Xuefu Zhuyu decoction, a blood regulating agent, has the effect of promoting blood circulation and removing blood stasis, promoting qi and relieving pain. Modern pharmacological studies have confirmed that Xuefu Zhuyu decoction has the effects of improving hemorheology, protecting vascular endothelial function, inhibiting platelet aggregation, treating coronary heart disease, improving myocardial infarction, lowering blood pressure, regulating blood lipids, anti-AS and so on. Utilizing network pharmacology, we found that the anti-AS effect of XZD involves a complex component-target-pathway network.
The theories to explain the pathogenesis of AS include vascular smooth muscle cell clone theory [13], lipid infiltration theory [14], oxidative stress theory [15], platelet hyperfunction theory [16], thrombosis theory [17], immune function abnormality theory [18], shear stress theory [19], injury reaction theory [20], inflammation theory and so on [21]. Among these theories, the inflammatory mechanism of AS is a relatively new theory, and a lot of research work has been carried out. According to the results of GO enrichment analysis, KEGG enrichment analysis, PPI analysis and network topology analysis, this paper discussed the mechanism of XZD in the treatment of AS.
The development of AS can be divided into three stages: early stage, progressive stage and terminal stage. In the early stage of AS, vascular endothelial cells (VECs) are injured for various reasons, which stimulate VECs to express adhesion molecules and promote monocytes and lymphocytes to adhere to vascular endothelium and migrate to the intima [22]. A large number of low-density lipoproteins are modified into oxidized low-density lipoproteins (oxLDL) and accumulate in the vascular wall, accelerating the process of AS [23]. After entering the vascular wall, monocytes differentiate into macrophages, engulf oxLDL and transform into foam cells [24], which participate in the process of AS together with T cells, B cells and other lymphocytes. The progression stage of AS is actually a proliferative inflammation of the vascular wall. Under the influence of cytokines, vascular smooth muscle cells migrate from media to the intima and proliferate [13]. In the terminal stage of AS, activated natural killer T cells can cause VECs death or apoptosis [25], coupled with the dissolution of collagen fibers in plaque extracellular matrix by matrix metalloproteinases [26], resulting in plaque rupture, bleeding and thrombosis. From the enrichment analysis of key targets, XZD has an influence on the early, progressive and terminal stages of AS.
4.1. Biomarkers
Atherosclerosis involves many biomarkers, such as cytokines, adhesion molecules, C-reactive protein, matrix metalloproteinases and so on. Interleukin-6 (IL-6) is a cytokine involves in the expression and regulation of the immunity system and serves to acute and chronic inflammatory responses[27]. Related clinical studies have shown that IL-6 is related to the incidence of cardiovascular events and mortality. The level of IL-6 in patients with AS was significantly higher than that in healthy people, and the level of IL-6 is positively correlated with the severity of the disease [28]. The level of IL-6 increased significantly in patients with plaque rupture [29]. Quercetin exerts an anti-inflammatory effect by inhibiting the neutrophil secretion of IL-6 and strongly inhibits mast cell secretion of IL-6 by inhibiting p38 signaling pathway and PKC phosphorylation [30,31]. Luteolin down-regulates the expression of IL-6 in monocytes by inhibiting MAPKs and NF-kB signaling pathways and reduces the secretion of IL-6 in microglia by inhibiting JNK phosphorylation and activating AP-1 [32,33].
When the inflammatory response system is activated, macrophages gather in the vascular endothelium and release a large amount of IL-6 and tumor necrosis factor-α, inducing the liver to synthesize a large amount of C-reactive protein (CRP)[34,35]. CRP is not present in healthy vascular walls, but it can be detected in the early stages of AS, binds to low-density lipoprotein, exists in atherosclerotic plaques and gradually accumulates [36,37]. CRP promotes the recruitment of monocytes to the vascular wall by increasing the secretion of endothelial cell adhesion molecules and promotes plaque instability by inducing the expression of matrix metalloproteinase (MMPs) [38–40]. CRP up-regulates the expression of vascular endothelial growth factor (VEGF) by activating MMP-2, which significantly promotes the proliferation of vascular endothelial cells [41]. CRP also contributes to thrombosis [42]. In addition, CRP is considered to be a predictor of cardiovascular events, and CRP levels can independently predict the risk of cardiovascular death in the general population [43]. A meta-analysis of available randomized controlled trials showed a significant reduction of circulating CRP levels with quercetin [44]. Quercetin inhibits the recruitment of monocytes by reducing the level of CRP in plasma, and inhibits the proliferation of vascular endothelial cells and stabilizes plaques by regulating the expression of matrix metalloproteinases induced by CRP. Its therapeutic effect runs through the early, progressive and terminal stages of AS.
