基于网络药理学与实验验证探讨络衡方防治冠状动脉微血管疾病的作用机制研究

Study on the mechanism of Luoheng Prescription in the prevention and treatment of coronary microvascular disease based on network pharmacology and experimental validation

关键词:  络衡方  冠状动脉微血管疾病  网络药理学  分子对接  炎症

Key words:  Luoheng Prescription  coronary microvascular disease  network pharmacology  molecular docking  inflammation

摘要:

[目的] 探讨络衡方防治冠状动脉微血管疾病（CMVD）的潜在机制。[方法] 1）通过网络药理学对络衡方的主要成分和治疗CMVD的靶点进行分析，运用分子对接预测核心成分与关键靶点的结合活性。2）将32只SD大鼠随机分为假手术组、模型组、络衡方组和尼可地尔组，每组8只。连续灌胃2周，再通过左心室注射月桂酸钠建立冠状动脉微血管损伤模型，采用超高分辨小动物超声仪检测冠状动脉血流储备（CFR）及心功能，苏木精-伊红（HE）染色观察心肌病理组织形态，透射电镜观察微血管内皮细胞和线粒体情况，酶联免疫吸附测定（ELISA）检测炎症因子白细胞介素-6（IL-6）和肿瘤坏死因子（TNF）-α水平。[结果] 1）得到络衡方治疗CMVD的44个潜在有效成分和253个潜在作用靶点；前6位关键成分为异鼠李素、槲皮素、山柰酚、华良姜素、亚麻酸，甘草素；前6位关键靶点为IL-6、TNF、信号转导和转录激活因子3（STAT3）、Jun原癌基因（JUN）、丝氨酸/苏氨酸激酶1（AKT1）、肿瘤蛋白p53（TP53）；分子对接结果显示，关键活性成分与目标靶点结合力较稳定。2）动物实验结果显示，与假手术组相比，模型组CFR明显降低（P<0.01），炎症因子IL-6和TNF-α水平明显升高（P<0.01）；心外膜大范围淋巴细胞、粒细胞浸润，局灶性间质淤血，心肌细胞坏死；微血管内皮细胞及线粒体肿胀，线粒体嵴溶解。与模型组相比，络衡方组CFR明显升高（P<0.01），炎症因子IL-6和TNF-α水平明显降低（P<0.01）；淋巴细胞、粒细胞浸润，局灶性间质淤血以及心肌细胞坏死得到改善；微血管内皮及线粒体细胞肿胀，线粒体嵴溶解均得到改善。[结论] 络衡方可以改善月桂酸钠诱导的冠状动脉微血管损伤，提高冠状动脉血流储备，其机制可能是通过下调IL-6、TNF-α表达水平，抑制炎性反应，减轻内皮细胞和线粒体肿胀，发挥心脏保护作用。

Abstract:

[Objective] To explore the potential mechanism of Luoheng Prescription for the treatment of coronary artery microvascular disease（CMVD）. [Methods] 1） We analyzed the main components and targets for the treatment of CMVD of Luoheng Prescription through network pharmacology and used molecular docking to predict the binding activities of the core components and the key targets. 2） Thirty-two SD rats were randomly divided into the sham-operation group，the model group，the Luoheng Prescription group，and the Nicorandil group，with eight rats in each group. These rats were gavaged continuously for 2 weeks. Then，the coronary microvascular injury model was established by left ventricular injection of sodium laurate. The coronary flow reserve（CFR） was detected by ultra-high-resolution small animal ultrasonography，and myocardial pathological histomorphology was observed by hematoxylin-eosin staining. Transmission electron microscopy was used to observe microvascular endothelial cells and mitochondria. Enzyme-Linked Immunosorbent Assay（ELISA） was used to detect the levels of the inflammatory factors interleukin-6（IL-6） and tumor necrosis factor-α（TNF-α）. [Results] 1）We obtained 44 active ingredients and 253 targets for the treatment of coronary artery microvascular disease by Luoheng Prescription；the top six core ingredients were isorhamnetin，Quercetin，Kaempferol，Jaranol，Linolenic acid，and Liquiritigenin；and the top six key targets were IL-6，TNF，signal transducer and activator of transcription 3（STAT3），Jun Proto-Oncogene（JUN），Serine/Threonine Kinase Proteins 1（AKT1），and tumor protein p53（TP53）；the molecular docking results show that the binding of the key active ingredients to the target targets is more stable. 2）The results of animal experiments showed that compared with the sham-operated group，the model group had significantly lower CFR（P<0.01） and higher levels of the inflammatory factors IL-6 and TNF-α（P<0.01）；extensive epicardial infiltration of lymphocytes and granulocytes，focal mesenchymal stasis，and necrosis of cardiomyocytes；and swelling of microvascular endothelium，mitochondria，and mitochondrial cristae lysis. Compared with the model group，CFR was significantly increased（P<0.01），and the levels of inflammatory factors IL-6 and TNF-α were significantly decreased（P<0.01） in the Luoheng Prescription group；lymphocyte and granulocyte infiltration，focal mesenchymal stasis，and necrosis of cardiomyocytes were ameliorated；and microvascular endothelial and mitochondrial cell swelling，and mitochondrial cristae lysis were all improved. [Conclusion] Luoheng Prescription can improve the coronary microvascular injury induced by sodium lauryl silicate and increase CFR. The mechanism may involve inhibiting the inflammatory response and reducing the swelling of endothelial cells and mitochondria by down-regulating the expression levels of IL-6 and TNF-α，thereby exerting cardioprotective effects.