Hypertension is a multifactorial disease affected by the complex interactions between genetic predisposition and environmental factors.[4] There have been several successful experiences in the treatment of cardiovascular diseases such as hypertension with GZD.[9, 10, 20] However, its material basis and potential mechanism have not been fully elucidated. In the present study, we initially identified key compounds, hub targets, and main biological processes and pathways of GZD against hypertension by network pharmacology analysis, and then examined the therapeutic effects of GZD on hypertension in the Dahl salt-sensitive rat model. Our integrative approach uncovered the potential mechanisms of GZD against hypertension from the systematic network perspective and demonstrated that GZD can effectively attenuate elevated blood pressure, improve both inflammatory cell infiltration and myocardial fibrosis, and inhibit the expression levels of IL-6, CCL2, IL-1β, MMP-2, and MMP-9 in the Dahl salt-sensitive rats.
In this study, 112 active compounds of GZD were identified from the TCMSP database, and we screened out three key compounds by the network analysis that might have potential anti-hypertensive function. The three key active compounds were β-sitosterol, kaempferol, and quercetin. Moreover, these three key compounds exhibited optimal binding affinity in molecular docking, indicating that they might play a crucial role in the anti-hypertensive effects of GZD. β-sitosterol is a type of phytosterol that exerts protective cardiovascular effects mainly by increasing the intracellular antioxidant defense, improving endothelial function, and inhibiting serum cholesterol levels.[21–23] Kaempferol has shown to be effective in maintaining blood pressure by effectively repressing the generation of inflammatory cytokines and apoptosis, stimulating the release of NO from the vascular endothelium, and decreasing myocardial fibrosis by inhibiting proliferation of cardiac fibroblasts.[24–26] Quercetin exerts remarkable effects in the treatment of hypertension by improving the endothelial function by increasing NO bioavailability and NO production, inhibiting the overactivation of RAAS, and reducing the generation of the adhesion molecules and other inflammatory factors.[27, 28] Overall, these observations suggest that the key compounds of GZD could be crucial in the treatment of hypertension.
In the PPI network, we observed that IL-6, CCL2, IL-1β, MMP-2, and MMP-9 were the hub targets of the GZD for improving hypertension. Notably, these hub targets were closely related to various compounds and biological processes and pathways based on the analysis of multi-layered networks, suggesting that these targets may be important in the role of GZD against hypertension. Several studies showed that a long-term inflammatory response can trigger the sympathetic activation and result in myocardial fibrosis and endothelial dysfunction.[29] Thus, the inhibition of inflammatory response can effectively delay or control the development of hypertension and severe complications.[30] The inflammatory process of hypertension is characterized by increased levels of local inflammatory cytokines such as IL-6, IL-1β, TNF-α, and ICAM-1, which are highly correlated with increased risk of hypertension, and they could be useful diagnostic tools for hypertension in the future.[4] Particularly, IL-6, a well-known pro-inflammatory cytokine, participates in the pathological process of hypertension by promoting endothelial dysfunction and inflammatory cell recruitment.[31] The inhibition of IL-1β could inhibit the overactivation of RAAS and decrease overproduction of other pro-inflammatory cytokines, thereby improving hypertension and cardiac fibrosis.[32, 33] CCL2 is a chemokine that contributes to progression of hypertension by recruiting the circulating monocytes to the blood vessel walls and promoting macrophage infiltration.[34] Myocardial fibrosis is a crucial pathological feature in the progression of hypertension, and it is predominantly related to an excessive accumulation of extracellular matrix proteins that contribute to increased ventricular wall stiffness and impaired diastolic function.[32, 35] MMPs participate in the development of myocardial fibrosis by regulating the degradation and production of collagen, and higher levels of MMP-2 and MMP-9 were considered as markers of cardiovascular risk and aberrant accumulation of collagen.[36, 37] As expected, our experimental results showed that GZD can reduce the degree of inflammatory infiltration and area of interstitial fibrosis, as well as downregulate the protein and mRNA levels of IL-6, IL-1β, CCL2, MMP-9, and MMP-2 in Dahl salt-sensitive rats. These results were in line with earlier reports that showed that the improvement of cardiac inflammation and fibrosis was associated with decrease in blood pressure.[10, 38]
