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曾千, 吴涛, 罗燕, 罗吉.肝喜片通过干扰线粒体动力学诱导肝癌细胞HepG2凋亡的机制研究[J].湖南中医药大学学报,2025,45(9):1636-1643[点击复制] |
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| 肝喜片通过干扰线粒体动力学诱导肝癌细胞HepG2凋亡的机制研究 |
| 曾千,吴涛,罗燕,罗吉 |
| (湖南中医药大学, 湖南 长沙 410208;湖南省中西医结合医院渊湖南省中医药研究院附属医院冤, 湖南 长沙 410006;湖南省中医药研究院, 湖南 长沙 410013) |
| 摘要: |
| 目的 探究肝喜片通过调控线粒体融合蛋白2(MFN2)、动力相关蛋白1(DRP1)介导的线粒体动态平衡诱导肝癌细胞HepG2凋亡的分子机制。方法 以HepG2细胞为模型，设置空白对照组和肝喜片低、中、高（10%、15%、20%含药血清）剂量组。采用实时无标记细胞分析动态监测细胞增殖；流式细胞术检测线粒体膜电位、活性氧（ROS）水平及细胞凋亡率；Western blot检测MFN2、DRP1、聚腺苷二磷酸核糖聚合酶1(PARP1)、裂解型PARP1(cleaved PARP1)、Bcl-2相关X蛋白（BAX）、细胞色素c(Cyt c)蛋白表达水平。结果 与空白对照组比较，肝喜片中、高剂量组细胞指数（CI）值降低（P<0.01）；与肝喜片中剂量组比较，肝喜片高剂量组CI值降低（P<0.05）。与空白对照组比较，肝喜片各剂量组线粒体膜电位降低（P<0.05,P<0.01），细胞内ROS水平升高（P<0.05,P<0.01）；与肝喜片低剂量组比较，肝喜片中、高剂量组线粒体膜电位降低（P<0.05），肝喜片高剂量组细胞内ROS水平升高（P<0.01）。与空白对照组比较，肝喜片各剂量组MFN2蛋白表达水平降低（P<0.05）、DRP1蛋白表达水平升高（P<0.05,P<0.01）；与肝喜片中剂量组比较，肝喜片高剂量组MFN2蛋白表达水平降低、DRP1蛋白表达水平升高（P<0.05）。与空白对照组及肝喜片低剂量组比较，肝喜片中、高剂量组凋亡率升高（P<0.01）；与肝喜片中剂量组比较，肝喜片高剂量组凋亡率升高（P<0.05）。与空白对照组比较，肝喜片各剂量组cleaved PARP1/PARP1比值及BAX蛋白表达水平均升高（P<0.05,P<0.01），肝喜片中、高剂量组Cyt c蛋白表达水平升高（P<0.05,P<0.01）。与肝喜片低剂量组比较，肝喜片中剂量组cleaved PARP1/PARP1比值及Cyt c蛋白表达水平升高（P<0.05），肝喜片高剂量组cleaved PARP1/PARP1比值及BAX、Cyt c蛋白表达水平升高（P<0.05,P<0.01）。结论 肝喜片通过抑制MFN2表达、激活DRP1介导的线粒体分裂，破坏线粒体动态平衡，诱导线粒体碎片化、氧化应激及膜电位降低，进而促进Cyt c释放、BAX升高和PARP1裂解，最终激活线粒体依赖性凋亡通路。 |
| 关键词: 肝癌 线粒体动力学 HepG2 线粒体融合蛋白2 动力相关蛋白1 凋亡 |
| DOI：10.3969/j.issn.1674-070X.2025.09.007 |
| 投稿时间：2025-07-04 |
| 基金项目:湖南省自然科学基金面上项目(2025JJ50499);湖南省自然科学基金医卫行业联合基金(2025JJ180792); 湖南省中医药科研计划重点项目(A2024018); 湖南中医药大学研究生创新课题(2023CX0116)。 |
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| Mechanism of Ganxi Tablet inducing apoptosis in hepatocellular carcinoma HepG2 cells by interfering with mitochondrial dynamics |
| ZENG Qian, WU Tao, LUO Yan, LUO Ji |
| (Hunan University of Chinese Medicine, Changsha, Hunan 410208, China;Hunan Provincial Hospital of Integrated Traditional Chinese and Western Medicine (The Affiliated Hospital of Hunan Academy of Traditional Chinese Medicine), Changsha, Hunan 410006, China;Hunan Academy of Chinese Medicine, Changsha, Hunan 410013, China) |
| Abstract: |
| Objective To investigate the molecular mechanism by which Ganxi Tablet induces apoptosis in hepatocellular carcinoma HepG2 cells through regulating the mitochondrial dynamics mediated by mitofusin 2（MFN2） and dynamin-related protein1（DRP1）. Methods HepG2 cells were used as the model, with a blank control group and low-, medium-, and high-dose（10%,15%, and 20% drug-containing serum, respectively） Ganxi Tablet groups established. Real-time label-free cell analysis was employed to dynamically monitor cell proliferation. Flow cytometry was used to measure mitochondrial membrane potential, reactive