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ORIGINAL ARTICLE
Year : 2023 | Volume
: 9
| Issue : 1 | Page : 53-60
Elucidation of the mechanism of Gualou-Xiebai-Banxia decoction for the treatment of unstable angina based on network pharmacology and molecular docking
Yu Tan1, Li Chen2, Hua Qu1, Da-Zhuo Shi1, Xiao-Juan Ma1
1 National Clinical Research Center for Chinese Medicine Cardiology, Xiyuan Hospital, China Academy of Chinese Medical Sciences, Beijing, China
2 National Clinical Research Center for Chinese Medicine Cardiology, Xiyuan Hospital, China Academy of Chinese Medical Sciences; Cardiovascular Department, Peking University Traditional Chinese Medicine Clinical Medical School (Xiyuan), Beijing, China
Correspondence Address:
Dr. Xiao-Juan Ma
National Clinical Research Center for Chinese Medicine Cardiology, Xiyuan Hospital, No. 1, Xiyuan Caochang, Haidian District, Beijing City 100091
China
Source of Support: None, Conflict of Interest: None
DOI: 10.4103/2311-8571.364411
Objective: The aim of this study was to identify the potential pharmacological mechanisms of Gualou-Xiebai-Banxia decoction (GLXBBX) against unstable angina (UA). Materials and Methods: The active compounds of GLXBBX were collected from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform, and their targets were predicted using the SwissTargetPrediction database. The targets associated with UA were obtained from the Online Mendelian Inheritance in Man, GeneCards, and Therapeutic Target Database. Individual targets associated with UA and GLXBBX were cross-checked to identify the targets of GLXBBX involved in the treatment of UA. A protein–protein interaction network was built using the STRING online database. Cytoscape 3.7.2 software was used to screen out hub genes. Additional gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses were performed using the clusterProfiler package in R. Results: A total of 28 bioactive compounds and 320 protein targets of GLXBBX associated with UA were screened out. Enrichment analysis indicated that the therapeutic effect of GLXBBX may be mediated through the PI3K/AKT, MAPK, and HIF-1 signaling pathways. Molecular docking suggested that the active compounds including Vitamin E, cavidine, and baicalein can bind to their protein receptors. Conclusions: This research confirmed the multifactorial effects of GLXBBX in the treatment of UA and laid the foundation for the experimental research on GLXBBX.
Keywords: Gualou Xiebai Banxia decoction, molecular docking, network pharmacology, unstable angina
Unstable angina (UA), a severe type of acute coronary syndrome, is the leading cause of death in industrialized western countries.[1] UA results from the rupture of unstable atherosclerotic plaques and the formation of a thrombus, which in turn keeps the ischemic cardiomyocytes in a state of low perfusion.[2] Patients with UA usually experience chest discomfort or pain, occurring frequently or unpredictably, and may develop acute myocardial infarction, which greatly affects their quality of life.[3] Treatments for UA include antiplatelet therapy, antithrombin therapy, nitrate administration, or interventional therapy.[4] However, these therapies are often associated with adverse secondary effects and high costs, imposing a heavy burden on patients.[5],[6] Therefore, a more economical and safer treatment, in combination with standard measures, is required to improve the therapeutic efficacy and quality of life for patients with UA.
Traditional Chinese Medicine (TCM) has been widely used to prevent cardiovascular diseases, including UA.[7],[8] Gualou Xiebai Banxia decoction (GLXBBX) consists of three herbs: Fructus Trichosanthis (dried ripen fruit of Trichosanthes kirilowii Maxim.), Bulbus Allii Macrostemonis (dried bulb of Allium macrostemon Bge), and Pinelliae Rhizoma (dry tuber of Pinellia ternata [Thunb.] Breit.). In TCM, GLXBBX has been confirmed to invigorate qi to relieve depression, dredge yang, and disperse knots, thereby eliminating phlegm and broadening the chest. However, the mechanism of action of GLXBBX against UA remains unknown.
Owing to the complex herbal formulations, it is difficult to investigate the interactions between ingredients and their mechanisms of action. Network pharmacology and molecular docking can reveal the synergistic effects of complex herbal formulations on the human systems. Previous studies suggested that network pharmacology and molecular docking are ideal tools to search for interactions among compounds, genes, proteins, and diseases.[9],[10],[11] These observations led us to analyze the potential mechanism by which GLXBBX attenuates UA.
