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ORIGINAL ARTICLE
Year : 2023 | Volume
: 9
| Issue : 2 | Page : 150-159
Using network pharmacology and molecular docking tools to investigate the potential mechanism of ephedra-gypsum in the treatment of respiratory diseases
Can Huang1, Ling Yuan2, Yang Niu3, Ya-Ting Yang1, Yi-Fan Yang1, Yi Nan3, Hong-Li Dou4, Joanna Japhet2
1 School of Chinese Medicine, Ningxia Medical University, Yinchuan, Ningxia, China
2 Pharmacy College of Ningxia Medical University, Yinchuan, Ningxia, China
3 Key Laboratory of Ningxia Minority Medicine Modernization Ministry of Education, Ningxia Medical University, Yinchuan, Ningxia, China
4 School of Marxism, Shaanxi University of Chinese Medicine, Xianyang, Shaanxi, China
Correspondence Address:
Dr. Hong-Li Dou
School of Marxism,Shaanxi University of Chinese Medicine, Xianyang, Shaanxi
China
Dr. Yi Nan
Key Laboratory of Ningxia Minority Medicine Modernization Ministry of Education, Ningxia Medical University, Yinchuan, Ningxia
China
Source of Support: None, Conflict of Interest: None
DOI: 10.4103/2311-8571.378172
Objective: The objective of this study was to investigate the potential mechanisms of ephedra-gypsum in the treatment of respiratory diseases (RDs) using network pharmacology and molecular docking techniques. Materials and Methods: The TCMSP and UniProt databases were used to mine the active components and targets of ephedra-gypsum, and the targets of RD were screened using the Online Mendelian Inheritance in Man (OMIM) and GeneCards databases. The protein-protein interaction network graph was created using the drug-disease intersection targets in the STRING database. The network diagram was analyzed using Cytoscape 3.9.1's topology function. The gene ontology (GO) and KEGG enrichment analyses were performed using the DAVID platform. Molecular docking bioactivity validation of the main active components and core targets was performed using AutoDock and PyMOL software. Results: Twenty-four compounds were screened, and 113 drug-disease targets overlapped. In total, 358 biological processes, 67 molecular functions, 38 cellular components of GO, and 139 pathways were identified. Molecular docking analysis demonstrated the strong binding ability of tumor protein 53 (TP53)-luteolin. Conclusion: The core components of ephedra-gypsum, such as quercetin, luteolin, kaempferol, and CaSO4·2H2O, act on key targets, such as tumor necrosis factor (TNF), interleukin-6 (IL-6), TP53, and IL-1 β through cytokine-mediated signaling pathways, inflammatory responses, cell proliferation, and apoptosis. This could be useful for the treatment of RD.
Keywords: Ephedra-gypsum, mechanistic studies, molecular docking, network pharmacology, respiratory diseases
Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2, is a highly contagious and dangerous disease that seriously endangers human life and health. The global scientific community is paying increased attention to the occurrence and development of respiratory diseases (RDs), as well as to the actions of medication. The main diseases include asthma, lung cancer, tuberculosis, and chronic obstructive pulmonary disease (COPD). Most lung diseases eventually lead to pulmonary ventilation dysfunction and respiratory failure, and their rapid progression and high mortality rates have caused massive global health problems.[1] Antibiotics, theophylline, and other drugs commonly used in clinical practice have some clinical adverse effects and resistance to long-term use, which is detrimental to the treatment and recovery from the disease. Therefore, there is an urgent need to discover drugs with a fewer side effects and higher efficacy.
The traditional Chinese medicine (TCM) herb-pair of ephedra-gypsum is derived from the treatise on febrile diseases, in which the combination of ephedra and gypsum is widely used for treating RD,[2] such as cold symptoms and bronchial asthma in the clinical practice of TCM. Ephedra-gypsum is also a high-frequency combination used in TCM treatment protocols for the treatment of COVID-19, issued by provincial and municipal health commissions in the chain.[3] Ephedra can release sweat and relieve exopathic symptoms, diffuse the lung, calm asthma, and reduce water retention and swelling. Gypsum can be used to clear heat and fire, quench thirst, and relieve dysphoria. Ephedra and gypsum are used together to enter the lung meridian, with gypsum helping ephedra to exorcise heat and diffuse the lung and ephedra gaining access to gypsum to diffuse stagnant heat and diuresis.[4] This herb-pair is used together for mutual regulation and benefit to the Lung Qi.
