Network Pharmacology and Molecular Docking Analysis of Shanhaidan granules in treatment of coronary heart disease angina pectoris

Objective To study the pharmacological mechanism of Shanhaidan granules for treatment of coronary heart disease angina pectoris(CHD-AP). Methods The active ingredients and their target genes of Shanhaidan granules were collected from TCMSP and TCMID. CHD-AP-related target genes were collected from GeneCards, OMIM and DurgBank database. The target PPI network was constructed by STRING. PPI network was used for topological analysis and core target screening. The hub target proteins and top core ingredients of Shanhaidan Granule on CHD-AP was analysed for the binding validation experiment by Molecular Docking. 198 active components and 320 potential targets were obtained from Shanhaidan granule, 1208 related targets and 158 joints of CHD-AP were screened. PPI network analysis showed that AKT1, IL-6, VEGF, TNF, MAPK, Caspase-3, MMP, and EGFR were the core and key teagets in treatment of CHD-AP with Shanhaidan granule. KEGG-GO analysis indicated that Shanhaidan granules treated CHD-AP by regulating the TNF signaling pathway, IL-17 signaling pathway, PI3K-Akt signaling pathway, MAPK signaling pathway and apoptosis-related signalling pathway. The results of the molecular docking showed that the core compounds and target proteins had strong binding activity. results predicted of Shanhaidan granule gainst CHD-AP from a holistic perspective, which provided theoretical support for rational clinical application of CHD-AP.


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The potential targets of active compounds were obtained from TCMSP and STITCH databases. All the target gene names were standardized by UniProt knowledgebase and only those of Homo sapiens were reserved for further analysis.

Construction of Chinese medicine-target Network
The Chinese medicine-target network and ingredient-target network by Cytoscape (3.6.0), which explored the relationship among the drugs of Shanhaidan Granule, active ingredients, and corresponding targets.

Collection of related genes associated with CHD-AP
Genes related to CHD-AP were obtained from the databases of GeneCard Database, OMIM Database and DurgBank by searching with the terms "Coronary heart disease angina pectoris" and "Homo sapiens".
The CHD-AP-target network was constructed by Cytoscape (3.6.0).

Protein-protein interaction (PPI) network and clustering analysis
The gene targets of Shanhaidan granule and CHD-AP were analyzed using the STRING database and "Homo sapiens" was limited. Two PPI network were obtained and merged by Cytoscape (3.6.0),which got the targets of Shanhaidan granule against CHD-AP. The network of Shanhaidan granule against CHD-AP was analyzed by Cytoscape (3.6.0). The 20 hub genes with plugcluster Cytohubba by maximal clique centrality (MCC) method were screened.
2.6 Core targets enrichment analysis GO annotation and KEGG pathway enrichment analysis of Shanhaidan Granule against CHD-AP were carried out through the Matescape online database, which got the potential targets, signal pathways, related biological processes, cell components and molecular functions of the effective components in treatment of CHD-AP. The results of GO enrichment analysis and the bubble diagram of KEGG enrichment pathways were drawn by Bioinformatics online tools.

Molecular Docking Veri cation
The 3D structures of hub target proteins were downloaded from the database of RCSB Protein Data Bank. The 3D structures of top 10 ingredients of Shanhaidan Granule were downloaded from the database of TCMSP. AutoDock software was used to modify the structure including removing H2O, adding hydrogen atoms, and calculating charge and atom type. Finally, AutoDock vina software was used for molecular docking.

Putative targets for Shanhaidan Granule
In this study, putative target genes of active ingredients in Shanhaidan Granule obtained from TCMSP database. The target genes of 198 ingredients were collected by TCMSP database and Uniport database. 1474 genes were identi ed, in which 1153 genes of the 15 Chinese medicine overlapped, and a total of 320 genes were identi ed after removing duplation.

Shanhaidan Granule ingredient-target network analysis
The Chinese medicine-target network contained 336 nodes and 1659 edges( Fig. 2A). The ingredienttarget network of Shanhaidan granule contained 519 nodes and 2185 edges (Fig. 2B). A median of 198 candidate compouds was 5 degrees which indicating that most compounds of Shanhaidan granule were affected by multiple target genes. The top 10 effective ingredient according were Quercetin, Kaempferol, Luteolin, Isorhamnetin, 7-O-methylisomucronulatol, Formononetin, Tanshinone iia, Beta-sitosterol, Arachidonic acid and Baicalein. Hence, they might be the crucial effective compounds of Shanhaidan granule according the network (Table 1).

