The occurrence of CHD seriously endangers human’s health worldwide. As is known to us, TCM has been used to treat CHD for thousands of years. Currently, TCM advances with the times and has enriched and developed the previous theories based on macroscopic syndrome differentiation and microscopic pathology, thus forming a unique treatment system for CHD [33]. However, due to the complexity of the active components in TCM, the specific mechanism through which it acts on CHD remains unclear. Fortunately, the emergence of network pharmacology makes up for the deficiency. It attempts to analyse the underlying mechasims of TCM acting on diseases through the virtual screening of potential active compounds, target proteins, and signaling pathways. Hence we conducted a comprehensive study on the molecular mechanism of ZD against CHD using the strategy of network pharmacology.
In the present study, a total of 62 compounds were screened by excavating the active ingredients of ZD. As shown in Figure 6, quercetin, kaempferol, luteolin, and tanshinone IIa were identified as representative compounds. Previous studies have reported that quercetin and kaempferol are flavonoids from Zhizi and have biological activities against CHD such as anti-oxidation, anti-inflammation, inhibition of platelet activation, and relaxation of blood vessels[36]. Meanwhile, luteolin is a polyphenolic compound from Danshen, which exerts cardiovascular protective effects for the roles of reducing oxidative stress, and inhibiting inflammation and apoptosis[37]. Tanshinone IIa is another compound existed in Danshen that can perform anti-ischemia, anti-arrhythmia, anti-atherosclerosis, and anti-coagulation[38]. The above active ingredients provided basis for the pharmacological activities of ZD acting on CHD.
Analysis of the PPI network based on the common targets of ZD and CHD indicated that TNF, IL6, AKT1, CXCL8, MAPK14, FOS, VEGFA, IL1B, IL4, MAPK8, and RXRA were selected as the core targets. These target genes were mainly linked to the release and inhibition of inflammation and regulation of angiogenesis, which explained the effects of anti-CHD at the molecular levels. For instance, TNF is a cytokine produced by activated macrophages. Further, TNF-α is an important pro-inflammatory cytokine which participates in the vasodilatation formation and mediates the recruitment of neutrophils and macrophages to sites of inflammation by stimulating endothelial cells to produce adhesion molecules[39]. IL6, an inflammatory mediator released by neutrophils, monocytes and cardiomyocytes, is associated with the thrombosis of atherosclerotic plaque. It has been reported that the elevated level of IL-6 is related to acute myocardial ischemia and serve as an indicator of recurrent CHD among patients[40]. Moreover, there are various pro-inflammatory cytokines including IL1B and CXCL8, which involved in the process of plaque formation and rupture via different pathways[41-42]. However, IL4 is an anti-inflammatory cytokine that inhibits the expression and release of inflammatory cytokines such as IL-1, IL-6, and TNF-α[43-44]. Szkodzinski et al.[45] have determined prognostic implications of IL-4 in developing severe cardiac dysfunction in the course of acute myocardial infarction (AMI). In addition, AKT1 is the main isomer in vascular endothelial cells, which plays a crucial role in cardiac growth, contraction and coronary angiogenesis[46]. MAPK14, also named as p38α MAPK, is a member of p38 MAPK family. Studies have found that cardiac myocyte p38α kinase regulates angiogenesis via myocyte-endothelial cell cross-talk during stress-induced remodeling in the heart[47]. VEGFA is a mitogen that promotes vascular endothelial cell proliferation and angiogenesis[48]. It has been suggested that endothelial dysfunction caused by VEGFA may increase the risk of CHD[49]. Furthermore, the combination of VEGFA and its receptor VEGFR2 can activate downstream signaling pathways such as MAPK, Akt to stimulate angiogenesis[50].
