基于网络药理学和实验验证探讨清营生脉汤对心肌缺血再灌注损伤的保护机制*

作者：武文硕，付 殷，张璐艳，谢 蝶，张 洋

单位：黑龙江中医药大学，黑龙江 哈尔滨 150040

引用：引用：武文硕,付殷,张璐艳,谢蝶,张洋.基于网络药理学和实验验证探讨清营生脉汤对心肌缺血再灌注损伤的保护机制[J].中医药导报,2025,31(6):7-14.

DOI：10.13862/j.cn43-1446/r.2025.06.002

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摘要：

目的：基于网络药理学和分子对接方法探究清营生脉汤对心肌缺血再灌注损伤（MIRI）的保护机制，并通过体外实验对部分关键靶点及信号通路进行验证。方法：应用中药系统药理学数据库与分析平台（TCMSP）、BATM-TCM数据库获得清营生脉汤中药物相关活性成分及作用靶点，通过GeneCards数据库、OMIM数据库筛选MIRI的疾病靶点，将药物的作用靶点与疾病的靶点通过Venny平台取交集。基于String数据库构建清营生脉汤治疗MIRI的交集靶点PPI网络，获取药物与疾病的核心作用靶点。利用Metascape数据库构建进行富集研究，并利用微生信平台对基因本体（GO）富集分析、京都基因和基因组百科全书（KEGG）通路富集分析的富集结果实现可视化，借助Cytoscape v3.9.1建立“成分-靶点-通路”互作网络。通过AutoDockTools和PyMol软件对核心成分进行分子对接。体外实验通过H2O2诱导H9c2心肌细胞构建氧化应激模型，通过CCK-8筛选最佳给药浓度，酶联免疫吸附试验（ELISA）检测乳酸脱氢酶（LDH）及心肌肌钙蛋白T（c-TnT）含量，激光共聚焦检测活性氧（ROS）及线粒体含量。Western blotting对预测通路进行验证，实时定量PCR技术检测核心靶点mRNA的水平。结果：清营生脉汤共有109个活性成分，对应1 002个作用靶点，其中122个作用靶点与治疗MIRI有关。GO富集分析共得到2 494个条目；KEGG富集分析共得到195条通路，主要涉及TNF信号通路、HIF-1信号通路、JAK-STAT信号通路等。分子对接结果显示槲皮素、木犀草素、山柰酚与TNF、IL-6、HIF-1α、JAK2、STAT3等关键靶点蛋白的分子对接结合力值均较稳定。细胞实验表明，清营生脉汤含药血清能降低LDH、c-TnT水平（P<0.05），抑制ROS的生成、提高线粒体含量（P<0.05），Western blotting和PCR结果显示含药血清能够下调HIF-1α、上调JAK2蛋白和mRNA的表达（P<0.05）。结论：清营生脉汤可通过多成分、多靶点、多通路治疗心肌缺血再灌注损伤，其含药血清可能通过调控HIF-1α和JAK2信号路通缓解氧化应激对心肌细胞造成的损伤。

关键词：心肌缺血再灌注损伤；清营生脉汤；网络药理学；分子对接；氧化应激；H9c2细胞

Abstract：

Objective: To explore the protective mechanism of Qingying Shengmai decoction on myocardial ischemia reperfusion injury (MIRI) based on network pharmacology and molecular docking methods, and to verify some key targets and signaling pathways through in vitro experiments. Methods: The active ingredients and targets of Qingying Shengmai decoction were obtained by traditional Chinese medicine pharmacology database and analysis platform (TCMSP) database and BATM-TCM database, and the disease targets of MIRI were screened by GeneCards database and OMIM database. The intersection of drug targets and disease targets was obtained through Venny platform. The intersection target PPI network of Qingying Shengmai decoction in the treatment of MIRI was constructed based on String database to obtain the core targets of drugs and diseases. The enrichment results of gene ontology (GO) enrichment analysis and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis were visualized using the bioinformatict platform. The "components-target-pathway" interaction network was established with Cytoscape v3.9.1. Molecular docking of core components was performed by AutoDockTools and PyMol software. In vitro, H9c2 cardiomyocytes were induced with H2O2 to construct oxidative stress model, and the optimal drug concentration was screened by CCK-8. The content of lactate dehydrogenase (LDH) and cardiac troponin T (c-TnT) was detected by enzyme-linked immunosorbent assay (ELISA), and the content of reactive oxygen species (ROS) and mitochondria was detected by laser confocal microscopy. Western blotting was used to verify the predicted pathway, and real-time quantitative PCR was used to detect the mRNA levels of core targets. Results: There were 109 active ingredients in Qingying Shengmai decoction, corresponding to 1 002 targets, of which 122 targets were related to the treatment of MIRI. GO enrichment analysis obtained a total of 2 494 items. A total of 195 pathways were obtained by KEGG enrichment analysis, mainly involving TNF signaling pathway, HIF-1 signaling pathway, JAK-STAT signaling pathway, etc. The results of molecular docking showed that the binding forces of quercetin, luteolin, kaempferol with TNF, IL-6, HIF-1α, JAK2, STAT3 and other key target proteins were stable. Cell experiments showed that the serum containing Qingying Shengmai decoction drug-containing serum could reduce the levels of LDH and c-TnT (P<0.05), inhibit the generation of ROS, and increase the content of mitochondria (P<0.05). The results of Western blotting and PCR showed that the Qingying Shengmai decoction drug-containing serum could down-regulate the expression of HIF-1α and up-regulate the protein and mRNA expression of JAK2 (P<0.05). Conclusion: Qingying Shengmai decoction has a multi-component, multi-target and multi-pathway therapeutic effect on myocardial ischemia reperfusion injury. The Qingying Shengmai decoction drug-containing serum may alleviate oxidative stress-induced myocardial injury by regulating HIF-1α and JAK2 signaling pathway.

Key words：myocardial ischemia reperfusion injury; Qingying sheng mai decoction; network pharmacology; molecular docking; oxidative stress; H9c2 cells

发布时间：2026-01-03

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