oxygen species（ROS） levels, and the apoptosis rate. Western blot analysis was performed to assess the protein expression levels of MFN2, DRP1, poly（ADP-ribose） polymerase 1（PARP1）, cleaved PARP1, Bcl-2-associated X protein（BAX）, and cytochrome c（Cyt c）. Results Compared with the blank control group, the cell index（CI） values of the medium-and high-dose Ganxi Tablet groups decreased（P＜0.01）; compared with the medium-dose Ganxi Tablet group, the CI value of the high-dose Ganxi Tablet group decreased（P＜0.05）. Compared with the blank control group, the proportion of cells with reduced mitochondrial membrane potential and intracellular ROS levels increased in all Ganxi Tablet groups（P＜0.05, P＜0.01）; compared with the low-dose Ganxi Tablet group,the proportion of cells with reduced mitochondrial membrane potential increased in the medium-and high-dose Ganxi Tablet groups（P＜0.05）, and intracellular ROS levels increased in the high-dose Ganxi Tablet group（P＜0.01）. Compared with the blank control group, MFN2 protein expression level decreased in all Ganxi Tablet groups（P＜0.05）, while DRP1 protein expression level increased（P＜0.05, P＜0.01）; compared with the medium-dose Ganxi Tablet group, MFN2 protein expression level decreased while DRP1 protein expression level increased in the high-dose Ganxi Tablet group（P＜0.05）. Compared with the blank control and lowdose Ganxi Tablet groups, the apoptosis rate increased in the medium-and high-dose Ganxi Tablet groups（P＜0.01）; compared with the medium-dose Ganxi Tablet group, the apoptosis rate increased in the high-dose Ganxi Tablet group（P＜0.05）. Compared with the blank control group, the cleaved PARP1/PARP1 ratio and BAX protein expression level increased in all Ganxi Tablet groups（P＜0.05, P＜0.01）, while Cyt c protein expression level increased in the medium-and high-dose Ganxi Tablet groups（P＜0.05, P＜0.01）.Compared with the low-dose Ganxi Tablet group, the cleaved PARP1/PARP1 ratio and Cyt c protein expression level increased in the medium-dose Ganxi Tablet group（P＜0.05）, and the cleaved PARP1/PARP1 ratio as well as BAX and Cyt c protein expression levels increased in the high-dose Ganxi Tablet group（P＜0.05, P＜0.01）. Conclusion Ganxi Tablet suppresses MFN2 expression and activates DRP1-mediated mitochondrial fission, thereby disrupting mitochondrial dynamics. This disruption induces mitochondrial fragmentation, oxidative stress, and membrane potential collapse, subsequently promoting Cyt c release, BAX upregulation, and PARP1cleavage, ultimately activating the mitochondrial-dependent apoptotic pathway. |
| Key words: liver cancer mitochondrial dynamics HepG2 mitofusin 2 dynamin-related protein 1 apoptosis |
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