QSYQP was approved for the treatment of angina pectoris of coronary heart disease by China State Food and Drug Administration in 2003. Network pharmacology analysis is becoming an important research method to study the effect of Chinese medicines on diseases to reveal the complex components and unknown targets of Chinese medicines. Although QSYQP has been used in the clinic for a long time, its specific mechanism is not fully understood. Through ETCM and TCM-Mesh screening, 333 compounds were recognized as active compounds of QSYQP, 158 of which were predicted to act on 674 specific targets.
Overlap analysis of the targets of the four herbs in QSYQP and construction of the herb-target network revealed that most targets were jointly regulated by multiple compounds and herbs. Usually, the potency of a single natural compound’s regulatory effect on a target is much lower than that of a specifically designed drug molecule because the interactions between natural compounds and proteins generally have low affinity. Our results suggest that QSYQP may produce therapeutic effects by acting on multiple proteins from multiple ingredients of different herbs to achieve synergetic efficacy.
An earlier network pharmacology study illustrated that the coexistence of disease genes and drug targets in the same network module suggested an underlying link between disease and drugs. We constructed a subnetwork influence by CVD genes and found that 40% of the targets of QSYQP appeared in this subnetwork, while 20 modules were identified in which target proteins and CVD genes coexisted. These results indicate that QSYQP may exhibit therapeutic effects on CVD.
By analyzing the topology of the subnetwork influenced by CVD, 42 targets of QSYQP were identified as key targets for the treatment of cardiovascular diseases. Furthermore, the important targets were found to be significantly enriched in 31 pathways, which were associated with the occurrence and development of cardiovascular diseases, including blood coagulation, inflammation, immunity and energy metabolism. KEGG pathway analysis identified that the important targets of QSYQP were involved in several pathways related to blood coagulation, such as complement and coagulation cascades, fluid shear stress and atherosclerosis, platelet activation, and the VEGF signaling pathway. Blood coagulation factors play important roles in cardiovascular diseases risk, participating in the initiation of the extrinsic coagulation process and causing the generation of thrombin[35–37]. A Previous publication indicated that Salvia miltiorrhiza ameliorated deep vein thrombosis by exerting antioxidative effects. Coagulation factors regulated by 2-isopropyl-8-methylphenanthrene-3,4-dione extracted from Salvia miltiorrhiza were associated with complement and coagulation cascade signaling pathways. As part of the cardiovascular phenotype in congestive heart failure (CHF), platelet activation is correlated with impaired endogenous platelet inhibition and endothelial dysfunction. Platelet activation is activated in atrial fibrillation (AF) and accelerates the progress of thrombin generation. However, the level of VEGF is downregulated in the pathophysiology of heart failure. Our results suggested that the active compounds JX, meliotocarpan c and odoricarpan, modulated the target TUBB. TUBB, which is specifically expressed by platelets and megakaryocytes, may be involved in the secretion and release of platelets. QSYQP’s targets ACTB, AKT1, NOS3, PIK3CA, PIK3R1, and SRC participated in platelet activation and VEGF signaling pathways, suggesting its function alleviating the symptoms of cardiovascular diseases by regulating blood coagulation.
The pathways correlated with inflammation and immunity, which are regulated by QSYQP, have also been identified, such as the PI3K-Akt signaling pathway, the Toll-like receptor signaling pathway (TLR), apoptosis, the HIF-1 signaling pathway, the T cell receptor signaling pathway, the chemokine signaling pathway, the TNF signaling pathway, the Jak-STAT signaling pathway, the Wnt signaling pathway, the Ras signaling pathway, the p53 signaling pathway, Th17 cell differentiation, the NF-kappa B signaling pathway, Th1 and Th2 cell differentiation, and the NOD-like receptor signaling pathway. The NF-kappa B signaling pathway is associated with immune deficiency, whereas dysregulated activation of this pathway contributes to the pathogenesis of various autoimmune and inflammatory diseases. Our data show that QSYQP can participate in individual validation and immune regulation via the chemokine signaling pathway and NF-kappa B signaling pathway. As a serine threonine kinase, Akt participates in the processes of cell proliferation and survival and decreases necrosis and apoptosis of cardiomyocytes[44–46]. Intracellular MAPK signaling cascades, probably play an important role in the pathogenesis of cardiovascular diseases. The Toll-like receptor signaling pathway, which accelerates the nuclear translocation of NF-κB and AP-1, recruits TRIF, and activates the transcription of proinflammatory cytokine genes.
