TCM is a complex, multi-component, multi-target hybrid system that has long been used for the prevention and treatment of various cardiovascular diseases (CVDs)[20–22]. RW can effectively treat AP, but its pharmacological mechanism of action is still unclear. Therefore, in this study, we used a pharmacological network approach to identify the bioactive compounds, potential targets, and pathways regulated by these compounds for RW treatment of AP.
Nine potential hub targets were identified based on selection and network topology analysis, including VEGFA, GAPDH, TP53, AKT1, CASP3, STAT3, TNF, MAPK1 and Jun. Numerous studies have shown that these targets above mentioned mainly involved in the protection of vascular endothelium, as well as the regulation of glucose metabolism, cellular processes, inflammatory responses, and cellular signal transduction. VEGF, a strong pro-angiogenesis cytokine, is secreted by vascular endothelial cells, which could increase the permeability of microvessels and venules, promote angiogenesis, thus improving myocardial hypoxia and relieving AP pectoris[23–25]. Recent studies have demonstrated that the inflammatory response is related to the occurrence of cardiovascular diseases such as coronary heart disease and AP, which may cause local endothelial activation, atherosclerotic plaque rupture, and then thrombosis form or rupture, leading to AP and myocardial infarction[26]. It is well-known that TNF-a, usually appearing in the early stage of inflammatory response, plays an important role in cell function regulation, immunity and inflammatory response, which could regulate atherosclerotic plaques and coronary heart disease by affecting vascular endothelial function and vascular remodeling[27]. Biasucci et al., Huang et al. studies have shown that proinflammatory cytokines were significantly increased in patients with AP compared to healthy individuals. However, it is important to note that this increased inflammatory activity may be related to the pathogenesis of AP. For example, TNF-α may increase the expression of monocyte/macrophage tissue factors, as well as cell apoptosis by improving thrombotic activity, leading to the increase of matrix metalloproteinases in atherosclerotic plaques[28–32]. AKT1 is an important protein in the PI3K pathway, which could regulate cell apoptosis, proliferation and antioxidant[33, 34]. Han et al.[35] demonstrated that hypericin can reduce the inflammatory response by activating phosphorylated AKT and reducing TNF-α and IL-6 activity, thus alleviating myocardial ischemia-reperfusion injury. The p53 gene is an important apoptosis-related gene, divided into two types: wild type (wp53) and mutant type (mp53). The mutant p53 gene can promote cell growth and participate in the occurrence of various tumors. The main function of the wild-type p53 gene is to participate in the negative regulation of cell growth, limiting cell growth and division. In recent years, studies have found that the p53 gene is not only related to the occurrence and development of many tumors but also participates in the occurrence of apoptosis in the cardiovascular system[36–38]. c-JUN is the heterodimer form of activating protein 1 (AP-1). Previous studies have demonstrated that c-JUN can induce the production of adhesion factors in endothelial cells, increase the expression of chemokines and the formation of foam cells, thus promoting the formation and development of atherosclerosis[39]. MAPK1 belongs to the Ser-Thr kinase protein family and has been previously reported in different features of cardiac modeling and regulation of inflammation, cell proliferation and differentiation[40, 41]. The high activity of Erk1/2 has been observed in T-lymphocytes from CAD patients, including ST-elevation myocardial infarction (STEMI), Non-STEMI and unstable AP[42]. A previous study demonstrated the EGFR-mediated cross-talk between MAPK and Akt1 signaling, which combined had an important role in abnormal vascular remodeling[43]. STAT3 is a latent transcription factor, initially identified as a cytokine signaling transductor[44],and is involved in a variety of biological processes such as cell proliferation[45], differentiation[46], and survival[47].
To understand the potential biological mechanism of RW against AP, GO and KEGG functional enrichment analysis of DAVID and KEGG were applied. Through the KEGG pathway analysis (p-value < 0.05), we recognized 21 AP-related signaling pathways, HIF-1, PI3K-Akt, MAPK, FoXO, TNF, Ras and toll-like receptor signaling pathway and so on. Accordingly, these pathways may be involved in the progress of AP. Based on P-value, we choose HIF-1 signaling pathway as most candidate signals for further study. HIF is a transcriptional complex that responds to changes in oxygen and provides a master regulator for cells to coordinate changes in gene transcription. HIF acts on all mammalian cell types and is ancient during evolution. At the molecular level, the HIF complex contains an alpha subunit and a beta subunit, both of which can be selected from several options. HIF-β subunits are composed and also participate in heterogeneous reactions. The alpha subunit is regulated and is unique to hypoxic reactions. Under hypoxic conditions, HIF-1α is induced and highly expressed, transferring from the cytoplasm to the nucleus and initiating downstream gene expression, such as erythropoietin and VEGF. HIF-1α could increase myocardial glucose intake and transportation to continuously provide the compensatory energy supply by regulating myocardial GLUT4 and PKM2 gene expression[48]. HIF-1α also facilitates the activation of PDK1 and PDK4 as well as UCP2 to enhance mitochondrial oxidative phosphorylation[49]. Moreover, NRF1 and TFAM play distinct roles in mitochondrial biogenesis[50] and the upregulation of NRF1 and TFAM promotes mitochondrial DNA synthesis in infarcted cardiac muscle[51]. Therefore, HIF-1 signaling pathway is activated in cardiomyocytes to produce continuous ATP in adaption to hypoxia, by shifting myocardial metabolism substrate to glucose intake and transportation[52].
In this study, GO enrichment analysis was adopted. The targets were connected with regulation of protein phosphorylation, nitric oxide biosynthetic, cell membrane region, platelet alpha granule, protein kinase and protein phosphatase et al. Therefore, the results suggest that RW treats AP by participating in these BP, CC and MF.
Molecular docking simulation analysis provides a visual interpretation of the interactions between key compounds and potential protein targets. For example, the Salidroside small molecule forms 4 hydrogen bonds primarily with Lys52, Asn152, Ser151 and Met106 residues on MAPK1. Salidroside has various pharmacological activities such as anti-fatigue, anti-oxidation, immune regulation and free radical scavenging. In recent years, in vivo and in vitro experiments have proved that the compound has positive effects on anti-cancer, anti-inflammatory, anti-oxidation, neuroprotection, myocardial protection, liver protection, kidney protection. It has extremely important application value in military, aerospace, sports and health medicine[53–56]. In addition, it is reported that salidroside can inhibit the release of LDH, CK and AST of human cardiomyocytes by increasing the expression of HIF-1α, increase the content of SOD, and at the same time increase the activity of human cardiomyocytes and reduce the death and apoptosis of cells[57]. Taken together, we speculate that the major components of RW may play an important role in the treatment of AP through hub targets in these top signaling pathways. However, there are some limitations to our study. For example, these results are only based on screening of known RW chemical components, associated targets and signaling pathways from the literature and existing databases. Therefore, more in-depth studies are needed to characterize the underlying mechanisms.