Chinese herbs have “multi-component, multi-pathway, multi-target, and overall control” characteristics, when acting on the human body, which is very consistent with the systematic analysis of “drug, target, pathway, and disease” in network pharmacology. This study explores the mechanism by which ephedra-gypsum components affect RD based on network pharmacology and molecular docking technology, screens the primary active components and predicts their targets action, analyzes the key targets and pathways, and offers a theoretical and scientific foundation for the intervention of ephedra-gypsum in RD. The graphical abstract is as follows [Figure 1].
Figure 1: The graphical abstract of the study. GO: Gene ontology, OMIM: Online Mendelian Inheritance in Man, DL: Druglikeness Materials and Methods Screening key active components and targets
The active components were first acquired in terms of oral bioavailability (OB) ≥30% and druglikeness ≥0.18. We identified the action target of ephedra on the TCMSP platform. Because gypsum was not found in the TCMSP, the key active components for “gypsum” were extracted from the TCMID database (http://www. tcmip.cn/). The screened compound target proteins were converted into target gene designations in the UniProt database (https:/www.UniProt.org).
Predicting potential targets of respiratory disease
The disease targets were searched using the search term “RDs” in the Online Mendelian Inheritance in Man (OMIM) and GeneCards databases, respectively, where RD-related targets with a relevance score >10 were searched in the GeneCards database. After combining all targets, duplicate data were deleted to obtain the disease target.
Drug-disease co-targets' acquisition
Both component and disease intersection targets were mapped through Venny 2.1.0 (https://bioinfogp.cnb.csic.es/tools/venny/index.html). Intersection is a co-target of drugs and diseases.
Constructing co-targets' protein-protein interaction network
Protein–protein interaction (PPI) was plotted after the co-targets were entered into the STRING database (https://string-db.org/). When conducting PPI analysis, the biological species was set as “Homo sapiens,” and the minimum interaction threshold as “confidence level >0.7.” Other settings were retained at the default values to obtain the interaction of the target. The obtained TSV file was imported into Cytoscape 3.9.1 software(This software was developed by the U.S. National Institute of General Medical Sciences (NIGMS), 45 Center Drive MSC 6200 Bethesda, MD.) for network topology analysis and visualization.
Analysis of gene ontology and KEGG enrichment
To better elucidate the potential molecular mechanisms of ephedra-gypsum, co-targets were identified using DAVID (https://david. ncifcrf.gov/). We selected the species as “Homo sapiens” for gene ontology (GO) and KEGG enrichment analysis. The aim of this study was to identify the biological functions and molecular pathways of these targets.
Constructed the “active component-respiratory disease target-pathway” network diagram
The above results were used to filter out the active components, core targets, and signaling pathways, and were inputted to Cytoscape 3.9.1 software to draw the “active component-targets-pathway” network diagram. The Network Analyzer function built into Cytoscape 3.9.1 was used to derive the network topology parameters of the active components and to infer the main active components that exerted their effects, thereby analyzing the mechanism of ephedra-gypsum in treating RD.
Molecular docking validation
The active components with a higher degree in the “active component-targets-pathway” network graph and targets with a higher degree in the PPI were verified by molecular docking. The specific steps are as follows: (1) download the SDF format files of the active components with high network degree from the PubChem website; (2) download the pdb format files of the target proteins with a high network degree from the PDB database (http://www.rcsb.org/); and (3) Use AutoDockTools 1.5.6 software to convert the standard proteins and core compounds into pdbqt file format and use the raw ligands to find the active pocket of the standard protein. Operations such as hydrogenation and charge calculation on ligands and receptors, and debug functional box sizes were performed. Docking was performed, and the docking score was used to evaluate how well the target and active components bind to each other. Finally, visualization was performed using PyMOL 2.5 software.
ResultsResults of key active components screening
The active components of ephedra were identified using TCMSP. TCMID acquired one active component of gypsum: calcium sulfate-containing water (CaSO4·2H2O). A total of 24 active components were identified in this manner [Table 1]. By combining and deduplication, 182 ephedra targets and nine gypsum targets were obtained from the UniProt protein database, making an aggregate of 191 targets.