Known therapeutic targets acting on CHD-AP
The therapeutic targets for CHD-AP were collected from the databases of GeneCard Database, OMIM Database and DurgBank. After eliminating the redundancy, a total of 1208 known therapeutic targets in the treatment of CHD-AP were collected in this study. The CHD-AP-target network ( Fig. 3) was constructed by Cytoscape (3.6.0).

PPI network
158 genes were eventually identi ed for Shanhaidan Granule against CHD-AP by PPI network analysis.
The PPI network of Shanhaidan Granule, CHD-AP, and Shanhaidan Granule against CHD-AP related target genes was separately constructed in this study. The network of Shanhaidan Granule putative target genes contained 311 nodes and 5946 edges (Fig. 4A), the network of CHD-AP related target genes contained 1121 nodes and 43958 edges (Fig. 4B), and the network of Shanhaidan Granule against CHD-AP contained 158 nodes and 3511 edges (Fig. 4C). The top 20 targets of core network were showed in Table 2 and Fig. 5. genes of Shanhaidan granule against CHD-AP (Fig. 8). The network showed that the core target genes had larger degree such as AKT1, IL-6, VEGF, TNF, MAPK, Caspase-3, MMP, and EGFR. They might be the key target genes using Shanhaidan granule in the process of treating CHD-AP. All of the above analysis could reveal a new strategy for drug development on CHD-AP.

Molecular Docking
The hub target proteins and top core ingredients of Shanhaidan Granule on CHD-AP was analysed for the binding validation experiment by Molecular Docking. Generally, the lower the b binding free energy, the more stable the binding between the ligand and protein receptor. In recently studies, the ingredient-target interaction with binding energy less than − 5 kcal/mol would deem the effective docking [11,12]. The result of binding energy showed in Fig. 9. According to the results, these targets have a good docking effect with the bioactive ingredients, which indirectly certifed the results of network pharmacology.

