| 摘要: |
| [目的] 通脉养心方在临床广泛用于治疗冠心病心绞痛。研究旨在探讨通脉养心方调控线粒体融合蛋白2（MFN2）/蛋白激酶R样内质网激酶（PERK）信号通路改善缺氧复氧（H/R）心肌细胞损伤的作用机制。[方法] 预先准备不同浓度的通脉养心方提取物，利用H9c2心肌细胞建立H/R模型，通过噻唑蓝（MTT）法确定通脉养心方乙醇提取物的最佳浓度；采用透射电镜（TEM）、Mito Tracker探针观察线粒体结构；JC-1染色观察线粒体膜电位改变；腺嘌呤核苷三磷酸（ATP）检测线粒体能量；Seahorse XF细胞能量代谢仪检测线粒体呼吸功能；采用荧光探针DCFHDA检测细胞内活性氧（ROS）水平；通过实时荧光定量聚合酶链式反应（Real-time PCR）和蛋白免疫印迹法（Western blot）检测MFN2/PERK通路关键基因和凋亡相关因子的表达。[结果] 结果显示，与模型组相比，通脉养心方给药干预后可显著改善H9c2心肌细胞缺血再灌注损伤（MIRI），降低ROS水平，改善线粒体结构损伤程度，增加细胞内ATP含量，升高线粒体膜电位水平，升高H9c2心肌细胞基础呼吸率、最大呼吸率和ATP合成含氧量；同时上调MFN2、B细胞淋巴瘤-2（Bcl-2）的基因和蛋白表达水平，并下调PERK、真核翻译起始因子2α（eIF2α）、激活转录因子4（ATF4）、C/EBP同源蛋白（CHOP）、Bcl-2相关X蛋白（Bax）、活化的半胱氨酸-天冬氨酸蛋白酶-3（Cleaved Caspase-3）的基因和蛋白表达水平，在低、中、高3种剂量通脉养心方组中，高剂量组的结果尤为显著。[结论] 研究结果表明，通脉养心方可能通过MFN2/PERK信号通路发挥抗心肌细胞线粒体损伤的作用。 |
| 关键词: 通脉养心方 线粒体 缺氧复氧 细胞凋亡 MFN2/PERK信号通路 |
| DOI：10.11656/j.issn.1672-1519.2026.07.11 |
| 分类号:R285.5 |
| 基金项目:国家自然科学基金项目（82104565）；天津中医药大学组分中药国家重点实验室青苗基金（QMJJ202403）。 |
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| Mechanism of Tongmai Yangxin Prescription in ameliorating hypoxia/reoxygenation-induced cardiomyocyte injury via the MFN2/PERK signaling pathway |
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LIU Shu1, LIU Yutong2, YU Lu1, YU Chunquan1, LI Lin1
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1.Tianjin University of Traditional Chinese Medicine, Tianjin 301617, China;2.Beijing University of Chinese Medicine Dongfang Hospital Qinhuangdao Branch(Qinhuangdao Traditional Chinese Medicine Hospital), Qinhuangdao 066000, China
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| Abstract: |
| [Objective] The Tongmai Yangxin Prescription is widely used in clinical practice to treat angina pectoris in coronary heart disease. This study aims to investigate the mechanism by which Tongmai Yangxin Prescription ameliorates hypoxia/reoxygenation（H/R）-induced injury in cardiomyocytes，focusing on the regulation of the mitofusin 2/protein kinase R-like ER kinase（MFN2/PERK） signaling pathway. [Methods] Various concentrations of Tongmai Yangxin Prescription extract were prepared in advance. An H/R model was established using H9c2 cardiomyocytes，and the optimal concentration of Tongmai Yangxin Prescription ethanolic extract was determined by the MTT assay. Mitochondrial morphology was examined using transmission electron microscopy（TEM）and MitoTracker probes. Changes in mitochondrial membrane potential were assessed by JC-1 staining. Mitochondrial energy metabolism was evaluated by measuring adenosine triphosphate（ATP）levels. Mitochondrial respiratory function was analyzed using a Seahorse XF Analyzer. Intracellular reactive oxygen species（ROS）levels were detected with the fluorescent probe DCFH-DA. The mRNA and protein expression of key genes in the MFN2/PERK pathway and apoptosis-related factors were measured by quantitative real-time PCR （qPCR）and Western blot analysis，respectively. [Results] The results demonstrated that，compared with the model group，treatment with Tongmai Yangxin Prescription significantly ameliorated H/R injury in H9c2 cardiomyocytes. Specifically，Tongmai Yangxin Prescription administration reduced intracellular ROS levels，attenuated mitochondrial structural damage，increased ATP content，and elevated the mitochondrial membrane potential. Furthermore，it enhanced mitochondrial respiratory function，as evidenced by increased basal respiration，maximal respiration，and ATP-linked oxygen consumption rate. At the molecular level，Tongmai Yangxin Prescription up-regulated the mRNA and protein expression of Mfn2 and Bcl-2，while down-regulating the expression of key mediators in the PERK pathway（PERK，eIF2α，ATF4，and CHOP）and pro-apoptotic factors（Bax and Cleaved Caspase-3）. These beneficial effects were observed in a dose-dependent manner，with the most pronounced outcomes in the high-dose Tongmai Yangxin Prescription group. [Conclusion] These findings suggest that Tongmai Yangxin Prescription confers protective effects against mitochondrial injury in cardiomyocytes，potentially through the modulation of the MFN2/PERK signaling pathway. |
| Key words: Tongmai Yangxin Prescription mitochondria hypoxia/reoxygenation apoptosis MFN2/PERK signaling pathwa |
