保元活络方改善心肌缺血再灌注损伤的作用机制预测及验证

Prediction and Verification of the Effect and Mechanism of Baoyuan Huoluo Prescription on Myocardial Ischemia-reperfusion Injury

关键词:  保元活络方  心肌缺血再灌注损伤  缺氧复氧  网络药理学  细胞凋亡

Key words:  Baoyuan Huoluo prescription  myocardial ischemia-reperfusion injury  hypoxia reoxygenation  network pharmacology  cell apoptosis

摘要:

目的 探讨保元活络方改善心肌缺血再灌注损伤（myocardial ischemia-reperfusion injury，MIRI）的可能作用和机制。方法 从TCMSP数据库获取保元活络方（丹参、三七、山楂、黄芪和西洋参）的成分和作用靶点，从GeenCard、OMIM数据库获取MIRI靶点，交集获得保元活络方改善MIRI的可能靶点；使用Cytoscape软件构建保元活络方作用MIRI的"成分-靶点"网络，拓扑分析得出关键化合物；使用STRING数据库构建蛋白质-蛋白质相互作用（protein protein interaction，PPI）网络，并导入Cytoscape软件进行优化，聚类分析得出关键靶标；使用ClueGo插件对核心靶标进行GO功能分析和KEGG通路富集分析。缺氧复氧（hypoxia and reoxygenation，H/R）处理大鼠心肌细胞（H9C2）建立MIRI模型，初步验证保元活络方含药血清调控NF-κB通路从线粒体途径抑制H/R诱导的心肌细胞凋亡的作用机制。结果 共筛选得到槲皮素、山柰酚、异鼠李素等11个关键化合物，作用靶标51个；PPI网络聚类分析得到2个子网络，涉及CASP3、NOS3、IL-6、ICAM1等16个靶标；GO分析得到体外凋亡信号通路、对活性氧的反应、血管内皮细胞迁移等239个生物学功能；KEGG富集得到TNF信号通路、NF-κB信号通路、VEGF信号通路、PPAR信号通路等63条通路。细胞试验显示，H/R处理后，各组H9C2心肌细胞的细胞活力降低，细胞凋亡增加，caspase-3、caspase-9活性增强，NF-κB p65、caspase-3和caspase-9蛋白表达水平增加，IκBα、Bcl-2蛋白表达水平降低；而保元活络方含药血清干预可逆转H/R诱导的上述损伤。结论 保元活络方可以通过多成分-靶点-通路的途径，发挥抗炎、抗氧化应激、抗细胞凋亡和改善冠脉血管内皮功能等作用从而改善MIRI，其中调控NF-κB通路，从线粒体途径抑制H/R诱导的心肌细胞凋亡可能是其关键机制。

Abstract:

OBJECTIVE To explore the possible effect and mechanism of Baoyuan Huoluo prescription in improving myocardial ischemia-reperfusion injury(MIRI). METHODS The components and targets of Baoyuan Huoluo prescription (Salvia Miltiorrhizae Radix et Rhizoma, Notoginseng Radix et Rhizoma, Crataegi Fructus, Astragali Radix and Panacis Quinquefolii Radix) were obtained in the TCMSP database. Targets of MIRI were obtained from GeneCards and OMIM databases. The targets of Baoyuan Huoluo prescription improving MIRI were intersected. The "composition-target" network of Baoyuan Huoluo prescription in the treatment of MIRI was constructed by using Cytoscape software, and the key compounds were obtained by topological analysis. The protein protein interaction(PPI) network was constructed by using STRING database and optimized by importing into Cytoscape software, and the key targets were obtained by cluster analysis. GO function analysis and KEGG pathway enrichment analysis were performed by using the plug-in of "ClueGo" of Cytoscape. Rat cardiomyocytes(H9C2) were treated with hypoxia and reoxygenation(H/R) to establish MIRI model, which was used to verify that Baoyuan Huoluo prescription containing serum inhibited H/R-induced cardiomyocyte apoptosis from mitochondrial pathway by regulating NF-κB pathway. RESULTS A total of 11 key compounds such as quercetin, kaempferol and isorhamnetin were screened and 51 action targets were obtained. Two sub-networks, involving 16 targets such as CASP3, NOS3, IL-6 and ICAM1 were obtained by clustering analysis of PPI network. The 239 biological functions including in vitro apoptotic signaling pathway, response to reactive oxygen species and migration of vascular endothelial cells were revealed by GO function analysis. The 63 pathways including TNF signaling pathway, NF-κB signaling pathway, VEGF signaling pathway and PPAR signaling pathway were enriched by KEGG pathway enrichment analysis. Cell experiments showed that after H/R treatment, the cell viability of H9C2 cardiomyocytes decreased, the apoptosis rate increased, the activity of caspase-3 and caspase-9 increased, the protein expression levels of NF-κB P65, caspase-3 and caspase-9 increased, and the protein expression levels of IκBα and Bcl-2 decreased. Moreover, Baoyuan Huoluo prescription containing serum could reverse all of the above injuries induced by H/R. CONCLUSION Baoyuan Huoluo prescription can be used to treat MIRI because of its effects on anti-inflammatory, anti-oxidative stress, anti-apoptosis and improve coronary vascular endothelial function, which through a multi-component- target-pathway way. Among them inhibition of H/R induced apoptosis by regulating NF-κB pathway may be the key mechanism.