**3. Discussion**

It is well known that TCM has complex component systems, and their mechanisms are often difficult to explain using existing techniques. This difficulty greatly limits the globalization of traditional Chinese medicine. Effective study of the holistic effects of complex component systems has become a huge challenge. However, "Network pharmacology" has offered a new way to solve this problem [23,24]. Using system biology and computer technology, network pharmacology builds a "disease–gene–target–drug" interaction network,

and systematically displays the impacts of drugs on the disease network [25]. Therefore, compared with the conventional single-target mode of action, network pharmacology is more suitable for studying the complex effects of TCM and has been rightly called the "next-generation drug development model". However, most of the network pharmacology studies in TCM are based on the results of network pharmacological analysis to explain the complexity of Chinese medicine, and there is a lack of mature theoretical guidance for subsequent experimental studies. Hence, this study proposes a Network pharmacology-driven strategy to investigate the molecular mechanism of TCM, with the hope of enhancing the precision of experimental research.

In this study, most of the components of DGS, including phenols, tanshinones, and flavonoids, were identified using HPLC-qTOF/MS. The phenolic compounds in Danshen have been reported to promote myocardial ischemic angiogenesis [26]. Mass spectrometry asserted the presence of salvianolic acid A, B, C, L, and other phenolic acids in DGS. Flavonoids are present in almost all plants and studies have shown that they can inhibit cardiac injury through a variety of mechanisms [27]. Mass spectrometric detection revealed the presence of rutin, hyperoside, and other flavonoids in DGS. The mechanisms by which these components acted in the treatment of CAD were investigated. Network pharmacology was used to analyze the action network of DGS, which indicated that the targets of DGS action are mainly enriched in biological processes related to cytokine regulation, protein phosphorylation, activation of the MAPK signaling pathway, and cell proliferation. Subsequently, KEGG pathway enrichment analysis revealed that the highest enrichment of targets was in the PI3K-Akt signaling pathway. A study has reported that the PI3K-Akt pathway plays a key role in the emergence, progression, and treatment of CAD, thereby activating downstream pathways that control cell survival, proliferation, migration, and other biological processes after receiving intracellular and extracellular feedback [28]. As an effector downstream of Akt, eNOS can be activated to control endothelial cell survival, proliferation, apoptosis, and intravascular environment stability after the development of coronary lesions in the heart [29,30]. Additionally, activation of downstream eNOS contributes to myocardial angiogenesis, which is similar to the effect of VEGF in promoting therapeutic angiogenesis in CAD [31]. It has been reported that Danhong injection can promote the growth of vessels in the myocardial infarct site by activating the ERK signaling pathway [32]. Therefore, the phenotypic and signaling pathway mechanisms were validated in subsequent studies.

The zebrafish is an emerging internationally recognized model animal. During zebrafish embryonic development, angiogenesis can be visualized under a microscope and is currently considered one of the best models for studies on angiogenesis [33,34]. Specifically, the transgenic Tg (flk1a:EGFP) zebrafish with green fluorescence of the vascular can be observed via video and imaged under a fluorescence microscope with great convenience to assess the effect of the drug on vessel growth [35]. All mammalian and zebrafish endothelial cells are extremely plastic and retain their plasticity even after reaching adulthood. After enduring cardiac injury, mice can form new coronary arteries from pre-existing endothelial cells [36,37]. The zebrafish vascular growth model is ideally suited as it mimics human vascular disease models for new drug screening and evaluation [38]. Our experimental results established that DGS can promote vascular growth in vivo by increasing the vessel length and the number of vascular outgrowths, without any effects on zebrafish development, at the effective dose.

The findings from network pharmacology and zebrafish experiments suggest that effective angiogenesis can enhance blood perfusion in the case of CAD and alleviate cardiac injury. Therefore, the angiogenic mechanisms of DGS in vitro were further investigated. Endothelial cells are involved in angiogenesis at the lesion site mainly via two pathways: proliferation and migration [39]. The CCk-8 assay demonstrated that DGS significantly increased the viability of HUVECs, which established that DGS could promote the proliferation of HUVECs. The ability of DGS to promote the migration of HUVECs was then assessed using the wound healing assay [40], transwell assay [41], and tube formation

assay [42]. The results showed that DGS significantly stimulated the migration of HU-VECs towards the scratch region and the lower transwell compartment and significantly stimulated the tube formation ability of HUVECs.

Furthermore, the levels of VEGFA and NO secretion in HUVECs were measured, and angiogenic and diastolic-associated proteins were quantified using Western blot. NO is a key cell-signaling molecule that is produced by activated eNOS catalyzing L-arginine[43]. Activation of eNOS and release of NO will induce the vasodilation of vessels, and NO is necessary to maintain endothelial cell function [44]. In this study, the secretion of NO and the phosphorylation level of eNOS in HUVECs were measured, which signified that DGS significantly upregulated p-eNOS and stimulated the secretion of NO. Moreover, NO is one of the main mechanisms that promote angiogenesis [45]. Our results suggest that DGS can stimulate the secretion of VEGFA to upregulate VEGFR2, thereby accentuating the binding of VEGFA to VEGFR2. ERK and Akt are located downstream of the VEGF/VEGFR2 pathway, and it has been shown that the phosphorylation of Erk and Akt can further mediate the proliferation and migration of HUVECs, thereby promoting angiogenesis [46]. The results allude that DGS may promote angiogenesis and vasodilation by promoting the phosphorylation of Akt and Erk. As shown in Figure 8, DGS activates the downstream Akt/Erk/eNOS pathway by promoting the binding of VEGFA to VEGFR2. This activation ultimately releases NO to expand blood vessels and mediates the proliferation, migration, and angiogenesis of HUVECs. The dynamic molecular docking results showed that the key compounds in DGS exhibit a good binding capacity for VEGFR2, specifically salvianolic acid C, which may be an important agonist of VEGFR2 and activate downstream signaling pathways.

**Figure 8.** DGS participates in the overall regulatory network of CAD inhibition through angiogenesis and vasodilation.
