The current study presents evidence that Tanshinone II, Neotanshinone C, Neocryptotanshinone Ii, Miltionone and Patchoulenone are the major compounds of DXTMG with potential pharmacological activity. Previous work at the cellular level has established that Tanshinone ⅡA greatly relieves myocardial inflammatory infiltration and oxidative damage caused by myocardial ischemia-reperfusion injury in rats and reduces leakage of lactate dehydrogenase and production of malondialdehyde, thereby protecting against myocardial ischemia-reperfusion injury[18]. The findings of Weng et al.[19] demonstrated that Tanshinone ⅡA has an estrogen-like effect resulting in protein kinase B (Akt) activation and suppression of cardiomyocyte apoptosis on activation by insulin-like growth factor II (IGF-II). This suggests a role for Tanshinone ⅡA as a potential selective estrogen receptor modulator in the prevention of myocardial apoptosis and treatment of cardiovascular diseases. Moreover, Li et al.[20] demonstrated a role for Tanshinone ⅡA in suppressing mitogen-activated protein kinase (MAPK) and down-regulating c-fos expression, thus reducing intimal hyperplasia and vascular smooth muscle cell proliferation in rats with carotid artery balloon injury. In addition, Wu et al.[21] showed that Neocryptotanshinone suppresses lipopolysaccharide-induced inflammatory factors, including TNF-α and IL-6, in rats. Neocryptotanshinone also reduces lipopolysaccharide-induced production of nitric oxide. The pharmacological and computer-based investigations of Maione et al.[22] demonstrated a concentration-dependent activity of Neocryptotanshinone in inhibiting platelet aggregation and antagonizing G protein-coupled P2Y12 platelet ADP receptor.
The PPI analysis of the current study identified INS, ALB, IL-6 and TNF as the major targets of DXTMG in CHD treatment. Accumulation of inflammatory cells around damaged vascular tissues may promote plaque formation through the secretion of inflammatory mediators. Therefore, inflammatory markers are considered risk factors for CHD[23, 24]. Insulin resistance (IR), a predisposing factor for cardiovascular diseases such as CHD[25], is characterized by increased levels of inflammatory cytokines and expression of adhesion molecules, vascular dysregulation and vascular endothelial injury, ultimately resulting in atherosclerosis[26]. INS enhances glucose uptake and utilization, thereby protecting the ischemic-hypoxic myocardium. Blood glucose elevation is a risk factor for cardiovascular diseases and the hypoglycemic effect of INS is central to blood glucose regulation[27]. High-levels of INS increase angiotensin Ⅱ, through the renin-angiotensin system, and promote production of reactive oxygen species, thereby facilitating inflammatory and oxidative stress responses[28]. Normal concentrations of albumin inhibit platelet activation and aggregation and apoptosis of vascular endothelial cells, thereby reducing CHD risk[29]. Moreover, activation of the Apelin/G protein-coupled receptor (APJ) causes blood vessel dilation, reducing blood pressure and increasing myocardial contractility and relaxation. Apelin inhibits the potential activity of AVP neurons and AVP release, reducing plasma osmotic pressure and ALB concentration and increasing CHD risk. Therefore, maintenance of stable plasma ALB concentrations can help reduce the occurrence of CHD[30, 31].Scheller J et al.[32] showed a positive correlation between IL-6 levels and incidence of severe coronary artery lesions. IL-6 promotes macrophage activation and formation of foam cells, stimulating expression of macrophage low-density lipoprotein receptors and triggering matrix metalloproteinases to destabilize atheromatous plaques, resulting in plaque rupture[33]. It is known that vascular endothelial injury stimulates inflammatory receptors and release of inflammatory factors, such as TNF-α and IL-6, followed by. thrombosis and plaque rupture[34]. The suppression of smooth muscle cell proliferation, an effect that correlates with down-regulated expression of TNF-α and IL-6, may improve the stability of atherosclerotic plaques[35]. TNF-α stimulates apoptosis through increasing protease activity which results in increased basal metabolism[36].
The current study used KEGG pathway enrichment analysis to demonstrate that fluid shear stress and atherosclerosis, the hypoxia-inducible factor-1 (HIF-1) signaling pathway, adrenergic signaling in cardiomyocytes and the AGE-RAGE signaling pathway in diabetic complications were implicated in the action of DXTMG in CHD treatment. Atherosclerosis is involved in the pathological development of CHD when plaque formation-induced changes in shear stress affect the normal function and phenotype of vascular wall endothelial cells[37]. It has been established that low shear stress up-regulates expression of intercellular adhesion molecule-1 on the endothelial cell surface, promoting activation of endothelial cells[38]. A further consequence is phosphorylation of platelet endothelial cell adhesion molecule-1, activating the MAPK pathway and worsening inflammation to promote CHD. HIF-1, a hypoxia-induced DNA-binding protein, induces increased expression of HIF-1α during myocardial ischemia and hypoxia, thereby protecting the myocardium[39]. Moreover, HIF-1 induces endothelial cell dysfunction, proliferation, angiogenesis and inflammation, thus playing a role in the pathogenesis of atherosclerosis via various pathways[40]. Via its action on the α1 receptor in vascular smooth muscle, epinephrine promotes vasoconstriction and increases aortic diastolic pressure, thereby raising coronary perfusion pressure[41]. Adrenergic signaling in cardiomyocytes enhances myocardial contractility, resulting in vascular dilation of both heart and liver and vascular contraction of both skin and mucosa, thereby exerting a therapeutic effect on CHD. Activated AGE/RAGE signaling which enhances inflammation, oxidative stress and vascular smooth muscle cell apoptosis, thus promoting the development of atherosclerosis, plays a significant role in CHD through the above mechanisms[42].
We present results of molecular docking experiments which revealed stable binding between components of DXTMG and receptor proteins with binding energy of the TNF-Neocryptotanshinone Ii interaction being the highest (-14.09 kcal/mol). Moreover, the molecular dynamics simulations of the current study show stable TNF-Neocryptotanshinone Ii binding. Binding sites had strong hydrophobicity and hydrophobic residues, such as Leu279, Val280 and Phe278, were involved. In addition, the ligand may undergo a hydrogen-bond interaction with Leu279.