Combined Network Pharmacology,Molecular Docking,and Experimental Verification Approach to Investigate the Potential Mechanisms of Polydatin Against COVID-19

Abstract

Background: Coronavirus disease 2019 (COVID-19) has posed a serious threat to human health and there is an urgent need for drug development. In this study, we explored the potential mechanisms underlying the efficacy of polydatin against COVID-19. Methods: A combined approach of network pharmacology, molecular docking, and experimental verification were employed in this study. Potential targets of polydatin for treating COVID-19 were obtained from multiple drug and disease databases. Protein–protein interaction and enrichment analyses were performed to predict the potential mechanism of action of polydatin against COVID-19. The binding potential of polydatin and key targets was evaluated through molecular docking. Furthermore, experimental methods including flow cytometry and luciferase assay were used to validate the results of computational analyses. Results: The main diseases identified as polydatin targets included metabolic diseases, lung diseases, inflammation, infectious diseases, and tumors. Polydatin may be used to treat COVID-19 through interventions that alter the immune and inflammatory responses, including IL-17 signaling pathway, T-cell activation, cytokines and inflammatory response, lipopolysaccharide-mediated signaling pathway, as well as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) innate immunity evasion and cell-specific immune response. Polydatin can potentially bind to the target proteins related to COVID-19, such as SARS-CoV-2 Mpro, RdRp, and human angiotensin-converting enzyme 2 (ACE2), while directly exerting its regulatory or therapeutic functions. The experimental results showed that polydatin decreased the infectivity of the SARS-CoV-2 spike pseudovirus in HEK293T-ACE2 cells. Accordingly, polydatin may retard the entry of SARS-CoV-2 into cells by competitively binding to human ACE2. Conclusion: The potential targets and signaling pathways of polydatin against COVID-19 were preliminarily identified. The findings may benefit the development and application of polydatin as a treatment for COVID-19.

Keywords

polydatin network pharmacology molecular docking COVID-19 ACE2

Introduction

Coronavirus disease 2019 (COVID-19), which is caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has rapidly spread worldwide since the end of 2019. The cumulative global number of confirmed cases is hundreds of millions, with the disease affecting most of the countries and regions. As COVID-19 poses a serious threat to human health, there is an urgent need for developing a drug against it.

Recent reports have highlighted that chemicals derived from medicinal plants, including traditional Chinese medicine, can play an important role in the fight against COVID-19.¹ The medicinal plant, Polygonum cuspidatum, has been helpful in preventing and controlling epidemics. The functions of P cuspidatum, as described in traditional Chinese medicine records, include clearing away of heat, detoxification, elimination of dampness, alleviation of jaundice, dispersing of stasis, resolution of pain, relieving cough, and resolving phlegm .² Modern pharmacological research has shown that this plant can also be used to treat viral infections and that it has antitussive and anti-asthmatic effects.² A number of medicines that may be effective in treating patients with COVID-19 have been discovered at Shanghai Institute of Materia Medica of Chinese Academy of Sciences and ShanghaiTech University using virtual screening and enzyme testing. Polydatin, a compound in P cuspidatum, was found to combine with the SARS-CoV-2 3CL hydrolase, leading to virus repression.³ Polydatin is also isolated from many plants, such as peanuts, grapes, red wines, and several daily diets ,⁴ which plays a role in prevalent diseases through their antioxidant and anti-inflammatory effects, regulation of autophagy and apoptosis, maintenance of mitochondrial function, and lipid regulation .⁵ Accordingly, the mechanism of polydatin against COVID-19 needs to be further explored.

With the accumulation of data and the progress in computational technology, several bioinformatics methods have come to prominence. Recently, they have been widely used in exploring the mechanism of phytochemicals against COVID-19. Phytochemicals derived from ayurvedic and African medicinal plants, including Picrorhiza kurroa, Cyperus rotundus, Aegle marmelos, Vetiveria zizanoides, Moringa olifera, Zingiber officinale, and Anacardium occidentale, have antiviral potential, which was validated through in silico methods, including molecular docking and molecular dynamic simulation.^6–10 As network pharmacology is an analytical method based on the interactive network of diseases, targets, and drugs, it was employed in the present study. This method integrates bioinformatics, systematic biology, molecular biology, and other disciplines.¹¹ We were able to explore the mechanism of action of polydatin in COVID-19 treatment using computational analysis and provide a reference for further research.