In addition, pathways related to cardiovascular contraction, blood pressure regulation and energy metabolism have been identified, which could explain the mechanism of QSYQP in the treatment of cardiovascular diseases. Ten targets were mapped to adherens junctions, and nine were mapped to focal adhesions. The cell matrix adhered and participated in the structural connection between membrane receptors and the actin cytoskeleton, and played an important role in a series of biological processes. The cell matrix is connected with gap junction channels and is the basis of many physiological events, including electrical coupling, metabolic transport, apoptosis and maintenance of homeostasis. Eight targets participated in tight junctions, in which transmembrane protein composition is critical for building a diffusion barrier for selective infiltration between adjacent cells, affecting many biological pathways. In addition, the relaxin signaling pathway, which is involved in vasodilation related to proteins targeted by compound components, plays a key role in the regulation of blood pressure, aldosterone-regulated sodium reabsorption, and sodium and potassium metabolism. The proportion of oxidative energy supplied to myocardial free fatty acids will increase, and myocardial remodeling is the main pathological feature in the progression of myocardial ischemic injury or heart failure. Some studies have shown that disorder of myocardial energy metabolism directly or indirectly promotes the process of myocardial remodeling and further aggravates heart failure. A large number of studies have shown that cardiac hypertrophy is related to cardiac energy metabolism. QSYQP can reduce cardiac hypertrophy and improve cardiac function by regulating cardiac energy metabolism[49; 50]. Multiple targets of the compound are mapped to the cGMP-PKG signaling pathway and cAMP signaling pathway, through which they improve and regulate myocardial energy metabolism, thus playing an important role in the treatment and symptom improvement of heart failure.
To validate the results from network pharmacological analysis, we investigated the protective effect of QSYQP from myocardial cell injury using an H/R model in vitro. Our results strongly suggest the cardioprotective effect of QSYQP from H/R injury, which was mainly manifested on the decreases of cell apoptosis and protective effect of cytoskeleton. Exposure of H9c2 cardiomyocytes to H/R caused a remarkable interrupt and exception aggregation, as evident by the significant confusion of red fluorescent signals in H/R group compared to the normal condition. However, H9c2 cardiomyocytes treated with QSYQP and its components showed a remarkable regulation.
At last, we applied Western blot experiment to validate the effects of QSYQP and its two active ingredients (DSS and ASIV) to three proteins, ACTC1, FoxO1, and DIAPH1. Our network pharmacological analysis suggested that ACTC1 and DIAPH1 were targets of QSYQP, and FoxO1 was CVD disease gene. These three proteins were in the sub-network influenced by CVD, as well as in important pathways associated with QSYSP’s treatment. Specifically, ACTC1 is associated with Hypertrophic cardiomyopathy signaling pathway, DIAPH1 is associated with Regulation of actin cytoskeleton and Focal adhesion signaling pathway, FoxO1 is correlated with the AMPK signaling pathway. A remarkable increase in expression showed that ACTC1, FoxO1, DIAPH1 might be involved in the cardiac injury induced by H/R. Our experiments showed that cardiomyocytes treated with QSYQP, DSS and ASIV had a remarkable increase in expression of ACTC1, FoxO1, DIAPH1.
One of cardiovascular diseases is hypertrophic cardiomyopathy (HCM), a group of related diseases characterized by hypertrophy of the ventricular myocardium. It was thought to be the result of a particularly group of mutations in the cardiac actin gene ACTC1. A variety of genes linked to HCM encode proteins related to the sarcomere, the fundamental contractile unit of the heart, formed from interacting filaments of cardiac actin. Increasing evidence has shown that FoxO1 is closely related to the occurrence and development of cardiac hypertrophy. FoxO1 can regulate cardiac growth, protein synthesis, calcium homeostasis, cell apoptosis, and autophagy. FoxO1 undergoes modification and transfers from the cytoplasm to nucleus, thus regulating the expression of series of target genes in myocardium in response to stress or external stimulation. FOXO1 is an essential regulator of vascular growth in endothelium. Recent studies suggest that DIAPH1 is involved in the regulation of actin filaments. It has revealed that DIAPH1 rotates along the F-actin. Therefore, our experiments suggested that QSYQP regulated the three genes and associated pathways to play a therapeutic role for cardiovascular diseases.
These results[52–54] may offer a reference for QSYQP’s further utilization or the drug repositioning on new indications. Integrating network and pathway analysis, we identified 7 key targets of QSYQP in the treatment of cardiovascular diseases. Among the seven key targets, CCND1 is the target of AMPK pathway (hsa04152), which is related to cardiomyocyte hypertrophy. SRC and EGFR are the targets of regulation of actin cytoskeleton (hsa04810) and play roles in the signal transduction of myocardial hypertrophy and alleviate myocardial hypertrophy.