Network pharmacology-based approach to investigate the mechanisms of Guizhi Fuling pill in the treatment of atherosclerosis

Chang-Lei Hu, Hong-Lin Xu, Zhen-Ye Luo, Tong Xu, Ying-Chun Zhou*

ARTICLE

Network pharmacology-based approach to investigate the mechanisms of Guizhi Fuling pill in the treatment of atherosclerosis

Chang-Lei Hu1,2, Hong-Lin Xu1,2, Zhen-Ye Luo1,2, Tong Xu1,2, Ying-Chun Zhou1,2*

1School of Traditional Chinese Medicine, Southern Medical University, Guangzhou 510515, China;2Department of Traditional Chinese Medicine, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China.

To investigate the mechanism of action of Guizhi Fuling pill in the treatment of atherosclerosis with network pharmacology.The active components of Guizhi Fuling pill were screened by specific ADME features on Traditional Chinese Medicine Systems Pharmacology platform, and their targets were obtained. Targets of atherosclerosis were extracted and screened from GeneCards database, OMIM database, DrugBank database and DisGeNET database, and the intersection of component targets and disease targets were regarded as potential targets. The protein-protein interaction network of potential targets was constructed by STRING database and Cytoscape 3.7.2. GO analysis and KEGG pathway analysis of potential targets were performed by Metascape platform. The “drugs-componenst-targets-pathways” network was constructed by Cytoscape 3.7.2. SwissDock was used for molecular docking of active components and key targets.The main components of Guizhi Fuling pill in the treatment of atherosclerosis were quercetin, beta-sitosterol, kaempfero, hederagenin, baicalein, etc. The key targets mainly included PGST2, RELA, AKT1, etc. Molecular docking analysis showed that the binding energy of the main active components to the core target was less than -7 kcal/mol. The associated pathways mainly included fluid shear stress and atherosclerosis, PI3K-Akt signaling pathway and AGE-RAGE signaling pathway.Through this study, we initially revealed the possible mechanism of Guizhi Fuling pill in treating atherosclerosis through anti-inflammatory, reducing blood lipids, and regulating endothelial function.

Guizhi Fuling pill, Atherosclerosis, Network pharmacology, Molecular docking, Target prediction

We predicted that the main components of Guizhi Fuling pill in the treatment of atherosclerosis were quercetin, kaempfero, hederagenin, baicalein, etc. The key targets mainly included PGST2, RELA, AKT1, etc. And the associated pathways were fluid shear stress and atherosclerosis, PI3K-Akt signaling pathway, AGE-RAGE signaling pathways, etc.

Background

Atherosclerosis (AS) is a chronic vascular disease characterized by lipid deposition and plaque formation. Modern medical treatment mainly includes lipid regulation, antiplatelet, anticoagulation and thrombolytic therapy, etc [1]. In traditional Chinese medicine (TCM), AS is classified as “chest pain”, “true heart pain”, etc. Some scholars summarized its pathogenesis as the mutual accumulation of phlegm and blood stasis, and the treatment is mostly to invigorate blood and nourish Qi, resolve phlegm and remove stasis [2, 3][2, 3].

The source of GuizhiFuling pill is(, an ancient Chinese medical book written by Zhong-Jing Zhang in the Eastern Han Dynasty)It is composed of Guizhi (); Fuling (); Chishao (); Mudanpi (); Taoren (). Pharmacological studies have shown that Guizhi Fuling pill can be anti-inflammatory and analgesic, and it can regulate endocrine, regulate smooth muscle, and regulate blood rheology, etc [4]. TCM colleagues have achieved good clinical efficacy in the treatment of AS with Guizhi Fuling pill, Lin et al. [5] found in a clinical study using Guizhi Fuling capsules to treat carotid AS that Guizhi Fuling capsules has no significant difference in curative effect with western medicine control group and Danshen dripping pill control group, and it is better than the control group and can stabilize plaque, reduce plaque area, improve endothelial function, and antiplatelet activity. But the current research on its mechanism of action is still unclear.

Network pharmacology is a new interdisciplinary subject based on high-throughput screening, computer technology and other methods, integrating system biology and network analysis of biological system [6]. Effect of TCM has the characteristics of multi-component, multi-target and multi-pathway. Network pharmacology coincides with Chinese medicine. Therefore, this study adopts the research method of network pharmacology to explore the mechanism of Guizhi Fuling pill in the treatment of AS, hoping to provide certain reference basis for related clinical and scientific research. The research flow chart was shown in Figure 1.

