海兰地嗪通过AMPK/mTOR信号通路诱导肝细胞癌自噬性死亡

doi:10.16139/j.1007-9610.2025.02.08

摘要/Abstract

摘要：

目的：探究海兰地嗪（HER）对肝细胞癌(HCC)的自噬激活效应以及对细胞自噬性死亡的诱导作用。方法：首先通过CCK-8实验分析HER对HCC细胞Hep 3B和Huh-7的增殖抑制作用。不同浓度梯度和时间梯度HER处理HCC细胞，蛋白质印迹法分析自噬标志蛋白LC3从Ⅰ型向Ⅱ型转化以及SQSTM1/p62的变化。红色荧光蛋白(RFP)-LC3慢病毒感染HCC细胞，构建稳定表达RFP-LC3的细胞株，经HER处理之后，共聚焦显微镜观察细胞中RFP-LC3荧光斑点的聚集情况。透射电镜观察HER处理后细胞内的自噬体。自噬抑制剂巴佛洛霉素A1（BafA1）或羟氯喹（HCQ）结合HER处理细胞，流式细胞术分析细胞死亡比例；蛋白质印迹法分析单磷酸腺苷激活的蛋白激酶(AMPK)和哺乳动物雷帕霉素靶蛋白(mTOR)磷酸化水平。结果：HER抑制HCC细胞Hep 3B和Huh-7增殖，促进LC3从Ⅰ型向Ⅱ型转化，自噬激活呈现出药物浓度依赖性和时间依赖性。共聚焦显微镜和透射电镜显示，经HER处理后，细胞内的自噬囊泡显著增多。HER对细胞死亡的诱导作用被自噬抑制剂BafA1或HCQ抑制；HER增加AMPK磷酸化水平且降低mTOR磷酸化水平。结论：HER通过AMPK/mTOR信号通路诱导HCC细胞自噬且导致细胞自噬性死亡。

关键词: 肝癌, 海兰地嗪, 单磷酸腺苷激活的蛋白激酶/哺乳动物雷帕霉素靶蛋白, 自噬性死亡

Abstract:

Objective To investigate the autophagy activation effect of hernandezine (HER) on hepatocellular carcinoma (HCC) cells and its role in inducing autophagic cell death. Methods The inhibitory effects of HER on the proliferation of HCC cell lines Hep 3B and Huh-7 were assessed using CCK-8 assay. HCC cells were treated with HER at different concentrations and time points gradient. Western blot analysis was performed to evaluate the conversion of autophagy marker LC3 from type Ⅰ to type Ⅱ and the levels of SQSTM1/p62. Stable red fluorescent protein(RFP)-LC3-expressing cell lines were established through lentiviral infection, and the accumulation of RFP-LC3 puncta following HER treatment was observed under confocal microscope. Autophagosomes in HER-treated cells were visualized using transmission electron microscopy. The effects of autophagy inhibitors, bafilomycin A1 (BafA1) or hydroxychloroquine (HCQ), in combination with HER were investigated by analyzing cell death rates via flow cytometry. The phosphorylation levels of adenosine 5′-monophosphate(AMP)-activated protein kinase(AMPK) and mammalian target of rapamycin(mTOR) were assessed by Western blot.Results HER inhibited the proliferation of HCC cells Hep 3B and Huh-7, promoted the conversion of LC3-Ⅰ to LC3-Ⅱ, and activated autophagy in a dose- and time-dependent manner. Confocal microscopy and transmission electron microscopy revealed a significant increase in autophagic vesicles in HER-treated cells. HER-induced cell death was attenuated by the autophagy inhibitors BafA1 or HCQ. HER treatment increased AMPK phosphorylation levels while decreasing mTOR phosphorylation levels.Conclusions HER induces autophagy and promotes autophagic cell death in HCC cells via the AMPK/mTOR signaling pathway.

Key words: Hepatocellular carcinoma(HCC), Hernandezine(HER), Adenosine 5'-monophosphate(AMP)-activated protein kinase(AMPK)/mammalian target of rapamycin(mTOR), Autophagic cell death

中图分类号:

R735.7

王军, 胡刚峰, 高伟陈, 王路兵. 海兰地嗪通过AMPK/mTOR信号通路诱导肝细胞癌自噬性死亡[J]. 外科理论与实践, 2025, 30(2): 138-145.

WANG Jun, HU Gangfeng, GAO Weichen, WANG Lubing. Hernandezine induces autophagic cell death in hepatocellular carcinoma cells via the AMPK/mTOR signaling pathway[J]. Journal of Surgery Concepts & Practice, 2025, 30(2): 138-145.

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