Materials and MethodsScreening of active ingredients
The active ingredients of GLXBBX were collected from the TCM Systems Pharmacology Database and Analysis Platform[12] (TCMSP, http://lsp.nwu.edu.cn/tcmsp.php).[13] Based on the thresholds of oral bioavailability (≥30%) and drug-likeness (≥0.18), repetitive compounds and compounds that lacked target prediction were eliminated.
Prediction of bioactive compound targets
The SwissTargetPrediction database[14] (http://www.swisstargetprediction.ch) was used to identify the protein targets of the bioactive compounds of GLXBBX.
Collection of unstable angina targets
The terms “Unstable angina” and “Angina, Unstable” were entered into Online Mendelian Inheritance in Man database[15] (http://www.omim.org), GeneCards[16] (http://www.genecards.org), and Therapeutic Target Database[17] (http://db.idrblab.net/ttd/). UniProt (https://www.uniprot.org/) was used to confirm information on UA-related targets, such as protein names and gene IDs.
Construction of a compound-target network
The active components of GLXBBX and UA-related targets were imported into the Cytoscape 3.7.2 platform (https://cytoscape. org/) to build a network for GLXBBX bioactive compounds and protein targets. The connection between nodes in the network was analyzed using the CytoNCA plug-in.
Construction of a protein–protein interaction network
Targets were imported to the STRING database (https://string-db.org/) in the “Homo sapiens” setting to create a protein–protein interaction (PPI) network, which was visualized using Cytoscape 3.7.2.[18] The information on nodes' interrelationships was displayed using the following parameters: degree centrality, betweenness centrality, and closeness centrality [Table 1].
Gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses
Gene ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed[19],[20] using the clusterProfiler 3.14.3 package[21] in R.
Molecular docking simulations
Molecular docking was performed using AutoDock Vina.[22] The structures of the bioactive compounds were collected from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/),[23] and the protein three-dimensional structures were obtained from the Protein Data Bank (http://www.rcsb.org/pdb/home/home.do).[24] Docking results were visualized using PyMoL.
ResultsCompound-target network analysis
A total of 28 candidate compounds were collected from the TCMSP [Table 2], and 320 protein targets of GLXBBX associated with UA were identified. The established “GLXBBX bioactive compound-target network” includes 348 nodes (28 bioactive compounds from GLXBBX and 320 UA-related targets) and 1123 edges, which indicate compound-target interactions [Figure 1]. As shown in [Table 2], the bioactive compounds had degree values >10. Among them, baicalein (degree = 64) had the largest number of associated targets, followed by cavidine (degree = 60) and Vitamin E (degree = 59).
Figure 1: The GLXBBX bioactive compound-target network. Yellow octagons represent Fructus Trichosanthis; red octagons, Bulbus Allii Macrostemonis; blue octagons, Pinelliae Rhizoma; cyan octagons, compounds appearing in different herbs; green triangles, targets of GLXBBX in the treatment of UA. GLXBBX: Gualou Xiebai Banxia decoction, UA: Unstable angina
Table 2: The information of the bioactive compounds of Gualou Xiebai Banxia decoction, Gualou (Fructus Trichosanthis), Xiebai (Bulbus Allii Macrostmonis), Banxia (Pinelliae Rhizoma)Protein–protein interaction network analysis
The obtained targets of GLXBBX involved in UA treatment were entered into the STRING online database (PPI combined score > 0.7) to establish a PPI network [Figure 2]a. The 320 UA-related targets were included in Cytoscape for the analysis. The network topology analysis plugin CytoNCA was used to screen the core PPI network [Figure 2]b. The following thresholds were set: Degree >60 (twice the median), BC >9.81546, and CC >0.96153843. A core PPI network containing 15 targets was established [Table 3]. Based on the degree screening principle, glyceraldehyde-3-phosphate dehydrogenase (GAPDH; degree = 195), RAC-alpha serine/threonine protein kinase (AKT1; degree = 192), interleukin-6 (IL-6; degree = 167), vascular endothelial growth factor A (degree = 159), and caspase-3 (CASP3; degree = 146) were selected as hub genes.