Materials and methods

Collecting and screening of targets of Guizhi Fuling pill

The chemical components of,,,andwere searched on the TCM Systems Pharmacology platform (TCMSP) (https://tcmspw.com/tcmsp.php) [7], which is a unique systems pharmacology platform of Chinese herbal medicines that captures the relationships between drugs, targets and diseases [8]. Based on two ADME features, drug-like index (DL) ≥ 0.18 and oral bioavailability (OB) ≥ 30%, the active components of 5 TCMs were screened and related protein targets were obtained. The protein names were standardized by UniProt database (https://www.uniprot.org) [9] and the corresponding gene targets were obtained.

Figure 1 Flow chart of research

Collecting and screening of targets of AS

The targets of AS were mainly collected in the GeneCards (https://www.genecards.org) and DisGeNET (https://www.disgenet.org). GeneCards, the Human Gene database can provide comprehensive information on all annotated and predicted human genes and DisGeNET is the one of the largest publicly available collections of genes and variants associated to human diseases. The targets of AS were further explored in OMIM database (http://www.omim.org) and DRUGBANK database (https://www.drugbank.ca) in order to complement the target of AS. Due to the huge number of AS targets, the targets collected in the GeneCards database were screened for targets whose Score value was greater than its median, and the targets collected in the DisGeNET database were screened for targets whose Score value was larger than its average. The number of targetswere appropriately reduced, and targets that were more relevant to diseaseswere screened out. Finally, the targets obtained from the 4 databases were integrated, and duplicate data was deleted.

Construction and analysis of protein-protein interaction network of potential targets

The intersections between targets of Guizhi Fuling pill and targets of AS were used as potential targetsand Venn diagram was drawn using bioinformaticst (http://www.bioinformatics.com.cn). The potential targets were submitted to the STRING database (https://string-db.org) to construct the protein-protein interaction (PPI) network, and explored the interactions between the targets. STRING is a database of known and predicted protein-protein interactions. The interactions include direct (physical) and indirect (functional) associations; they stem from computational prediction, from knowledge transfer between organisms, and from interactions aggregated from other (primary) databases [10]. In order to ensure the reliability of the data, the highest confidence level (highest confidence > 0.9) was selected, and the species was selected as Homo sapiens. PPI network was imported into Cytoscape 3.7.2, and cluster analysis was performed by MCODE to obtain potential function module (sub-networks), and described its function through analyzing its biological process.

Enrichment analysis of potential targets

Metascape integrates more than 40 gene function annotation databases, which can efficiently identify rich biological pathways [11]. The potential targets were submitted to the Metascape platform (http://metascape.org/gp/index.html) and selected< 0.01 for GO analysis and KEGG pathway analysis. The GO analysis includes three parts: biological process (GO-BP), molecular function (GO-MF), and cellular component (GO-CC). The data were used on intelligent online drawing platform (http://www.ehbio.com) to draw the enrichment bubble diagram.

Construction of “drugs-components-targets-pathways” network diagram

The data of drugs, components and signal pathways corresponding to the intersection targets were imported into Cytoscape 3.7.2, and the network diagram of “drugs-components-targets-pathways” was constructed. Network Analyzer of Cytoscape 3.7.2 was used to analyze the parameters of the network nodes to obtain the core components and core targets in the network.

Molecular docking verification

According to the node parameters obtained from “drugs-components-targets-pathways” network, three targets closely related to AS were selected in the top ten degrees of value ranking to conduct molecular docking with the top ten active components. The PDB ID of these three targets and the mol.2 files of the top ten components were found and submitted to swissdock (http://www.swissdock.ch) for docking. Swissdock is an online docking website based on EADock. It adopts a blind docking method, and evaluates the docking effect of components and targets based on the binding energy (ΔG).

Results

Acquirement of targets of active components in Guizhi Fuling pill

Under the screening conditions of specific ADME features, 7 components of, 15 components of, 23 components of, 11 components of, and 29 components ofwere collected, including quercetin, beta-sitosterol, kaempferol, hederagenin, and baicalein etc., from TCMSP. A total of 39 components were obtained, except for the components without targets on TCMSP (Table 1). Among them, 48 targets were obtained for, 22 targets for, 48 targets for, 165 targets for, 95 targets for, and 208 targets were obtained after integration and deletion of duplicate targets.