Through KEGG pathway enrichiment analysis, seven signaling pathways including fluid shear stress and atherosclerosis, AGE-RAGE signaling pathway in diabetic complications, HIF-1 signaling pathway, IL-17 signaling pathway, TNF signaling pathway, Toll-like receptor signaling pathway, and relaxin signaling pathway were relevant to vascular endothelial function regulation, inflammatory effects, and hormone regulation. It is worth mentioning that endothelial dysfunction plays a significant role in the progression of atherosclerotic plaque which is a main factor resulting in developing CHD. Endothelial shear stress heavily influences endothelial function, and low shear stress with the proatherogenic effect can be independent predictor of revascularization[51]. Additionally, low shear stress may drive epicardial endothelial dysfunction and atherosclerosis progression, thereby causing more advanced phenotypic manifestations of CHD[52]. Studies have shown that low shear stress activated the expression of vascular cell adhesion molecule 1 (VCAM-1) which was induced by cytokines such as IL-1 and TNF-α, making the markedly greater monocyte binding to the carotid[53-54]. Nevertheless, the activation of AKT1 mediated by fluid shear stress can promote cell survival in endothelial cells[55]. Advanced glycation end products (AGEs) interacting with a cell surface receptor, RAGE was reported to evoke inflammatory and thrombogenic reactions, which contributed to the development of CHD[56]. Research found that AGE/RAGE induced the activation of the ERK/MAPK signaling pathway, ultimately leading to activate nuclear factor NF-κB, thus increasing production of proinflammatory and proatherogenic mediators [57]. Moreover, regulating the AGE/RAGE pathway may be able to treat cardiac ischemic–reperfusion injury, diabetic cardiomyopathy, and inflammatory heart diseases[58]. Hypoxia-inducible factor 1 (HIF-1) signaling pathway plays an important regulatory role in the hypoxia response. It can upregulate the expression of angiogenic factors such as vascular endothelial growth factor (VEGF) to promote angiogenesis. Besides, HIF-1 activates inducible nitric oxide to increase blood flow and reduce ischemic injury[59-60].
In addition, recent studies supported the involvement of chronic inflammation in the progression and initiation of atherosclerosis, which resulted in various cardiovascular diseases[61]. IL-17, derived by T helper-17 (Th17) cells, is involved in inflammatory pathology of various diseases [62]. It promotes the release of proinflammatory cytokines (IL6, TNF-α, IL1B) and chemokines (IL8, CXCL1) to mediate inflammation. Studies found IL-17 signaling activated downstream pathways such as NF-kB and MAPK to stimulate cytokines with relevance to atherosclerosis [63]. Further, it was identified that downregulating IL-17 expression could suppress inflammation and improve heart function[64]. TNF signaling also induces inflammatory response by activating the expression of proinflammatory cytokines and transcription factors[65]. For example, TNF combined with TNF receptor 1 (TNFR1) mediated the activation of NF-kB, which was involved in the pathological process of cardiovascular disease such as ischemia-reperfusion, heart failure, and ventricular remodeling[66]. Moreover, Toll-like receptor (TLR), belongs to pattern recognition receptors, can activate expression of a series of genes related to inflammation and immune response[67]. TLRs have eleven types, of which TLR4 plays an extremely important role in CHD[68]. It was reported that TLR4 interacting with some other pathways such as PI3K/Akt and MAPK activated a variety of downstream transcriptional factors, and initiated inflammatory response. [69].
Finally, relaxin signaling is a pathway associated with hormone regulation. Produced by the heart, relaxin coupling with its cognate receptor mediates the production of cyclic adenosine monophosphate (cAMP), and activation of nitric oxide (NO) pathway, PI3K/Akt pathway, MAPK pathway, and nuclear factor NF-κB, of which NO production plays a central role in cardioprotection[70]. Some experiments have found that relaxin is able to promote vasodilatation and angiogenesis, inhibite inflammation, and ameliorate ischemia/reperfusion injury because of the upregulated expression in ischemic heart disease [71]. Besides, in cardiac ischemia model, relaxin was verified to reduce interstitial collagen accumulation and cardiac hypertrophy, thus exerting antifibrotic effects[72].