Methods

Collection of Drug Targets and Analysis of Their spectrum of Disease

The two-dimensional (2D) and three-dimensional (3D) structural models of polydatin were obtained from PubChem (CID:5281718),¹² wherein the polydatin targets were identified from TCMSP,¹³ TCMIP v2.0,¹⁴ and CTD¹⁵ databases; the targets were named in accordance with those in UniProt.¹⁶ After target collection, Metascape¹⁷ was used to analyze the disease spectrum associated with the polydatin targets.

Determining Targets of Polydatin Against COVID-19

Using DisGeNET (N.PMIDs≥20),¹⁸ Genecards (relevance score>5),¹⁹ and CTD¹⁵ databases (top 100), the targets of novel coronavirus pneumonia or COVID-19 were identified and named according to those in UniProt.¹⁶ Moreover, in concordance with existing reports,²⁰ genes (P value<0.05) that correlated with the angiotensin-converting enzyme 2 (ACE2) and are expressed in colonic epithelial cells were also identified and named according to those in UniProt.

Venny v2.1 software²¹ was used to generate a Venn diagram of the shared targets between polydatin and those of COVID-19. STRING v11²² was used to analyze the protein–protein interactions (parameter settings: minimum required interaction score, 0.40), Cytoscape 3.7.2²³ was used to draw the target diagrams, and to obtain the hub targets of polydatin against COVID-19. Venny v2.1 was also used to produce a Venn diagram depicting the common targets between polydatin and those correlated with ACE2 expression in colonic epithelial cells; STRING v11 was used to draw diagrams of the common targets (parameter settings: minimum required interaction score, 0.40).

Enrichment Analysis of Drug–Disease Targets

To elucidate the mechanism of action of polydatin against COVID-19, enrichment analyses were performed using the Metascape database, with the following ontology sources: KEGG Pathway, GO Biological Processes, Reactome Gene Sets, Canonical Pathways, and WikiPathways.¹⁷ To analyze the common targets between polydatin and those correlated with ACE2 expression in colonic epithelial cells, KEGG pathway analysis was conducted using STRING v11 (parameter settings: minimum required interaction score, 0.40).²²

Molecular Docking

To validate further the polydatin targets in COVID-19, we analyzed the docking scores of polydatin, with the following potential target proteins: SARS-CoV-2 Mpro (PDB: 6LU7), SARS-CoV-2 RdRp (PDB: 6M71), and human ACE2 (PDB: 6M17). The crystalline structures of the candidate proteins were obtained from the Protein Data Bank,²⁴ and were then modified by removing water, adding hydrogen, and optimizing the structure. The protein structures were subjected to a series of restrained minimizations to relieve any static clashes using an OPLS2005 force field up to 0.30 Å RMSD. The molecular docking was performed using Schrödinger Maestro software v11.4.²⁵

Cell and Pseudovirus Preparation

HEK293T-ACE2, a stable cell line overexpressing human ACE2, was obtained from Obio Technology (Shanghai) Corp., Ltd. The cell line was constructed via lentiviral infection combined with puromycin (Amresco) screening. Cells were cultured using DMEM (Gibco) containing 10% fetal bovine serum (FBS) (Gibco) and 1% penicillin/streptomycin (Gibco). The cells were maintained at 37 °C in a 5% CO₂ atmosphere. SARS-CoV-2 spike pseudovirus was obtained from Obio Technology (Shanghai) Corp., Ltd; it can be used to infect cells expressing ACE2, takes lentivirus as the basic vector, and replaces the VSV-G envelope protein of lentivirus with the spike protein of SARS-CoV-2. The pseudovirus also carries enhanced green fluorescent protein (EGFP) gene and luciferase reporter gene, which facilitate the detection in the subsequent experiment.

CCK8 Assay

To assess cell viability, HEK293T-ACE2 cells were seeded in 96-well plates. The cells were incubated with different concentrations of polydatin (MedChemExpress) for 48 h. The CCK8 reagent (Dojindo) was then added to each well and cells were incubated for 1 to 4 h. During incubation, the absorbance of cells in each well at 450 nm was measured using a microplate reader (Tecan).