Acquisition of atherosclerotic targets

In the GeneCards database, 4,346 targets were searched with “Atherosclerosis” as the keyword, and 2,218 targets were obtained with “Atherosclerotic Cardiovascular Diseases” as the keyword. Setting “Score value ≥ the median” as the condition, a total of 3,398 targets were screened. Searched for “Atherosclerosis” in the “diseases” column of the DisGeNET database, and a total of 4,153 targets were obtained. Setting “Score value ≥ the average”, a total of 2,044 targets were screened. 522 related targets were searched from the OMIM database and 88 targets were found in the DRUGBANK database. Integrating all targets and deleting duplicate targets, a total of 3,096 targets of AS were obtained.

Table 1 Components of Guizhi Fuling pill

Table 1 Components of Guizhi Fuling pill (continued)

Note: GZ, Guizhi (); FL, Fuling (); CS, Chishao (Radix Paeoniae Rubra); DP, Mudanpi (Cortex Moutan); TR, Taoren (Persicae Semen); DL, drug-like index; OB, oral bioavailability.

Construction and analysis of protein-protein interaction network of potential targets

Taking the intersection of disease targets and component targets, a total of 151 potential targets (Guizhi Fuling pill-AS targets) were obtained, including APOB, PPARG, CCL2, CRP, PON1, etc. (Figure 2). Then the potential targets were submitted to the STRING platform, and the PPI network was obtained (Figure 3). Imported PPI into Cytoscape 3.7.2 for cluster analysis, and 5 internal sub-networks were obtained (Figure 4), which are mainly concentrated in the biological processes such as transcription factor binding, protein domain specific binding, etc.

Enrichment analysis of Guizhi Fuling pill-AS targets

The potential targets were submitted on the Metascape platform, and the biological process (GO-BP), molecular function (GO-MF), cellular component (GO-CC), KEGG path analysis was carried out respectively, and the enrichment bubble diagram was drawn. The results showed the biological processes that Guizhi Fuling pill mainly involved in the treatment of AS including cytokine-mediated signaling pathway, cellular response to lipid, response to lipopolysaccharide, apoptotic signaling pathway, response to oxidative stress, response to reactive oxygen species, as shown in Figure 5 (A). The molecular functions of targets regulating AS were transcription factor binding, protein domain specific binding, oxidoreductase activity, lipid binding, cytokine receptor binding, as shown in Figure 5 (B). The cell components regulating AS were mainly distributed in membrane region, membrane microdomain, membrane raft, as shown in Figure 5 (C). The related pathways involved in the regulation of AS were fluid shear stress and AS, PI3K-Akt signaling pathway, AGE-RAGE signaling pathway, IL-17 signaling pathway, apoptotic, TNF signaling pathway, as shown in Figure 5 (D).

Figure 2 Venn diagram of Guizhi Fuling pill-AS targets

Figure 3 PPI network of Guizhi Fuling pil-AS targets

Construction of “drugs-components-targets-pathways” network

The relevant data were imported into Cytoscape 3.7.2 to construct a “drugs-components-targets-pathways” network, as shown in Figure 6. Network Analyzer was used to analyze network node parameters. The parameters closeness centrality (0.57526882), degree (116), betweenness centrality (0.45439557) of quercetin were the maximum values, followed by beta-sitosterol with closeness centrality (0.40225564), degree (83), betweenness centrality (0.09069072); then by kaempfero with closeness centrality (0.42971888), degree (49), betweenness centrality (0.10301424) and hederagenin with closeness centrality (0.3590604), degree (28), betweenness centrality (0.03534885). Among the target node parameters, with closeness centrality (0.48089888), degree (29), betweenness centrality (0.10432894), PTGS2 is the target with the best parameters, followed by PGR with closeness centrality (0.36332767), degree (21), betweenness centrality (0.0471069), then RELA with closeness centrality (0.44123711), degree (21), betweenness centrality (0.024454844), and then AKT1 with closeness centrality (0.43407708), degree (21), betweenness centrality (0.01465326). NCOA2, MAPK1, PTGS1, BCL2, CASP3, JUN are all important targets.

