State Key Laboratory on Technologies for Chinese Medicine Pharmaceutical Process Control and Intelligent Manufacture, Jiangning Industrial City, Economic and Technological Development Zone, Lianyungang, Jiangsu 222001, China; Kanion School of Chinese Materia Medica, Nanjing University of Chinese Medicine, 138 Xianlin Avenue Qixia District, Nanjing 210046, PR China; Jiangsu Kanion Pharmaceutical Co Ltd, Jiangning Industrial City, Economic and Technological Development Zone, Lianyungang, Jiangsu 222001, China.
State Key Laboratory on Technologies for Chinese Medicine Pharmaceutical Process Control and Intelligent Manufacture, Jiangning Industrial City, Economic and Technological Development Zone, Lianyungang, Jiangsu 222001, China; Jiangsu Kanion Pharmaceutical Co Ltd, Jiangning Industrial City, Economic and Technological Development Zone, Lianyungang, Jiangsu 222001, China.
Phytomedicine. 2024 Nov;134:155993. doi: 10.1016/j.phymed.2024.155993. Epub 2024 Aug 30.
Chinese dragon's blood, the red resin of Dracaena cochinchinensis (Lour.) S. C. Chen., is widely used to treat cardiovascular and cerebrovascular diseases in China. Longxuetongluo Capsule (LTC) is a total phenolic compound extracted from Chinese dragon's blood, currently used in treating ischemic stroke. Myocardial injury can be aggravated after reperfusion of ischemic myocardium, which is called myocardial ischemia-reperfusion injury (MIRI), and the mechanism of MIRI is complex. However, the exact effect and mechanism of LTC on MIRI are still unclear. We explore the effect of LTC on alleviating MIRI based on mitochondrial dysfunction and oxidative stress.
To explore the cardioprotective mechanism of LTC against MIRI.
A rat MIRI model was constructed through ligation of the left anterior descending coronary artery, and LTC was given continuously for 28 days before surgery. The H9c2 cardiomyocyte injury model was induced by oxygen-glucose deprivation/reperfusion (OGD/R), and LTC was given 24 h before OGD. Myocardial ischemia areas were detected with 2,3,5-triphenyltetrazolium chloride (TTC) staining. Cardiac histopathological changes were detected with hematoxylin-eosin (HE) staining. And biochemical indexes were detected with serum biochemical kit. Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL) staining and flow cytometry were used to detect apoptosis. Fluorescent probes were used to observe reactive oxygen species (ROS), mitochondrial membrane potential (ΔΨm), Caand other indexes. MitoTracker staining and immunofluorescence were used to observe the morphology of mitochondria and translocation of dynamin-related protein 1 (Drp1). Finally, immunohistochemistry and Western blotting were used to examine the expression of proteins related to apoptosis, mitochondrial fission and fusion and oxidative stress.
LTC could ameliorate cardiac pathological changes, decrease myocardial infarct area and the content or level of relevant serum cardiac enzymes, indicating that LTC could alleviate MIRI. Meanwhile, LTC could inhibit cardiomyocyte apoptosis via regulating apoptosis-related protein expression, and it could restore mitochondrial morphology, maintain ΔΨm, inhibit mitochondrial ROS generation and Ca accumulation, increase the expression of mitochondrial fusion protein 2 (Mfn2), decrease the level of phosphorylation dynamin-related protein 1 (p-Drp1), and regulate ATP synthesis, thereby significantly ameliorating mitochondrial dysfunction. Moreover, LTC significantly reduced the expression of NADPH oxidase 2 (NOX2), NADPH oxidase 4 (NOX4) and neutrophil cytosolic factor 2 (NOXA2/p67), and reduced ROS production.
The study demonstrated that LTC could inhibit MIRI induced cardiomyocyte apoptosis by inhibiting ROS generation and mitochondrial dysfunction, and these fundings suggested that LTC can be used to alleviate MIRI, which provides a potential therapeutic approach for future treatment of MIRI.
龙血竭，是龙舌兰科植物柬埔寨龙血树 Dracaena cochinchinensis（Lour.）S. C. Chen. 的红色树脂，在中国被广泛用于治疗心脑血管疾病。龙血通络胶囊（LTC）是从龙血竭中提取的总酚类化合物，目前用于治疗缺血性中风。缺血心肌再灌注后会加重心肌损伤，称为心肌缺血再灌注损伤（MIRI），其机制复杂。然而，LTC 对 MIRI 的确切作用和机制仍不清楚。我们基于线粒体功能障碍和氧化应激来探讨 LTC 对减轻 MIRI 的作用。
探讨 LTC 对 MIRI 的心脏保护作用机制。
通过结扎左前降支冠状动脉构建大鼠 MIRI 模型，术前连续给予 LTC 28 天。通过氧葡萄糖剥夺/再灌注（OGD/R）诱导 H9c2 心肌细胞损伤模型，LTC 在 OGD 前 24 小时给予。用 2,3,5-三苯基四氮唑氯化物（TTC）染色检测心肌缺血区。用苏木精-伊红（HE）染色检测心肌组织病理学变化。采用血清生化试剂盒检测生化指标。末端脱氧核苷酸转移酶介导的 dUTP 缺口末端标记（TUNEL）染色和流式细胞术检测细胞凋亡。荧光探针用于观察活性氧（ROS）、线粒体膜电位（ΔΨm）、Ca 等指标。线粒体追踪染色和免疫荧光用于观察线粒体形态和动力相关蛋白 1（Drp1）的转位。最后，免疫组织化学和 Western blot 用于检测与细胞凋亡、线粒体分裂和融合以及氧化应激相关的蛋白表达。
LTC 可改善心脏病理变化，减少心肌梗死面积和相关血清心肌酶含量或水平，表明 LTC 可减轻 MIRI。同时，LTC 可以通过调节凋亡相关蛋白的表达抑制心肌细胞凋亡，恢复线粒体形态，维持ΔΨm，抑制线粒体 ROS 生成和 Ca 积累，增加线粒体融合蛋白 2（Mfn2）的表达，降低磷酸化动力相关蛋白 1（p-Drp1）水平，并调节 ATP 合成，从而显著改善线粒体功能障碍。此外，LTC 显著降低 NADPH 氧化酶 2（NOX2）、NADPH 氧化酶 4（NOX4）和中性粒细胞胞浆因子 2（NOXA2/p67）的表达，减少 ROS 生成。
该研究表明，LTC 通过抑制 ROS 生成和线粒体功能障碍抑制 MIRI 诱导的心肌细胞凋亡，这些研究结果提示 LTC 可用于减轻 MIRI，为未来治疗 MIRI 提供了一种潜在的治疗方法。
