In this study, we attempted to explore the mechanism of action by which CR treating CHD, with the method of network pharmacology.
In the active components of CR-disease targets of CHD network, quercetin, C09367 and stigmasterol are key ingredients as they are closely related to the most target genes compared to other ingredients of CR. Also, quercetin relates to the majority of hub genes as we concluded in Table 6, while C09367 relates to the rest of hub genes. Quercetin presents significant functions as inhibition of LDL oxidation, reduction of inflammatory markers, endothelium-independent vasodilator effects, protection on endothelial function and nitric oxide, and antiplatelet effect, proved by vitro and some animal models, showing the potential to treat cardiovascular diseases[31, 32, 33]. Also, quercetin showed the analgesic property in various models of inflammation[34]. It is worth mentioning that stigmasterol is related to almost the same genes as C09367, excepting the AR gene. Mouse experiments demonstrated that stigmasterol prevented the HFWD-induced elevation of some di- and triacylglycerol species, decreased serum levels of ceramides, inhibited intestinal absorption of cholesterol and plant sterol, and suppressed hepatic cholesterol[35, 36]. Therefore, these key ingredients can achieve a good effect on CHD by lowering cholesterol, inhibiting platelet aggregation, suppressing inflammatory response and relieving the pain.
After analysis of the active components of CR-disease targets of CHD network, we found out that, PTGS1, CHRM3 and AR are the top three most significant genes, for a total of 40 “CR-CHD” common target genes, with the most relative ingredients of CR. Prostaglandin G/H synthase 1 protein corresponding to PTGS1 gene, is involved in the generation of thromboxane A2 (TXA2), which promotes platelet activation and aggregation, vasoconstriction and proliferation of vascular smooth muscle cells. Therefore, we expect a modulating effect of CR on the expression of PTGS1 encoding the Prostaglandin G/H synthase 1, proving in future experiments. CHRM3 encodes Muscarinic acetylcholine receptor M3 protein proved to cause an endothelium-independent vasodilatation in a mouse experiment[37]. The muscarinic acetylcholine receptor mediates various cellular responses, including inhibition of adenylate cyclase, breakdown of phosphoinositide and modulation of potassium channels through the action of G proteins[38]. As to AR gene, which encodes androgen receptor protein, is verified in some investigations directly that it has atheroprotective effects through both AR-dependent and AR-independent mechanisms, causing different incidence of cardiovascular disease between men and women[39, 40]. When analyzing the PPI network, genes with the node degree equal or more than 8 were selected as key genes, including CASP3, IL6, MYC, ERBB2, AR, FOS. A member of caspase family, caspase-3, encoded by CASP3 gene, in neuronal cells, has been identified as a key mediator of apoptosis[41]. Encoded by IL6, interleukin-6 is increased in a number of cardiovascular diseases, and also associates with a higher incidence of future cardiovascular events, which effects on activity and expression of endothelial nitric oxide synthase and increases vascular superoxide, thus inactivating NO thereby and limiting NO bioavailability[42]. A Swedish cohort indicated that interleukin-6 trans-signaling driven by the IL6 and soluble IL6 receptor binary complex, could be a promising marker of cardiovascular events risk and possibly be used for anti-inflammatory therapy[43]. Expression of ERBB2 relates to mitochondrial function in cardiomyocytes, according to a mouse experiment[44], encoding receptor tyrosine-protein kinase which has a protective effect on cardiomyocytes[45] as well as regulates outgrowth and stabilization of peripheral microtubules. FOS gene could be one of the indexes reflecting myocardial ischemia[46]. By analyzing the target gene-molecular function and pathway network, there were 11 genes, including CASP3, PPARG, NOS3, CASP8, FOS, GSTP1, ERBB3, CYP1A1, AR, AHR, HIF1A, found out to act important roles in the network, as each of them associating with equal or more than 10 molecular functions and pathways. PPARG might increase the risk of CHD in Asian population, as suggested in a meta-analysis[47], as well as PPARGC161T CT/TT was associated with lower levels of blood