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黄舒淳, 张秋雁, 杨漾, 李静, 匡慧芳, 王明韵.基于线粒体钙稳态探讨血府逐瘀汤对冠心病血瘀证模型大鼠的作用机制[J].湖南中医药大学学报英文版,2024,44(6):951-959.[Click to copy
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| 基于线粒体钙稳态探讨血府逐瘀汤对冠心病血瘀证模型大鼠的作用机制 |
| 黄舒淳,张秋雁,杨漾,李静,匡慧芳,王明韵 |
| (湖南中医药大学, 湖南 长沙 410208) |
| 摘要: |
| 目的 基于线粒体钙稳态探讨血府逐瘀汤治疗冠心病血瘀证的作用机制。方法 150只SD大鼠随机均分为正常组、假手术组（只穿线不结扎）、模型组、阿托伐他汀钙组（0.90 mg/kg·d，以下简称他汀组）以及血府逐瘀汤低、中、高剂量组[3.51、7.02、14.04 g/（kg·d），以下简称低、中、高剂量组]，每组6只。除正常组和假手术组外，其他组均通过结扎大鼠冠状动脉左前降支制备冠心病血瘀证模型，造模成功24 h后灌胃给药，连续灌胃7 d，取各组大鼠左室缺血区心肌组织。HE染色观察样本坏死病变情况；线粒体钙离子探针检测线粒体内钙离子的荧光强度；定磷法检测心肌细胞Ca2+-Mg2+-ATP酶活性；免疫组织化学检测心肌组织线粒体钙单向转运蛋白（mitochondrial calcium uniporter, MCU）、心肌肌浆网钙离子ATP酶（sarco/sndoplasmic reticulum Ca2+-ATPase 2a, SERCA2a）、瞬时受体电位通道3（transient receptor potential canonical 3, TRPC3）、钙释放激活钙通道1（calcium release-activated calcium channel protein 1, ORAI1）、半胱氨酸（cysteinyl aspartate specific proteinase, Caspase）-3、Caspase-9的蛋白表达。结果 与正常组相比，模型组大鼠心肌组织可见细胞排列紊乱，横纹模糊，大量心肌细胞坏死，线粒体Ca2+平均荧光强度、MCU、TRPC3、ORAI1、Caspase-3、Caspase-9的AOD值明显升高（P<0.01），Ca2+-Mg2+-ATP酶活性、SERCA2a的AOD值明显下降（P<0.01）；与模型组相比，他汀组以及血府逐瘀汤各剂量组，心肌细胞水肿程度减轻，炎性细胞浸润减少，细胞排列相对紧密，细胞病理损伤程度有所缓解，他汀组以及血府逐瘀汤各剂量组的SERCA2a的AOD值显著上升（P<0.01），线粒体Ca2+平均荧光强度、MCU、TRPC3、ORAI1、Caspase-9的AOD值显著下降（P<0.05，P<0.01），血府逐瘀汤各剂量组的Ca2+-Mg2+-ATP酶活性显著上升（P<0.05，P<0.01）以及Caspase-3的AOD值显著下降（P<0.05，P<0.01）。结论 血府逐瘀汤可以调控冠心病血瘀证模型大鼠心肌线粒体钙离子转运，调节心肌细胞线粒体钙稳态，可能与上调SERCA2a的蛋白表达，下调MCU、TRPC3、ORAI1、Caspase-3、Caspase-9的蛋白表达相关。 |
| 关键词: 血府逐瘀汤 冠心病 血瘀证 线粒体 钙超载 |
| DOI：10.3969/j.issn.1674-070X.2024.06.004 |
| Received:October 31, 2023 |
| 基金项目:湖南省教育厅科学研究重点项目（21A0253）；湖南中医药大学2022年度学科建设“揭榜挂帅”项目（22JBZ005）；湖南中医药大学中药粉体与创新药物省部共建国家重点实验室培育基地开放基金项目（21PTKF1012）；湖南中医药大学2021年度中医学一流学科（2021ZYX35）。 |
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| Mechanism of action of Xuefu Zhuyu Decoction on model rats of coronary heart disease with blood stasis pattern based on mitochondrial calcium homeostasis |
| HUANG Shuchun, ZHANG Qiuyan, YANG Yang, LI Jing, KUANG Huifang, WANG Mingyun |
| (Hunan University of Chinese Medicine, Changsha, Hunan 410208, China) |
| Abstract: |
| Objective To explore the mechanism of action of Xuefu Zhuyu Decoction (XFZYD) in treating coronary heart disease (CHD) with blood stasis pattern based on mitochondrial calcium homeostasis. Methods A total of 150 SD rats were randomized into normal group, sham-operated group (only threading without ligation), model group, atorvastatin calcium group (0.90 mg·kg-1·d-1, hereinafter referred to as statin group), and low-, medium-, and high-dose XFZYD groups (3.51, 7.02, 14.04 g·kg-1·d-1, hereinafter referred to as low-, medium-, and high-dose groups), with six rats in each group. Except for the normal and sham-operated groups, the other groups were prepared for CHD with blood stasis pattern models by ligating the left anterior descending coronary artery of the rats. After 24 hours of successful modeling, the rats were given continuous intragastric administration for 7 d. The myocardial tissue of left ventricular ischemic area in rats of each group was taken. HE staining was used to observe the necrosis of the samples. The fluorescence intensity of calcium ions in mitochondria was determined by mitochondrial calcium ion probe. The Ca2+-Mg2+-ATPase activity of myocardial cells was examined by phosphorus determination method. Immunohistochemistry was used to check the protein expressions of mitochondrial calcium uniporter (MCU), sarco/endoplasmic reticulum Ca2+-ATPase 2a (SERCA2a), transient receptor potential canonical 3 (TRPC3), calcium release-activated calcium channel protein 1 (ORAI1), cysteinyl aspartate specific proteinase-3 (Caspase-3), and cysteinyl aspartate specific proteinase-9 (Caspase-9). Results Compared with the normal group, the model group showed disordered cell arrangement, blurred transverse stripes, necrosis of a large number of myocardial cells, a significant increase in mitochondrial Ca2+ average fluorescence intensity and AOD values of MCU, TRPC3, ORAI1, Caspase-3, and Caspase-9 (P<0.01), and an obvious decrease in Ca2+-Mg2+-ATPase activity and AOD value of SERCA2a in the myocardial tissue of rats (P<0.01). Compared with the model group, the statin group and low-, medium-, and high-dose groups showed reduced myocardial cell edema, inflammatory cell infiltration, and cell pathological damage as well as relatively tight cell arrangement, meanwhile, the AOD value of SERCA2a in those groups significantly increased (P<0.01), and the average fluorescence intensity of mitochondrial Ca2+, AOD values of MCU, TRPC3, ORAI1, and Caspase-9 decreased significantly (P<0.05, P<0.01). In addition, the Ca2+-Mg2+-ATPase activity in low-, medium-, and high-dose groups increased significantly (P<0.05, P<0.01), while the AOD value of Caspase-3 decreased significantly (P<0.05, P<0.01). Conclusion XFZYD can regulate myocardial mitochondrial calcium ion transport and mitochondrial calcium homeostasis in rats of CHD with blood stasis pattern, which may be related to up-regulating the protein expression of SERCA2a and down-regulating the protein expressions of MCU, Caspase-3, and Caspase-9. |
| Key words: Xuefu Zhuyu Decoction coronary heart disease blood stasis pattern mitochondria calcium overload |
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