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黄玉, 张涛, 丁琳, 孙克伟.基于PINK1/Parkin通路探讨温阳解毒化瘀方含药血浆对肝细胞损伤的影响[J].湖南中医药大学学报英文版,2025,45(5):845-855.[Click to copy
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| 基于PINK1/Parkin通路探讨温阳解毒化瘀方含药血浆对肝细胞损伤的影响 |
| 黄玉,张涛,丁琳,孙克伟 |
| (湖南中医药大学第一中医临床学院, 湖南 长沙 410007;湖南中医药大学第一附属医院, 湖南 长沙 410007) |
| 摘要: |
| 目的 基于PTEN诱导激酶1（PINK1）/E3泛素连接酶（Parkin）通路探讨温阳解毒化瘀方含药血浆对肝细胞损伤的影响。方法 采用D-氨基半乳糖（D-GalN）+脂多糖（LPS）构建肝细胞损伤模型，实验设置对照组、模型组、空白血浆组、中药血浆组、3-MA组、中药+3-MA组。CCK-8法筛选含药血浆最佳干预浓度及时间；Annexin V-APC/PI双染色法检测细胞凋亡情况；JC-1染色检测线粒体膜电位；MitoSOX Red染色检测活性氧（ROS）水平；生物化学法检测超氧化物歧化酶（SOD）、丙二醛（MDA）水平；透射电镜观察肝细胞超微结构；RT-qPCR法检测PINK1、Parkin、微管相关蛋白1轻链3（LC3）、螯合体1（SQSTM1/P62）mRNA水平；Western blot法检测PINK1、Parkin、LC3、P62、B淋巴细胞瘤-2基因（Bcl-2）、Bcl-2相关X蛋白（Bax）、裂解的胱天蛋白酶-3（cleaved-Caspase-3）和胱天蛋白酶-3（Caspase-3）蛋白表达水平。结果 CCK-8结果显示，15%含药血浆干预12 h为实验的最佳干预浓度及时间。与对照组相比，模型组细胞各期凋亡率升高（P＜0.01）；线粒体膜电位下降（P＜0.01）；细胞内ROS、MDA水平上升（P＜0.01）；SOD水平下降（P＜0.01）；线粒体结构明显紊乱、自噬溶酶体数量较少；PINK1、Parkin、LC3 mRNA表达下降（P＜0.01），P62 mRNA表达上升（P＜0.01）； PINK1、Parkin、Bcl-2蛋白及LC3Ⅱ/Ⅰ比值下降（P＜0.01），P62、Bax蛋白表达及cleaved-Caspase-3/Caspase-3比值上升（P＜0.01）。与模型组比较，中药血浆组细胞各期凋亡程度减轻（P＜0.01）；线粒体膜电位升高（P＜0.01）；细胞内ROS、MDA水平下降（P＜0.01）；SOD水平上升（P＜0.01）；与模型组比较，中药血浆组线粒体结构紊乱程度减轻、自噬溶酶体增加； PINK1、Parkin、LC3 mRNA表达升高（P＜0.01），P62 mRNA表达下降（P＜0.01）； PINK1、Parkin、Bcl-2蛋白及LC3Ⅱ/Ⅰ比值升高（P＜0.01，P＜0.05），P62、Bax蛋白表达及cleaved-Caspase-3/Caspase-3比值下降（P＜0.01，P＜0.05）。与中药血浆组比较，中药+3-MA组细胞凋亡率均明显上升（P＜0.01）；线粒体膜电位下降（P＜0.01）；ROS、MDA水平显著升高（P＜0.01），SOD水平显著下降（P＜0.01）；电镜显示线粒体结构紊乱加重，自噬溶酶体数量减少；PINK1、Parkin、LC3 mRNA表达下降（P＜0.01），P62 mRNA表达升高（P＜0.01）；PINK1、Parkin、Bcl-2蛋白及LC3Ⅱ/Ⅰ比值下降（P＜0.01），P62、Bax蛋白表达及cleaved-Caspase-3/Caspase-3比值升高（P＜0.01，P＜0.05）。结论 温阳解毒化瘀方含药血浆可减轻ROS蓄积和氧化应激反应，抑制线粒体内源性途径诱导的肝细胞凋亡在D-GalN/LPS诱导的肝细胞损伤中发挥保护作用，其机制可能与其促进PINK1/Parkin通路介导的线粒体自噬、改善线粒体功能有关。 |
| 关键词: 慢加急性肝衰竭 肝损伤 温阳解毒化瘀方 PINK1/Parkin通路 线粒体自噬 |
| DOI：10.3969/j.issn.1674-070X.2025.05.009 |
| Received:January 07, 2025 |
| 基金项目:国家自然科学基金项目（81973833）；国家中医药管理局高水平中医药重点学科项目；湖南中医药大学双一流学科建设项目（2018〔03〕）。 |
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| Effects of plasma containing Wenyang Jiedu Huayu Formula on hepatocyte injury based on the PINK1/Parkin signaling pathway |
| HUANG Yu, ZHANG Tao, DING Lin, SUN Kewei |
| (The First Clinical School of Chinese Medicine, Hunan University of Chinese Medicine, Changsha, Hunan 410007, China;The First Hospital of Hunan University of Chinese Medicine, Changsha, Hunan 410007, China) |
| Abstract: |
| Objective To investigate the effects of plasma containing Wenyang Jiedu Huayu Formula (WYJDHYF) on hepatocyte injury based on the PTEN-induced kinase 1 (PINK1)/E3 ubiquitin ligase (Parkin) pathway. Methods A hepatocellular injury model was established using D-galactosamine (D-GalN) combined with lipopolysaccharide (LPS). Experimental groups included control group, model group, blank plasma group, WYJDHYF plasma group, 3-MA group, and WYJDHYF+3-MA group. The optimal concentration and intervention duration of plasma was determined via CCK-8 assay. Cell apoptosis was assessed using Annexin V-APC/PI double staining. Mitochondrial membrane potential was measured by JC-1 staining, and reactive oxygen species (ROS) levels were checked using MitoSOX Red