TC and LDL-C in Han population[48]. NOS3 is significantly altered in patients with CHD[49], of which expression reduces in patients with atherosclerosis[50]. Caspase-8, encoded by CASP8 gene controls apoptosis, necroptosis and pyroptosis as a switch[51]. CYP1A1 showed an increased expression in females compared to males under the situation of ischemic heart disease[52]. AHR regulates the expression of members in CYP1 family, including CYP1A1 and CYP1A2. It was demonstrated that the AHR system could induce the reporter gene expression by acute hypoxia, of which induction was transient, in an ischemic hind limb model[53]. Hypoxia inducible factors, including HIF1A, are key oxygen sensors that mediate the ability of the cell to deal with hypoxia[54]. Moreover, 3 modules derived from MCODE analysis, which are module 1 consisting of ERBB2, IGF2, HSPB1, module 2 consisting of CHRM3, CRP, and module 3 consisting of DUOX2, PTGS1. IGF2 may be relevant to the regeneration of the mammalian heart after injury[55], of which expression is induced to increase by hypoxia in rat hearts[56]. An experiment suggested that HSPB1 acts a role that reduced inflammation and healed wound after myocardial infarction(MI), expected to be a target for myocardial repair in MI patients[57]. CRP, which is one of the inflammatory biomarkers, is considered to be an indicator for evaluating severity and prognosis of CHD, as lipoproteins inflammation is considered significant in the pathogenesis of CHD[58]. The key genes mentioned above reveal mechanisms underlying the therapeutic effect of CR in the treatment of CHD, participating, to varying degrees, in the process of platelet aggregation, vasoconstriction and vasodilatation, proliferation and repair of vascular endothelial cells and cardiomyocytes, lipid metabolism and in inflammation in the heart. CR may play a full part in the treatment by acting on these key genes. Furthermore, taking these results together, it was concluded that AR, FOS, CASP3 were the 3 most critical genes that played roles in the underlying mechanisms for CR treatment on CHD.
We scanned out the biological process of DNA-binding transcription activator activity, RNA polymerase II-specific with the highest enrichment score, and the next most critical processes, which were RNA polymerase II transcription factor binding, kinase regulator activity, ubiquitin-like protein ligase binding after GO enrichment analysis. GO enrichment analysis showed that CR may achieve effects on CHD through the bi-directional regulation of DNA-binding transcription activator activity, RNA polymerase II-specific, RNA polymerase II transcription factor binding, kinase regulator activity, ubiquitin-like protein ligase binding, though regulation mechanisms still waiting for strong scientific evidence. After KEGG enrichment analysis and removal of wide range of metabolic pathways, we concluded 5 top pathways with the highest enrichment scores, including fluid shear stress and atherosclerosis, TNF signaling pathway, apoptosis, MAPK signaling pathway, PI3K-Akt signaling pathway. Sensing of fluid shear stress and atherosclerosis is considered important in processes of vascular development and remodeling[59, 60]. Studies demonstrated that TNF antagonists has a potent effect of anti-inflammatory and antioxidant[61, 62]. MAPK signaling pathway is activated to regulate apoptosis of cardiac myocytes and angiogenic response of microvascular endothelial cells by related factors, for example, nicotine and protein phosphatase 2A, demonstrating that activation of the MAPK signaling pathway appeared to be a key process in microvascular endothelial cells and cardiac myocytes life-death decisions[63]. The PI3K/Akt signal pathway regulates survival, apoptosis, cell morphology, protein synthesis, and integration of metabolism in cardiomyocytes[64], involved in regulating inflammatory responses, playing a critical role in cardioprotection of preconditioning against ischemia injury[65]. These researches indicate that CR acts on CHD at multiple levels through multiple biological processes and mainstream signaling pathways, mainly regulating vascular development and remodeling, inflammatory process, oxidation, endothelial cell and cardiacmyocyte apoptosis and protein synthesis. Furthermore, we can understand the advantages of CR in the treatment of CHD and its potential in new drug development by this study.