Results of access to potential targets for disease
Two thousand six hundred and eighteen disease targets were acquired by searching the GeneCards database for RD-related targets with a relevance score >10, after combining and deduplicating the targets retrieved from the OMIM database.
Drug-disease co-targets' acquisition
A Venn graph was created by intersecting the ephedra-gypsum active component targets with the RD targets on the Venny 2.1.0 website [Figure 2]. A total of 113 drug-disease co-targets were identified.
Figure 2: Venn graph of “component-target” and “disease-target” intersectionThe protein-protein interaction network of co-targets was constructed
We imported 113 drug-disease intersection targets into the STRING database to obtain a target PPI network. The PPI information was submitted to Cytoscape 3.9.1 software as tsv files to draw the PPI network [Figure 3]. The larger the number of nodes and thicker the lines, the more grounded the objective connection relationship. PPI network data were analyzed using the built-in plug-in of Cytoscape 3.9.1 software, and the strongest relationships were found for tumor necrosis factor (TNF), AKT1, interleukin-6 (IL-6), tumor protein 53 (TP53), and IL-1 β in terms of Degree, Betweenness, and Closeness thresholds [Table 2], which were distinguished as the core targets.
Figure 3: The PPI network of 113 target genes. PPI: Protein-protein interaction, IL: Interleukin, TP53: Tumor protein 53, TNF: Tumor necrosis factor
Table 2: Topological properties of top 20 core targets in protein interactionResults of GO and KEGG enrichment analysis
In total, 113 co-targets were entered into the DAVID website for GO gene enrichment analysis. With P < 0.01, a total of 358 biological process were obtained, mainly involved in signaling pathways, positive regulation of transcription from RNA polymerase II promoter, positive regulation of gene expression, regulation of DNA-templated transcription, negative regulation of apoptosis process, signal transduction, regulation of cell proliferation, drug response, and inflammatory response; molecular function: 67 were obtained, mainly involved in protein binding, enzyme binding, macromolecular complex binding, and DNA binding; and cellular component: 38, mainly involved in the cytosol, nucleus, cytoplasm, and extracellular region. The top 10 results were ranked from the highest to the lowest based on the number of related genes from high to low [Figure 4]. A total of 139 pathways were mapped and arranged in descending order by the number of related genes, including pathways in cancer; PI3K-Akt, AGE-RAGE, MAPK, and TNF signaling pathways, among others, were ranked in the top 20 pathways [Table 3]. The results are shown as a bubble plot [Figure 5].
Figure 5: KEGG pathway analysis results bubble chart (top 20 terms). IL: Interleukin
Table 3: The Kyoto Encyclopedia of Genes and Genomes enrichment result (top 20 terms)Create the “active component-respiratory disease target-pathway” network diagram
The top 20 pathways were selected from KEGG enrichment analysis. The network diagram of the “active component-core target-disease-pathway” was created using Cytoscape 3.9.1 [Figure 6]. The larger the area of each graph, the stronger the regulatory role of the node in the network. The built-in analysis tool Cytoscape 3.9.1 was used to analyze the topological parameters of the ephedra-gypsum intervention RD network to obtain the core components [Table 4].
Figure 6: Network diagram of “Active component-RD target-pathway.” (○: Active components,□: RD target,△: pathway). RD: Respiratory diseaseMolecular docking results
The core components quercetin, luteolin, kaempferol, and CaSO4·2H2O obtained from network pharmacology screening were validated by molecular docking with the key targets TNF, AKT1, IL-6, TP53, and IL-1 β [Table 5]. The PyMOL software was used to visualize a portion of the outcomes with a high binding energy [Figure 7]. According to molecular docking, the binding energy of the target and core components was <−4.25 kcal·mol−1, accounting for 65%, indicating that both the components and the targets had a certain binding activity.