Flow Cytometry

HEK293T-ACE2 cells were treated with SARS-CoV-2 spike pseudovirus and polydatin. HEK293T-ACE2 cells in the pseudovirus group only were infected with SARS-CoV-2 spike pseudovirus for 8 h. After 8 h of pseudovirus infection, the medium was replaced with a culture medium without pseudovirus and polydatin. HEK293T-ACE2 cells in the polydatin + pseudovirus group were pretreated with polydatin at different concentrations for 4 h, and then infected with SARS-CoV-2 spike pseudovirus for 8 h. After 8 h of pseudovirus infection, the medium was replaced with a culture medium containing polydatin. HEK293T-ACE2 cells in the pseudovirus + polydatin group were first infected with SARS-CoV-2 spike pseudovirus for 8 h, and then polydatin at different concentrations was added after 4 h of pseudovirus infection. After 8 h of pseudovirus infection, the medium was replaced with a culture medium containing polydatin. After incubation for 48 h in a 5% CO₂ cell incubator at 37 °C, the cells were gently harvested and prepared as a single cell suspension for counting. To validate whether or not polydatin decreases SARS-CoV-2 spike pseudovirus infection in HEK293T-ACE2 cells, the level of green fluorescence in the cells was determined using flow cytometry. The cells were transferred to the tubes for flow cytometry and evaluated using FACSCalibur (BD).

Luciferase Assay

Cell preparation and treatments for the luciferase assays were the same as those described for the flow cytometry analysis. Luciferase activity was detected using a dual-luciferase reporter assay system (Promega) according to the manufacturer's specifications. After the appropriate pseudovirus and/or polydatin treatment, the cells were gently harvested at a density of 1 × 10⁶ cells of each sample and resuspended in 100 μL of 1 × passive lysis buffer (PLB), which was provided with the assay system. Thereafter, 10 μL of lysis buffer was transferred into 96-well plates and 100 μL of luciferase assay reagent II (LAR II) was added. Fluorescence was detected using a microplate reader (Tecan). The experiment was performed in the dark.

Statistical Analysis

Experimental data are presented as the mean ± SEM. The data were analyzed by Student's two-tailed t-test using GraphPad Prism 8.0 (www.graphpad.com). Error bars in the figures represent SEM. Statistical significance was accepted at *P < 0.05, **P < 0.01,***P < 0.001, and **** P < 0.0001.

Results

Molecular Structure, Targets, and Disease Spectrum of Polydatin

The 2D and 3D structures of polydatin are shown in Figure 1, and the top 20 diseases targeted by polydatin ranked according to log₁₀(P) or log₁₀(q) are summarized in Figure 2 and Supplementary Table 1. Log₁₀(P) is the P value in log base 10, whereas log₁₀(q) is the multi-test adjusted P value in log base 10. The spectrum of diseases that can be treated using polydatin includes diabetes mellitus, fatty liver disease, lung diseases, respiratory distress syndrome, endothelial dysfunction, cerebral infarction, pancreatitis, hepatitis C, Helicobacter pylori infection, and malignant neoplasm of the skin, mouth and kidney. Overall, these diseases involve aspects of metabolism, lung, blood vessels, inflammation, infection, and cancer.

Figure 1.

(A) Two- and (B) three-dimensional (2D and 3D) structural models of polydatin obtained from PubChem.

Figure 2.

Top 20 potential diseases targeted by polydatin according to the enrichment analysis based on Metascape.

Analysis of the Polydatin Mechanism of Action Against COVID-19

Targets of polydatin and COVID-19 were identified. The Venn diagram of the shared targets of polydatin and COVID-19 is shown in Figure 3. The network diagram of the common targets is shown in Figure 4 with the targets listed in Supplementary Table 2. In addition, hub 10 targets and hub 6 targets were obtained based on the degree values of protein–protein interactions. These targets, including MAPK3, TP53, TNF, IL6, IL1B, PTGS2, CCL2, RELA, TGFB1, and CXCL8, are important potential targets of polydatin against COVID-19.

Figure 3.

Venn diagram of the shared targets of polydatin and COVID-19 generated using Venny.

Figure 4.