Figure 4 Sub-networks in PPI network of GuizhiFuling pil-AS targets

Figure 5 Enrichment analysis of Guizhi Fuling pil-AS targets. (A: GO-BP analysis; B: GO-MF analysis; C: GO-CC analysis; D: KEGG analysis)

Verification of molecular docking

In Swissdock, the active components were used as ligands and the proteins corresponding to PTGS2, AKT1 and RELA related to AS were used as receptors. In this study, the least binding energy of docking sites (ΔG) was selected for statistics. The ΔG of each group was less than -7 kcal/mol in 40 times of molecular docking. The smaller the ΔG, the more energy released, which indicates that the ligand is more tightly bound to the receptor. It is generally believed that the docking is stable when ΔG is less than -5kcal/mol. Therefore, it suggested that the active components in the treatment of AS of Guizhi Fuling pill have good binding activity with three targets. The results were shown in Figure 7.

Discussion

The TCM treatment of AS has unique effect. Based on literature data excavating, some scholars found that the three most supportive drug pairs in Chinese herbs used in both medicine and food in the treatment of carotid AS have[12]. Based on data filtering, Hou et al. [13] found 16 commonly used single herbs in clinical treatment of AS, including,,. Through literature review, Liu [14] found that common drugs for coronary disease includeand; common drugs for arteriosclerotic cerebral infarction includeand; common drugs for lower extremity atherosclerotic disease includeand. Animal experiments also confirmed that Guizhi Fuling pill can effectively improve the levels of serum endothelin-1 (ET-1), nitric oxide (NO), high-sensitivity C-reactive protein (hs-CRP) and the expression of aortic intercellular adhesion molecule-1 (ICAM-1) in AS model rats, optimize the expression of serum oxidized low density lipoprotein (ox-LDL), malondialdehyde (MDA) and superoxide dismutase (SOD) levels, so as to exert the anti-oxidation and anti-AS effect [15].

Figure 6 Network of “Drugs-Components-Targets-Pathways”. (Circle represents Chinese medicine, triangle represents composition, inverted triangle represents pathway, and diamond represents target. Graphic area represents degree.)

Figure 7 Thermogram of molecular docking results

In the above network analysis, quercetin, beta-sitosterol, kaempfero, hederagenin, baicalein are predicted as the major active components. Liang [16] has systematically summarized the relevant research on quercetin in the treatment of AS up to 2018. Various studies have shown that quercetin can reduce blood lipid and anti lipid peroxidation through various ways, so as to achieving the anti-AS effect. Recently, it has been found that quercetin can regulate the activity of liver X receptor (LXR) [17], LXR is a nuclear receptor involved in many physiological processes, and its function is mainly to control cholesterol homeostasis. Oral quercetin treatment can change the composition of gut microbiota and reduce the level of lipid metabolites causing AS [18]. Quercetin may also inhibit the formation of ox-LDL induced foam cells by regulating Mst1 mediated autophagy in RAW264.7 cell [19]. Given quercetin in ApoE gene knockout mice could decrease the lipid accumulation in arterial cavity, serum sICAM-1, IL-6 and aorta VCAM-1 and increase SIRT1 density, it can also inhibit the development of AS by regulating the expression of ABCA1, LXR-α and PCSK9 [20, 21]. Studies have shown that beta-sitosterol is an effective substance for the treatment of hypercholesterolemia, which can reduce cholesterol synthesis at the level of hydroxymethyl cellulose CoA reductase gene expression [22, 23]. Kaempferol can reducegene and protein levels of adhesion molecules such as VCAM-1, integrin, and MCP-1 to inhibit the inflammatory response and combat the progression of AS [24]. Hederagenin can adjust the imbalance of endothelial function by reducing the release of inducible nitric oxide synthase (iNOS) and increasing the content of endothelial nitric oxide synthase (eNOS), inhibit the IKKβ/NF-κB signaling pathway, reduce the release of IL-6, IFN-γ, TNF-α and other inflammatory factors, and ultimately improve the pathological state of AS [25]. Baicalein can reduce the expression of RGS5, PPARδ, MCP-1, NF-κB, inhibit the proliferation of VSMC and promote its apoptosis, which plays a role in antagonizing AS in a certain extent [26, 27]. In molecular docking, the binding energies ΔG of quercetin, beta-sitosterol, kaempferol, Ivy saponin and baicalein with the three targets were all less than 7kcal/mol. Based on the above results, we can conclude that the main components of Guizhi Fuling pill in treating AS are quercetin, beta-sitosterol, kaempfero, hederagenin and baicalein. The mechanism of action was summarized as shown in Figure 8.