Discussion
In this study, 158 targets of Shanhaidan granule on CHD-AP and 355 pathways were identi ed by network pharmacology. The results showed that Shanhaidan granule act a multi-target and multi-pathway on CHD-AP. The most ingredients of Shanhaidan granule were affected by multiple target genes, such as Quercetin, Kaempferol and Luteolin acted on 169, 68 and 64 target genes, respectively. Research shows that quercetin, kaempferol and luteolin are avonoid antioxidants with 2-or 3-phenylchroman structures, which may reduce coronary disease and cancer [13]. Quercetin exhibits the capacity for cardioprotective as inhibition of LDL oxidation, endothelium-independent vasodilator effects, reduction of adhesion molecules and other in ammatory markers, the protective effect on nitric oxide and endothelial function under conditions of oxidative stress [14]. Kaempferol possesses a wide range of therapeutic properties such as antioxidant, anticancer, and anti-in ammatory, which could act as a therapeutic agent in the treatment of cardiovascular diseases [15]. Luteolin possesses antioxidant activity, which may control of both NAFLD and cardiovascular risk [16]. These study shows that these active ingredients are signi cantly associated with CHD-AP, suggesting Shanhaidan granules in treatment of CHD-AP through these.
According to the results of the PPI network, GO and KEGG analysis, AKT1, IL-6, VEGF, TNF, MAPK, Caspase-3, MMP, EGFR and other targets have important implications in the treatment of CHD-AP with Shanhaidan granules. Shanhaidan granule could improve the CHD-AP by acting on in ammation, oxidative stress, apoptosis, vascular development and generation, circulation system and other processes, which regulating by the TNF signaling pathway, IL-17 signaling pathway, PI3K-Akt signaling pathway, MAPK signaling pathway and apoptosis signalling pathway etc..
The basic pathology of CHD involves the dysfunction of both vascular and myocardial processes which was induced and exacerbated by oxidative stress, in ammation, and apoptosis [17]. IL-6 and TNF-α were important risk factors of coronary heart disease, which connects with the severity of coronary heart disease[18]. In coronary atherosclerosis, IL-6 could stimulate the expression of TNF-α, IL-1β and other in ammatory factors which enhanced the in ammatory response and promoted the development of coronary heart disease [19]. In addition, IL-6 could accelerate the development of atherosclerosis by activating JAK-STAT signalling pathway and enhancing the proliferation of vascular smooth muscle cells [19]. In coronary heart disease, TNF-α might increase the expression of monocyte/macrophage tissue factor which could enhance thrombus activity. In atherosclerotic plaques, TNF-α cause plaque instability by enhancing apoptosis and degradation of matrix metalloproteinases (MMP), which aggravated the development of atherosclerosis and angina pectoris [20]. In patients with CHD and heart failure, TNF-α activated the transcription nuclear factor-kappa B which further induce in ammatory and oxidative stress responses [21]. Caspase-3 acted as the convergence of apoptosis-related signal pathways, and activation of Caspase-3 thereby amplify the apoptotic signalling pathway irreversibly [17]. Several studies showed that TNF-α, IL-6 and caspase-3 were closely related to the occurrence of angina pectoris, which can induce in ammatory factors, apoptosis pathway, oxidative stress, and aggravate the myocardial injury [19,20,22]. In the treatment of CHD-AP, Shanhaidan granule might improve the level of in ammation, apoptosis, and oxidative stress by affecting the expression of IL-6, TNF-α, and caspase-3.
VEGF played a key role in the development of angina pectoris, such as promoting in ammation and thrombosis, improving vascular permeability, and promoting angiogenesis. VEGFA could activate downstream signalling pathways, such as MAPK, Akt and eNOS to stimulate angiogenesis [23], and it can improve ischemic myocardium by affecting VEGF signalling pathway [24]. VEGF participated in myocardial cell proliferation, cardiac regeneration and prevent myocardial infarction through activating eNOS activity and promoting the synthesis of NO [25]. Rotllan, N et al. found that VEGF inhibited apoptosis through PI3K-Akt signalling, and promoted endothelial cell proliferation and angiogenesis through mTORc2 and FoxO1 [26].
The concentration of MMP in patients with CHD increased signi cantly [27]. The increase of MMP activity causes progressive cardiac expansion through degraded collagen, which eventually leads to ventricular remodeling, myocardial infarction, and heart failure [28]. MMP-9 could increase the thickness and in ammation of atherosclerotic plaques which caused plaque instability and rupture [29].
MAPK signalling pathway is the common pathway of various extracellular signal transmission including ERK, p38 MAPK and JNK signalling pathways, which could regulate proliferation, apoptosis, in ammation, immune response and other processes [30]. Previous study showed that p38 MAPK was involved in the ischemia, reperfusion, in ammation and apoptosis of myocardial cells, and the activation of p38 MAPK was implicated in the regulation of myocardial cell apoptosis [31,32]. In the pathogenesis of coronary heart disease, MAPK was involved in myocardial hypertrophy, proliferation and apoptosis induced by various stimuli, and the drug targeting MAPK may be useful candidates for the treatment of myocardial hypertrophy [33].
PI3K/Akt signalling pathway played an important role in regulating cardiomyocyte survival and function [34]. Activation of PI3/Akt signalling pathway regulated glucose and lipid metabolism, and protected the heart which affected the occurrence and development of coronary heart disease [35]. In addition, Activation of PI3/Akt could reduce the activities of in ammatory factors such as TNF-α and IL-6, which reduced the in ammatory reaction in cardiovascular diseases [36]. Akt also increased VEGF secretion and mediated eNOS phosphorylation, vasorelaxation and angiogenesis [37].
It has been clinically con rmed that Shanhaidan granule had the effect of promoting blood circulation, removing blood stasis, and replenishing qi and nourishing blood, and could effectively treat CHD [38,39].
A study showed that Shanhaidan granule could relieve the symptoms of coronary heart disease and improve left ventricular diastolic function, which had a good effect on angina pectoris of coronary heart disease [7]. These study showed that Shanhaidan granules had the good effect in treatment of CHD.
Collectively, these results revealed that Shanhaidan granules may decrease in ammation, oxidative stress, and apoptosis, improve vascular endothelial function, and protect cardiovascular. The network and docking analysis showed that Shanhaidan granules may regulate TNF signaling pathway, IL-17 signaling pathway, PI3K-Akt signaling pathway, MAPK signaling pathway and apoptosis signalling pathway by acting on AKT1, IL-6, VEGF, TNF, MAPK, Caspase-3, MMP, and EGFR to treat CHD-AP. The results of molecular docking veri ed that the present network pharmacology method was reasonable, however, the prediction of targets and mechanism require further clinical validation.

Conclusions
This study revealed the potential pharmacological mechanism of Shanhaidan granule in the treatment of CHD-AP at a system level. The results showed that the anti-angina effect of Shanhaidan granule was played through regulating multi-targets by multi-components, which provided theoretical support for rational clinical application of Shanhaidan granule against CHD-AP. Moreover, we hope that our study will be useful for Anti-angina drug discovery.     The top 20 hub genes of PPI network. Top 20 targets-pathway network. The blue hexagon represent the signal pathways, the pink diamond represent the the targets. The color of nodes represents the value of degree centrality