Figure 2: The process of screening core targets. (a) PPI network of GLXBBX against UA was constructed using STRING, (b) Core PPI network of related targets of GLXBBX preventing UA extracted from A, applying the cutoff values indicated in red. DC: Degree centrality, BC: Betweenness centrality, CC: Closeness centrality, PPI: Protein–protein interaction, GLXBBX: Gualou Xiebai Banxia decoction
Table 3: Core targets of treating unstable angina of Gualou Xiebai Banxia decoction and its topological propertiesGene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses
GO enrichment analysis of the 320 UA targets indicated that they are involved in biological processes such as peptidyl-tyrosine/serine phosphorylation and positive regulation of protein serine/threonine kinase activity. The identified cellular components included membrane rafts, microdomains, and regions. The molecular function results demonstrated that these targets may exhibit protein serine/threonine and protein tyrosine kinase activities and bind to phosphatases. KEGG enrichment analysis suggested that the targets were mainly associated with the PI3K/AKT, MAPK, and HIF-1 signaling pathways [Figure 3] and [Figure 4].
Figure 3: Gene ontology enrichment analysis. (a) Bar chart of the top 20 biological processes; (b) bar chart of the top 20 cellular components; (c) bar chart of the top 20 molecular functions
Figure 4: KEGG pathway enrichment analysis. Bar chart of the top 20 KEGG signaling pathways. KEGG: Kyoto Encyclopedia of Genes and GenomesMolecular docking simulations
As shown in [Figure 5], the results indicated that baicalein binds to CASP3, AKT1, and HSP90AA1 (docking scores of −8.3, −8.4, and −9.3, respectively). Cavidine showed a good correlation with CASP3, AKT1, and EGFR (docking scores of −10.2, −9.4, and −8.8, respectively) [Figure 6]. Furthermore, all bioactive ingredients of GLXBBX proved good binding with three hub proteins, GAPDH, CASP3, and AKT1, suggesting that GLXBBX may exert its therapeutic actions in UA prevention through these targets.
Figure 5: Heat map of the docking scores of core targets integrated with key compounds from GLXBBX. GLXBBX: Gualou Xiebai Banxia decoction
Figure 6: The three-dimensional structures of the protein-compound complexes of the three key compounds and the three hub proteins, as obtained by molecular docking. (a) GAPDH-baicalein; (b) GAPDH-cavidine; (c) GAPDH-Vitamin E; (d) AKT1-baicalein; (e) AKT1-cavidine; (f) AKT1-Vitamin E; (g) CASP3-baicalein; (h) CASP3-cavidine; and (i) CASP3-Vitamin E complexes. GAPDH: Glyceraldehyde-3-phosphate dehydrogenase, AKT1: Alpha serine/threonine protein kinase, CASP3: Caspase-3 Discussion GLXBBX is a TCM prescription comprising three herbs: Fructus Trichosanthis, Bulbus Allii Macrostemonis, and Pinelliae Rhizoma. In TCM, UA is considered to be caused by blood stasis that obstructs the heart pulse. GLXBBX has been widely prescribed for the treatment of UA in TCM.[25] However, the mechanism through which this formula alleviates UA progression remains unclear. In recent years, network pharmacology has provided a new approach for exploring potential pharmacological molecular mechanisms in TCM. In this study, 28 active compounds and 320 protein targets of GLXBBX were identified, which may be able to promote its scientific application in clinical settings.
We employed the Cytoscape software to construct a GLXBBX compound-target network for interrelationship analysis. The results demonstrated that most active compounds of GLXBBX acted on numerous targets. For instance, baicalein, cavidine, and Vitamin E interacted with 64, 60, and 59 targets, respectively. These key compounds may play important roles in the pharmacological process. Baicalein, a flavonoid derived from the roots of Scutellaria baicalensis Georgi., has different therapeutic effects, such as an antioxidant action.[26] A previous study reported that baicalein effectively prevents peripheral neuropathy induced by oxidative stress.[27] Cavidine, an active alkaloid isolated from Corydalis impatiens, has been shown to have high anti-inflammatory and antioxidant properties. One study indicated that cavidine ameliorates experimental colitis by negatively regulating the expression of oxygen metabolites and NF-κB and reducing the production of pro-inflammatory cytokines.[28] Vitamin E is an important fat-soluble vitamin and a good antioxidant. Recent investigations have shown that Vitamin E plays a crucial role in reducing inflammation and fibrosis.[29] Moreover, all these ingredients have good oral bioavailability and drug-likeness. Therefore, they may play a critical role in the pharmacological process.
