂氯喹(chloroquine, CQ)后发现,THP可进一步促进mtROS产生并增加凋亡率。进一步实验表明,THP可以抑制线粒体呼吸水平,导致线粒体基础呼吸、三磷酸腺苷(adenosine triphosphate, ATP)产量及最大呼吸能力下降。同时,THP显著降低了肝癌细胞的增殖指数和线粒体膜电位,进而诱导细胞发生凋亡达到抑制细胞增殖的目的,而NAC则缓解这种细胞凋亡的产生。结果表明,THP上调mtROS水平可显著促进人肝细胞癌细胞自噬,引发的自噬为保护性自噬,并通过重编程能量代谢表型进而抑制线粒体呼吸,激活线粒体凋亡途径而引发细胞凋亡。Mitochondrion is an important organelle that maintains cellular homeostasis and plays a crucial role in determining cell fate. The present study investigated the effect of levo-tetrahydropalmatine(THP) on autophagic flux and energy metabolism phenotype of human hepatocellular carcinoma(HCC) SMMC-7721 and BEL-7402 cells. SMMC-7721 and BEL-7402 cells were treated with THP(100 μmol·L^(-1)) with or without N-acetyl-L-cysteine(NAC, 10 μmol·L^(-1)) for 24 h. The mitochondrial reactive oxygen species(mtROS) was detected by flow cytometry(FCM) with MitoSOX probe and fluorescence microscopy, respectively. Thereafter, autophagic flux was detected by FCM with CYTO-ID probe, and the protein levels of microtubule-associated protein 1 A/1 B-light chain 3-Ⅰ(LC3Ⅰ), LC3Ⅱ, and phosphorylated AMP-activated protein kinase(p-AMPK)/AMPK were measured by Western blot. Mitochondrial respiration was examined by Seahorse XFp assay and cell proliferation by a system. Annexin V-FITC and PI/RNase staining was employed to detect apoptosis of SMMC-7721 and BEL-7402 cells treated with THP and/or NAC. Subsequently, membrane potential was measured with MitoTracker Red CMXRos. Compared with the control group, THP promoted mtROS production and THP combined with NAC attenuated the autophagic flux increase induced by THP alone in SMMC-7721 and BEL-7402 cells. When cells were co-treated with THP and chloroquine(CQ, an autophagy inhibitor), THP further increased mtROS and apoptosis. In addition, THP significantly reduced mitochondrial respiration in terms of mitochondrial basal respiration, ATP production, and maximal respiration. Meanwhile, THP significantly reduced the proliferation index and mitochondrial membrane potential of HCC cells accompanied by the increased apoptosis. This study demonstrates that the up-regulation of mtROS by THP significantly promotes HCC cell autophagy(protective autophagy) and impairs mitochondrial respiration through reprogramming energy metabolism, ultimately inducing the mitochondria-mediated apoptosis of SMM

关 键 词：延胡索乙素 肝细胞癌 自噬 能量代谢 线粒体呼吸 

分 类 号：R285[医药卫生—中药学]