Figure 7: Molecular docking. (a) TNF-luteolin; (b) TP53-kaempferol; (c) TP53-luteolin; (d) TP53-quercetin; (e) AKT1-kaempferol; (f) AKT1-quercetin. TP53: Tumor protein 53, TNF: Tumor necrosis factor Discussion From these results, it is clear that quercetin, luteolin, kaempferol, CaSO4·2H2O, and other important active components can be helpful in the treatment of RD. Respiratory symptoms such as cough, sputum, and wheezing are associated with chronic inflammation of the airways. Quercetin, the active component of ephedra, has great expectorant, cough-suppressing, and wheezing effects and is generally utilized in the treatment of chronic bronchitis. Quercetin can prevent the expression of inflammatory factor IL-6, restrain the spread of inflammatory cells,[5],[6] reduce COPD and systemic inflammatory response,[7] and repress lung fibrosis[8] by downregulating the PI3K-AKT and NF-κB signaling pathways. The proliferation and apoptosis of lung cancer cells are related to the PI3K-AKT signaling pathway, and quercetin can exert anti-lung cancer effects by modulating the PI3K-Akt and MAPK pathways, inducing apoptosis of lung cancer cells,[9] inhibiting enzyme activity, blocking the cell cycle, and reversing tumor drug resistance.[10] Luteolin also has anti-inflammatory, cough suppressant, and expectorant effects. RD generally causes lung epithelial cell damage,[11] promotes lung epithelial cell proliferation, and inhibits apoptosis by increasing epithelial sodium channel protein expression in acute lung injury,[12] thus providing protection against acute lung injury,[13] which can better control serum inflammatory cytokines in stable COPD patients, and has a good clinical effect in the recuperation of lung function.[14] Luteolin can inhibit the proliferation, migration, and apoptosis of nonsmall cell lung cancer and plays an anticancer role.[15] Kaempferol has cough suppressant, enzyme inhibitor, antioxidant, antibacterial, antiviral, anti-inflammatory, and antitumor activities, and can protect normal cells and promote their regeneration and development. Research has found that kaempferol modulates the oxidative/antioxidative lopsidedness induced by acute lung injury and inhibits NF-κB and MAPK signaling pathways,[16] thereby restricting the expression of NF-κB, IL-1 β, and TNF-α to alleviate acute lung injury,[17] improving inflammatory response,[18] fibrous deposition, oxidative activity, etc., thus slowing down the process of lung fibrosis.[19] Kaempferol is also a dietary flavonoid that inhibits tumorigenesis, inhibits the formation of lung cancer cells, and repairs lung damage. All the above three active components are flavonoids, which can inhibit respiratory inflammation through the TNF signaling pathway, promote cell growth, differentiation, and apoptosis, and reduce mucus secretion, thus relieving cough and expectorant effects. Moreover, the TNF signaling pathway can obstruct the binding of angiotensin-converting enzyme 2 (ACE2) to the COVID-19 spike protein receptor and can inhibit novel coronavirus pneumonia.[20] Pneumonia is one of the most common infectious diseases, and the most well-known type of fever is infectious fever, which is the primary manifestation of pneumonia. Gypsum is made from CaSO4·2H2O; the calcium reduces vascular permeability and has anti-inflammatory and anti-allergic effects. Gypsum can inhibit the hyperactivity of the thermoregulatory center, which in turn produces a powerful antipyretic effect, regulating the signaling of fever cells and inhibiting the sweating center, thus relieving fever without sweating or harming fluid.[21] In summary, the main active components of ephedra-gypsum possess anti-inflammatory, antioxidant, and cough suppressant properties.
In the PPI data, the core proteins TNF, IL-6, and IL-1 β are pro-inflammatory cytokines, and severe inflammation causes TP53 to induce apoptosis. The TNF family directly kills tumor cells and some virus-infected cells, resulting in cell death or apoptosis. It also exerts antitumor effects by stimulating the production and protection of normal cells while activating the immune system.[22] TNF can cause several target genes to circulate positively with pro-inflammatory responses after clinical validation.[23] Lung injury is further exacerbated by the production of increased levels of TNF-α, a member of the TNF family, which activates caspases to fulfill their biological functions of antiviral, immunomodulatory, and apoptotic induction. The upregulation of TNF-α plays a key role in lung inflammation,[24] which can induce the secretion of various inflammatory mediators such as IL-1 and IL-6, thereby damaging cells and tissues.[25] Therefore, blocking TNF-α function using antibodies can reduce the damage caused by a lack of alveolar surface-active substances.[26] IL-6 is released during inflammatory infections and mediates the febrile response to raise body temperature, which can prompt the body to repair itself and achieve antipyretic and anti-inflammatory aims, and can be used as a target for nonsteroidal anti-inflammatory drugs.[27] IL-6 expression is helpful for the early assessment of viral pneumonia because its levels are closely related to the severity of infection.[28] Studies have shown that ephedra-gypsum has a pronounced inhibitory effect on IL-6 release.[29] IL-1 β, a key pro-inflammatory cytokine, is essential for cell defense and repair in almost all tissues, and is mainly involved in autoimmune inflammatory responses, cell proliferation, differentiation, and apoptosis. Reduced IL-1 β and TNF-α expression can lessen lung tissue damage, effectively relieve inflammation in viral pneumonia, and hinder the transition from COPD to lung cancer.[30] Ephedra-gypsum inhibits the release of TNF-α by promoting the expression of the IL-1 β gene, thereby achieving anti-inflammatory and antiseptic actions and treating bronchitis.[31] LTP53 is a significant oncogene that regulates the cell cycle and apoptosis, and preserves genomic stability. When the body experiences a severe inflammatory response, TP53 protein is involved in the initial stage, provoking this cell inducing apoptosis. Some studies have shown that if RD causes an inflammatory response and cell necrosis, TP53 participates in the transcriptional regulation of DNA repair genes.[32] TP53 is an oncogene with high tumor relevance and is prone to gene mutations in lung cancer, which disrupts the function of tumor suppressors and has guiding significance for clinical use and prognosis.[33] Luteolin, the active component of ephedra-gypsum, binds well and stably to the TP53 protein according to the results of molecular docking.