GO enrichment analysis showed that the UA-related targets of GLXBBX may play a significant role in biological processes such as the positive regulation of protein serine/threonine kinase activity, protein autophosphorylation, and protein modification. Both MAPKs and AKTs are members of the serine/threonine protein kinase family and mediate signal transduction from the cell surface to the nucleus. Therefore, the positive regulation of protein serine/threonine kinase activity can lead to the abnormal activation of the signaling pathways mediated by these molecules.[30],[31] Previous studies have reported that once the MAPK/AKT signaling pathway is activated, numerous inflammatory cytokines, crucial for the regulation of inflammation, are released.[32],[33] Inflammation impairs normal endothelial function, accelerates the formation of unstable atherosclerotic plaque, and contributes to plaque rupture and thrombosis.[34],[35] Fibrous cap thickness is one of the most important determinants of plaque vulnerability. Vulnerable atherosclerotic plaques are heavily infiltrated by macrophage foam cells, which release multiple inflammatory mediators and cytokines, such as plasminogen activators, cathepsins, and matrix metalloproteinases; this finally results in matrix degradation, fibrous cap thinning, and plaque rupture, leading to a severe cardiovascular event.[36] It has been shown that monocyte/macrophage differentiation and recruitment are negatively regulated by the selective Mas receptor agonist AVE0991, which suppresses inflammation in the perivascular space in ApoE−/−mice, thus exerting a cardioprotective action.[37] A previous study has found that atherosclerosis exhibits identical properties of disorders of lipid metabolism, protein phosphorylation, and protein modification,[38],[39] consistent with the present study. In addition, GLXBBX might be associated with molecular functions such as serine/threonine protein kinase activity, phosphatase binding, and protein tyrosine kinase activity in the treatment of UA.
KEGG enrichment analysis suggested that the MAPK, PI3K/AKT, and HIF-1 signaling pathways had large numbers of gene enrichment. A previous study has demonstrated that the MAPK signaling pathway is a main mediator of endothelial inflammation and plays a crucial role in the pro-inflammatory maturation of endothelial cells after stimulation by TNF-α.[40] Research has suggested that the PI3K/AKT signaling pathway is closely associated with inflammation and that its activation promotes the expression of pro-inflammatory cytokines.[41] Furthermore, several studies have reported that MAPK and AKT crosstalk with NF-κB signaling pathways, suggesting that they are involved in NF-κB activation.[42],[43] In this study, the HIF-1 signaling pathway was also significantly enriched. One study has shown that hypoxia-induced reduction of cell viability can be reversed by HIF-1-mediated autophagy in H9c2 cells, proving that HIF-1 protects the ischemic myocardium.[44] Another study has suggested that HIF-1 can afford cardioprotection against myocardial infarction by reducing infarcted heart size, improving heart function, and increasing capillary density, in a rat model of myocardial infarction.[45] In addition, GLXBBX may act in other pathways, including apoptosis, platelet activation, and the FoxO and Ras signaling pathways.
According to PPI network analysis, three genes may confer cardioprotection, namely, GAPDH, CASP3, and AKT1. GAPDH is involved in nuclear events, such as apoptosis, transcription, RNA transport, DNA replication, and cytoskeleton assembly.[46] Previous research has shown that apoptotic stimuli, including oxidative or genotoxic stress, result in GAPDH translocation and accumulation into the nucleus, thereby inducing apoptosis.[47],[48] CASP3 plays a crucial role in apoptosis.[49],[50] AKT exerts its anti-apoptotic effects by phosphorylating its target proteins through a variety of downstream pathways.[51] In addition, these three key targets had binding energies lower than −5.0 kJ/mol upon docking with the 13 key compounds, which indicates that these proteins have good affinity and structural stability when bound to these compounds.
ConclusionsOur research revealed potential mechanisms of action of GLXBBX in the treatment of UA. These results highlight the potential for subsequent experimental research.
Financial support and sponsorship
This study was financially supported by the National Natural Science Foundation of China (No. 81774141).
Conflicts of interest
The authors declare that they have no conflicts of interest.
References
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