The network showed that therapeutic targets in the treatment of AS of Guizhi Fuling pill mainly focused on PGST2, AKT1, RELA and other targets. PTGS-2 is the coding gene of COX-2, the production of COX-2 is one of the core segments in the inflammatory process, and AS is a chronic inflammatory disease. Sun et al. [28] systematically summarized the close relationship between COX-2 and acute coronary syndrome in the aspects of related inflammatory factors, and the pathological basis of acute coronary syndrome is AS. RELA encodes transcription factor P65, which belongs to a substructure of NF-κB family. NF-κB regulates the expression of inflammatory factors such as IL-8 and MCP1. The imbalance of NF-κB signaling pathway is closely related to AS [29]. AKT1 is an expression gene of serine/threonine protein kinase AKT1. AKT1 can promote the secretion of vascular endothelial growth factor, mediate the migration of VSMCs to intima, maintain the stability of plaque, increase the expression of NO, protect the function of vascular endothelial and so on. It participates in the process of AS in many aspects [30]. The results of pathway enrichment also showed that the main pathway of Guizhi Fuling pill in the treatment of AS included PI3K-Akt signaling pathway. In addition, the results of pathway enrichment included fluid shear stress and AS. Qiao et al. [31] had discussed the role of endothelial shear stress in plaque formation, progression and rupture. AGE-RAGE signaling pathway was also one of the results of pathway enrichment. Studies have shown that hypercholesterolemia is positively related to serum AFE and AGE/sRAGE. AS caused by hypercholesterolemia may be induced by AGE-RAGE signaling pathway [32]. Based on the above discussion, we conjecture that Guizhi Fuling pill can treat AS by pathways including fluid shear stress and AS, PI3K-Akt signaling pathway, AGE-RAGE signaling pathway, and targets including PGST2, RELA and AKT1. The mechanism summary was shown in Figure 9. In addition, we submitted the top 15 core targets to STRING website and conducted GO enrichment analysis, all the settings were defaul. 13 targets were enriched in response to lipid, 7 targets were enriched in inflammatory response, and 4 targets were enriched in positive regulation of smooth muscle cell promotion. The result was shown in Figure 10, which was consistent with the mechanism summary in Figure 9.

Figure 8 Summary of the anti-AS mechanism of the main components

Figure 9 Summary of the mechanism of core targets and key pathways in the treatment of AS

Conclusion

Through this network pharmacology study, we can find that the main components of Guizhi Fuling pill can inhibit inflammation, regulate endothelial function, and reduce blood lipids through various mechanisms, so as to achieve the effect of treating AS. In addition, the core targets and key pathways of Guizhi Fuling pill are also closely related to anti-inflammatory, reducing blood lipids and regulating endothelial function. However, limitations exist in network pharmacology. The components of TCM are not simply one added the other. In the process of decocting, we cannot ascertain the interactions between the components; we also ignore the content of drug composition, which hasinevitable impacts on the efficacy. The results of this study need to be verified by further experiment to clarify the effective components, targets and pathways of Guizhi Fuling pill in the treatment of AS.

Figure 10 Red parts represent GO: 0033993, response to lipid. Blue parts represent GO: 0006954, inflammatory response. Green parts represent GO: 0048661, positive regulation of smooth muscle cell proliferation.

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This study was supported by National Science Foundation of China (81973645, 81673805, 81704058, and 81774100), Natural Science Foundation of Guangdong (2019A1515011560), and Traditional Chinese Medicine Bureau of Guangdong Province (20161167, 20201392).

:

AS, atherosclerosis; TCM, traditional Chinese medicine; PPI, protein-protein interaction; ET-1, endothelin-1; NO, nitric oxide; hs-CRP, high-sensitivity C-reactive protein; ICAM-1, intercellular adhesion molecule-1; ox-LDL, oxidized low density lipoprotein; MDA, malondialdehyde; SOD, superoxide dismutase; LXR, X receptor.

:

Chang-Lei Hu, Hong-Lin Xu, Zhen-Ye Luo, et al. Network pharmacology-based approach to investigate the mechanisms of Guizhi Fuling pill in the treatment of atherosclerosis. Drug Combination Therapy 2020, 2 (4): 157–170.

: Shan-Shan Lin.

: 21 August 2020,

30 October 2020,

:05 November 2020

*Ying-Chun Zhou, School of Traditional Chinese Medicine, Southern Medical University, 1838 North Guangzhou Avenue, Jingxi, Baiyun District, Guangzhou 510515, China. Email: zhychun@126.com.

10.12032/DCT2020A029