In the KEGG analysis, PI3K-Akt, MAPK, and TNF signaling pathways were important signaling pathways in the treatment of RD with ephedra-gypsum. The PI3K/AKT signaling pathway regulates cell apoptosis, transformation, and proliferation, participates in the inflammatory reaction of lungs and airways, and is closely related to RD, such as COPD, bronchial asthma, asthma, acute pneumonia, and pulmonary fibrosis.[34] MAPK is essential for mediating the activation of the inflammatory response, participating in the production, division, death of cytokines, and functional synchronization between cells,[35] and often causes respiratory tract infection. Studies have found that repeated respiratory syncytial virus infection causes significant inflammatory reactions and airway hyper-responsiveness in the lungs of young Sprague dawley (SD) rats and increases the activation level of the extracellular signal-regulated kinase pathway in the MAPK cascade reaction pathway.[36],[37] The TNF signaling pathway can promote the activation of AP-1 through the MAPK signaling pathway; thus activating IL-1 β regulates the proliferation, apoptosis, and other biological processes of pneumonia cells, and participates in the occurrence and development of RD.[38],[39] The TNF signaling pathway can regulate vascular endothelial growth factor and MAPK signaling pathways and involves the synthesis of inflammatory mediators, cell growth and differentiation, and angiogenesis to promote the regeneration of repaired damaged lung tissue and pulmonary vessels.[40] The core components quercetin, luteolin, and kaempferol are flavonoids, which can inhibit respiratory inflammation, promote cell growth, differentiation, and apoptosis, and reduce mucus secretion through the TNF signaling pathway, thus playing a role in cough relief and expectoration. The TNF signaling pathway can block the binding of ACE2 to the COVID-19 spike protein receptor, thus inhibiting novel coronavirus pneumonia.[41] Therefore, the active components of ephedra-gypsum can inhibit the inflammatory reaction through the PI3K-Akt, MAPK, and TNF signaling pathways, which are involved in the occurrence, development, and prognosis of RD.
ConclusionIn summary, quercetin, luteolin, kaempferol, and CaSO4•2H2O are the core components of ephedra-gypsum, act on key targets such as TNF, IL-6, TP53, and IL-1 β mainly through cytokine-mediated PI3K-Akt, MAPK, and TNF signaling pathways, inflammatory responses, and apoptosis. This suggests that ephedra-gypsum can regulate the respiratory system by multiple components, targets, and pathways, thereby reducing inflammation and the expression of inflammatory mediators in lung illnesses. The outcomes of molecular docking further confirm that the key active components of ephedra-gypsum may be essential for the treatment of RD. These findings provide an idea and theoretical foundation for further exploration of the active components of ephedra-gypsum in the treatment of RDs and their possible mechanisms of action.
Data availability
All the data generated and analyzed during the study are included in this article and its supplementary information files.
Acknowledgment
Nil.
Financial support and sponsorship
This study was supported by Grants from the National Natural Science Foundation of China (grant no.U20A20404); National College Students' Innovation and Entrepreneurship Training Program (202210752014).
Conflicts of interest
There are no conflicts of interest.
References
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