staining. Biochemical methods were employed to evaluate superoxide dismutase (SOD) and malondialdehyde (MDA) levels. Hepatocyte ultrastructure was observed by transmission electron microscopy (TEM). RT-qPCR was used to quantify mRNA levels of PINK1, Parkin, microtubule-associated protein 1 light chain 3 (LC3), and sequestosome 1 (SQSTM1/P62). The protein expression levels of PINK1, Parkin, LC3, p62/SQSTM1, B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), cleaved-Caspase-3, and Caspase-3 were analyzed by Western blot. Results The CCK-8 assay indicated that intervention with 15% drug-containing plasma for 12 hours was the optimal concentration and duration for the experiment. Compared with the control group, the model group showed increased apoptosis rate of cells at all stages (P<0.01); decreased mitochondrial membrane potential (P<0.01); elevated levels of intracellular ROS and MDA (P<0.01) and reduced SOD level (P<0.01). TEM revealed severely disordered mitochondrial structures and fewer autolysosomes. The mRNA expression levels of PINK1, Parkin, and LC3 decreased (P<0.01), while P62 mRNA expression increased (P<0.01). The protein expressions of PINK1, Parkin, Bcl-2, and the ratio of LC3Ⅱ/Ⅰ decreased (P<0.01), whereas the P62 and Bax protein expressions and the cleaved-Caspase-3/Caspase-3 ratio were elevated (P<0.01). Compared to the model group, the WYJDHYF plasma group exhibited reduced apoptosis rates at all stages (P<0.01), increased mitochondrial membrane potential (P<0.01), decreased intracellular ROS and MDA levels (P<0.01), and increased SOD levels (P<0.01). Alleviated mitochondrial structural disruption and increased autolysosomes were revealed. Upregulated PINK1, Parkin, and LC3 mRNA (P<0.01) and downregulated P62 mRNA (P<0.01) levels were shown. Protein expressions of PINK1, Parkin, and Bcl-2, as well as the LC3Ⅱ/Ⅰ ratio, were demonstrated elevated (P<0.01 or P<0.05), whereas the expressions of P62 and Bax proteins and the cleaved-Caspase-3/Caspase-3 ratio decreased (P<0.01 or P<0.05). Compared to the WYJDHYF plasma group, the WYJDHYF+3-MA group exhibited markedly increased apoptosis rate (P<0.01), decreased mitochondrial membrane potential (P<0.01), elevated ROS and MDA levels (P<0.01), reduced SOD levels (P<0.01). Electron microscopy showed worsened mitochondrial disorganization and reduced autolysosome numbers. mRNA expressions of PINK1, Parkin, and LC3 decreased (P<0.01), while P62 mRNA expression increased (P<0.01). Protein expressions of PINK1, Parkin, Bcl-2 protein, and LC3-II/I ratio reduced (P<0.01), whereas expressions of P62 and Bax proteins and Cleaved-Caspase-3/Caspase-3 ratio increased (P<0.01 or P<0.05). Conclusion The plasma containing WYJDHYF can alleviate ROS accumulation and oxidative stress, inhibit mitochondrial intrinsic apoptosis, and exert a protective effect against D-GalN/LPS-induced hepatocyte injury. Its protective mechanism may involve enhancing PINK1/Parkin pathway-mediated mitophagy and improving mitochondrial function. |
| Key words: acute-on-chronic liver failure liver injury Wenyang Jiedu Huayu Formula PINK1/Parkin pathway mitophagy |
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