Based on the results of the network analysis and molecular docking, several targets such as AKT1, VEGFA, eNOS, ICAM-1, PTGS2, and ALB could be associated with the potential effect of GZD on hypertension. For example, AKT1 directly participates in the phosphorylation of eNOS at serine 1177, which could increase enzyme activity, NO production, and angiogenesis.[39] Vascular endothelium is implicated in the regulation of vascular tone and structure, and the abnormal vascular endothelial function could be a major contributor to the adverse outcomes of hypertension.[4, 40] VEGFA serves as a homologue of the VEGF family and regulates cell migration, division, and angiogenesis regulation in normal microvascular endothelial cells.[41] Several studies have provided evidence that eNOS is a significant contributor in the maintenance of vascular function and cardiovascular homeostasis.[42] NO, produced by eNOS, mediates control of the inflammatory process as well as regulation of neoangiogenesis and vasodilatation. The decrease in bioavailability of NO has been implicated as a major cause of endothelial dysfunction in hypertension.[4] ICAM-1 promotes the adhesion of leukocytes and vascular endothelial cells, and subsequently leukocyte activation, which probably triggers the endothelial dysfunction, inflammatory response, and blood-vessel remodeling.[32, 43] PTGS2-derived products have been proven to be extensively linked with the regulation of fluid balance, endothelial function, and ROS production.[44]As a major protein in human serum, the low level of serum ALB is associated with hypertension, increased risk of cardiovascular disease, and carotid atherosclerosis.[45] These targets need to be verified in follow-up experiments.
In the KEGG pathway analysis, the overlapping targets were related to multiple pathways, such as TNF signaling pathway, HIF-1 signaling pathway, TLR signaling pathway, insulin resistance, PI3K-AKT signaling pathway, and NF-ĸB signaling pathway. These signaling pathways play a significant role in the pathogenesis and management of hypertension. The activation of the TNF signaling pathway is an important contributor to inflammatory processes, which plays an essential part in modulating the gene expression of many cytokines and chemokines involved in vascular inflammation and remodeling.[46] In the inflammatory process associated with hypertension, the inappropriate activation of the NF-kB pathway facilitates disease progression by inducing inflammatory cytokine release, vascular dysfunction, and generation of reactive oxygen species.[32, 47] The TLR signaling pathway, a critical upstream mechanism activating inflammatory signaling, regulates the inflammatory response by directly promoting the release of a variety of inflammatory mediators, inducing the migration of immune cells to inflammatory sites, and increasing the adhesion and infiltration ability of inflammatory cells.[29, 48] The HIF-1 pathway is highly correlated with energy metabolism and angiogenesis, and participates in the pathophysiology of inflammation and ischemia. HIF-1 is the chief hypoxia-regulated transcription factor that regulates cellular responses in hypoxic and ischemic conditions.[49, 50] The PI3K/AKT pathway plays a seminal role in regulating multiple biological effects, including cell growth and proliferation, apoptosis, and angiogenesis.[51] The PI3K/AKT pathway could activate the phosphorylation and activation of eNOS, and maintain blood pressure homeostasis, endothelium function, and vascular integrity.[52] Insulin resistance is a risk factor in hypertensive patients that closely correlates with the activation of RAAS and SNS, resulting in increased peripheral vascular resistance and circulating plasma volume.[53] Therefore, the improvement of insulin resistance would be significant in the management of hypertension and its complications.[54] However, the effects of these signaling pathways in the mechanisms of GZD against hypertension need to be validated through rigorous investigations.
Because of the limitations regarding screening conditions of database and statistical software, several ingredients and targets of GZD against hypertension may have been missed during the screening process. Despite the limitations of this study, our results revealed the potential mechanism of GZD against hypertension, and provided scientific basis and valuable enlightenment for guiding future in-depth research and clinical applications.