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

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.