Adhesion molecules play an important role in the process of AS. [45]. Adhesion molecules are proteins that mediate cell-cell and cell-extracellular matrix contact and adhesion. They can be divided into selectin family, integrin family and immunoglobulin superfamily (IgSF) which can be regulated by XZD [46]. Selectin is the first step in leukocyte adhesion in inflammatory, and E-selectin (SELE) supports leukocyte adhesion to vascular endothelial cells [45]. Vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1), which can promote the early adhesion of monocytes to vascular endothelial cells, are members of the immunoglobulin superfamily and ligands of integrins [47,48]. The expression of VCAM-1 and ICAM-1 is induced by cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-1 (IL-1) [49,50]. The components of XZD, including quercetin, luteolin and kaempferol, interfere with the adhesion of monocytes to vascular endothelial cells in the early stage of AS by regulating the expression of SELE, VCAM1 and ICAM1. Quercetin and kaempferol have direct effects on the three adhesion molecules.
Vascular endothelial growth factor A (VEGFA) plays an important role in vascular development and neovascularization in physiological and pathological processes. VEGFA and its receptor VEGFR are involved in the progression of AS. In the early stage of AS, VEGFA secreted by macrophages acts on both VEGFR-1 and VEGFR-2 receptors [51]. VEGFR-1 induces monocyte migration [52], thus enabling macrophages differentiated by monocytes to gather in the plaque, while VEGFR-2 accelerates the process of AS by stimulating vascular growth [53]. Many studies have shown that intimal neovascularization is related to the development and stability of plaques [54–57]. Quercetin, beta-carotene, luteolin, baicalein and ellagic acid can regulate the expression of VEGFA, thus affecting monocyte recruitment and vascular endothelial cell proliferation, delaying the early stage of AS.
4.2. Signaling pathways
In the results of KEGG enrichment analysis, we found that three signaling pathways have an important impact on the process of AS, namely Toll-like receptor (TLR) signaling pathway, Nuclear factor-kappa B (NF-kB) signaling pathway and Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway. In vascular smooth muscle cells, exogenous lipopolysaccharides activate the TLR4 signaling pathway, which activates NF-kB and promotes the release of cytokines such as monocyte chemoattractant protein-1 (MCP-1) and interleukin-6 (IL6) [58,59]. TLR4 can also induce local lipid accumulation by down-regulating the expression of ATP-binding cassette sub-family G member 1 (ABCG1) [60]. Although TLR4 is not directly regulated by the effective components of XZD, it is of great significance to the progress of AS and is an ideal target to interfere with the progression of AS. NF-kB signaling pathway is associated with inflammation and apoptosis in the progression of AS [61], which can be activated by inflammatory cytokines, adhesion molecules (Such as VCAM-1 and ICAM-1) and chemokines [62]. Down-regulation of NF-kB leads to a decrease in foam cell formation [63], while activation of the TLR4/NF-kB signaling pathway promotes plaque growth and reduces plaque stability [64]. Mice with high NF-kB expression increase the risk of atherosclerotic plaque formation in the aorta [65]. Although the enrichment of XZD targets in the NF-kB signaling pathway was not significant, the NF-kB signaling pathway plays an important role in the progression of AS and is an important way to interfere with AS. JAK-STAT signaling pathway is an important signaling pathway regulating the early and terminal stages of AS [66]. JAK-STAT signaling pathway is activated by cytokines and participates in immune regulation in the progression of AS [67]. STAT6 activated by IL-4 promotes the differentiation of T helper (Th) cells into Th2. STAT4 activated by IL-12 promotes the differentiation of Th cells into Th4. Th2 has the activity of anti-atherogenesis [68], while Th1 has the activity of pro-atherogenesis, which mediates the activation of macrophages and promotes the expansion of atherosclerotic plaque [69]. JAK-STAT signaling pathway plays a role in the bidirectional regulation of AS.
4.3. Estrogen receptor in the network topology analysis
In the network topology analysis, ESR1 was in the most critical position in the network with a Degree of 764. We found that ESR1 can regulate some biological processes in the early stage of AS. Estrogen receptor (ESR) inhibits the promoter of IL6 by disrupting the transactivation of NF-kB [70,71], thereby inhibiting the synthesis of C-reactive protein and the proliferation of vascular endothelial cells [72]. ER46, the splice isomer of estrogen receptor, can activate endothelial nitric oxide synthase (eNOS) and promotes NO production [73]. The biological processes such as oxidative modification of low-density lipoproteins, a proliferation of vascular endothelial cells and infiltration of macrophages in AS are inhibited by NO [74]. ESR1 not only occupies the most critical position in topology analysis but also plays an important role in the component-target network. Eighty-five of the 101 effective components of XZD have a direct effect on ESR1. According to the results of network topology analysis, some of the nodes with higher Degree values were not the direct targets of the effective components of XZD. They have protein-protein interaction with the targets of effective components and thus participate in the regulation of AS by XZD. NTRK1 participates in the regulation of angiogenesis and the positive regulation of NF-kB transcription factor activity [75]. FN1 participates in the process of cell adhesion and cell-matrix adhesion [76,77]. However, the correlation between more targets in topological analysis and the pathogenesis of AS needs to be further studied.
The effective components and targets of XZD came from TCMSP and DrugBank databases, and the related genes of AS came from DisGeNET and GEO databases. The research in this paper was based on the known information in the database, and unknown compounds, targets and disease-related genes were not discussed in this paper. Therefore, this paper has certain timeliness.