(A) Protein–protein interaction network of potential targets of polydatin against COVID-19, identified from STRING and visualized using Cytoscape. Protein–protein interaction network of (B) hub 10 targets and (C) hub 6 targets of polydatin against COVID-19 generated using Cytoscape. Rectangular nodes indicate targets, and the links represent different interactions.

The results of enrichment analysis regarding the mechanism of action of polydatin against COVID-19 are shown in Figure 5 and Supplementary Table 3. The main potential mechanisms involve the IL-17 signaling pathway, regulation of protein phosphorylation, folate metabolism, lipopolysaccharide-mediated signaling pathway, FOXO signaling pathway, T-cell activation, cytokines and inflammatory response, SARS-CoV-2 innate immunity evasion, and cell-specific immune response.

Figure 5.

Top 20 terms identified from the pathway and process enrichment analyses of polydatin against COVID-19 based on Metascape.

Pathway Analysis Revealed the Common Targets Between Polydatin and Those Correlated With ACE2 Expression in Colonic Epithelial Cells

The identified common targets between polydatin and those correlated with ACE2 expression in colonic epithelial cells are shown in Figure 6A, whereas the network diagram of the shared targets including MAPK3, EGFR, IL4, and IFNB1 is shown in Figure 6B. The top 20 terms identified from the KEGG pathway analysis of the common targets are summarized in Table 1. The main enrichment terms involve cAMP signaling pathway, cytomegalovirus infection, tuberculosis, Rap1 signaling pathway, pertussis, shigellosis, Ras signaling pathway, bile secretion, pancreatic secretion, C-type lectin receptor signaling pathway, and T cell receptor signaling pathway.

Figure 6.

(A) Venn diagram depicting the targets of polydatin and the targets correlated with ACE2 expression in colonic epithelial cells generated using Venny. (B) The network diagram of the shared targets between polydatin and those correlated with ACE2 expression in colonic epithelial cells generated using STRING.

Table 1.

Top 20 Terms Identified From KEGG Pathway Analysis of the Common Targets Between Polydatin and Those Correlated With ACE2 Expression in Colonic Epithelial Cells, Which are Ranked Using the False Discovery Rate (FDR).

Rank	Term ID	Term description	FDR
1	hsa04024	cAMP signaling pathway	0.004
2	hsa05163	Human cytomegalovirus infection	0.004
3	hsa05200	Pathways in cancer	0.004
4	hsa05231	Choline metabolism in cancer	0.004
5	hsa04921	Oxytocin signaling pathway	0.0066
6	hsa05152	Tuberculosis	0.0087
7	hsa04520	Adherens junction	0.0097
8	hsa04015	Rap1 signaling pathway	0.0104
9	hsa05133	Pertussis	0.0104
10	hsa05214	Glioma	0.0104
11	hsa05210	Colorectal cancer	0.0109
12	hsa05131	Shigellosis	0.0115
13	hsa04014	Ras signaling pathway	0.0117
14	hsa04912	GnRH signaling pathway	0.0117
15	hsa04976	Bile secretion	0.0117
16	hsa04972	Pancreatic secretion	0.0125
17	hsa04625	C-type lectin receptor signaling pathway	0.0128
18	hsa04660	T cell receptor signaling pathway	0.0128
19	hsa04928	Parathyroid hormone synthesis, secretion, and action	0.0128
20	hsa04722	Neurotrophin signaling pathway	0.0154

Molecular Docking Analysis of Polydatin and its Targets

The molecular docking results of 2D and 3D structural models of polydatin and potential target proteins, SARS-CoV-2 Mpro, RdRp, and human ACE2 are shown in Figure 7. Table 2 presents the docking score, which refers to the binding affinity between the compound and the target protein. The higher the absolute docking score, the greater the binding affinity. As can be seen from the analysis results, polydatin exhibited the potential to bind to all three of these target proteins related to COVID-19.

Figure 7.

(A) Two- and (B) three-dimensional (2D and 3D) molecular docking models depict polydatin binding to the target proteins generated using the Schrödinger Maestro software.

Table 2.

Molecular Docking Results of Polydatin and its Candidate Target Proteins, Determined Using the Schrödinger Maestro Software.

Compound	PDB ID	Candidate target	Docking score
Polydatin	6LU7	SARS-CoV-2 Mpro	−8.904
	6M71	SARS-CoV-2 RdRp	−5.736
	6M17	Human ACE2	−4.503

Effects of Polydatin on the Viability of HEK293T-ACE2 Cells

In the CCK8 assay, we detected the viability of HEK293T-ACE2 cells with an increase in polydatin dose. The results showed that the cell viabilities of the Group Polydatin (PLD) 0.781 μM, PLD 3.125 μM, PLD 12.5 μM, PLD 50 μM, PLD 200 μM, and PLD 400 μM were 102.95% (P > 0.05), 96.69% (P > 0.05), 95.93% (P < 0.05), 83.72% (P < 0.05), 73.66% (P < 0.05), and 58.69% (P < 0.05), respectively relative to the Control (Figure 8). This suggested that 12.5 μM polydatin exerted a slight inhibitory effect on the viability of HEK293T-ACE2 cells; the higher the concentration of polydatin, the more significant is the inhibitory effect.

Figure 8.

Effects of polydatin on the cell viability of HEK293T-ACE2 cells. Cells were cultured with different concentrations of polydatin for 48 h.

Effects of Polydatin on HEK293T-ACE2 Cells Infected With SARS-CoV-2 Spike Pseudovirus

The green fluorescence of HEK293T-ACE2 cells was detected using flow cytometry. The results showed that polydatin could decrease the infection of SARS-CoV-2 spike pseudovirus in HEK293T-ACE2 cells (Figure 9A and B). The infection rate in the groups that were pretreated with 3.125 and 12.5 μM polydatin decreased by 20.80% and 24.32%, respectively, compared with that in the positive control group, which was treated with the pseudovirus only. Moreover, the infection rate in the groups that were treated with 3.125 and 12.5 μM polydatin after 4 h of pseudovirus infection decreased by 7.45% and 11.76%, respectively, compared with that in the positive control group.

Figure 9.

Effects of polydatin on HEK293T-ACE2 cells infected with SARS-CoV-2 spike pseudovirus detected using (A, B) flow cytometry and (C) luciferase assay.

The evaluation of luciferase fluorescence led to a similar conclusion (Figure 9C). The infection rate in the groups pretreated with 3.125 and 12.5 μM polydatin decreased by 26.24% and 45.34%, respectively, compared with that in the positive control group. Moreover, the infection rate in the groups treated with 3.125 and 12.5 μM polydatin after 4 h of pseudovirus infection decreased by 28.07% and 41.32%, respectively, compared with that in the positive control group.

Discussion

SARS-CoV-2 is a newly identified coronavirus strain that was previously not observed in humans. The genome of SARS-CoV-2 shares approximately 80% identity with that of SARS-CoV.²⁶ P cuspidatum has a long history of use in traditional Chinese medicine, with its active components reported to inhibit the replication of the influenza virus.²⁷ In the current study, the enrichment analysis of potential targets showed that polydatin may be effective against metabolic diseases, lung diseases, inflammation, infectious diseases, and tumors, which are also closely related to COVID-19. Acute respiratory distress syndrome (ARDS) is a common complication as a result of severe COVID-19 infection caused by SARS-CoV-2, which can induce hyperinflammation and cytokine storm, thereby, causing lung damage.²⁸ The severity and death due to COVID-19 are also related to cardiovascular disease, obesity, and metabolic disorders.^29,30 In addition, the expression of ACE2 is higher in cervical squamous cell carcinoma, endocervical adenocarcinoma, pancreatic adenocarcinoma, rectum adenocarcinoma, kidney renal papillary cell carcinoma, kidney renal clear cell carcinoma, and other tumor tissues relative to that in the surrounding tissues. This has led to the hypothesis that patients with any one of these cancers may be susceptible to SARS-CoV-2 infection.³¹ This hypothesis was partially supported by our analysis of the spectrum of diseases targeted by polydatin.

The overactivation of T cells may occur in patients with COVID-19, leading to severe immune-related damage.³² The cytokine storm induced by SARS-CoV-2 infection also attacks healthy cells in the lungs, leading to respiratory failure. In the current study, we predicted therapeutic targets of polydatin against COVID-19, including MAPK3, TP53, TNF, IL6, IL1B, PTGS2, CCL2, RELA, TGFB1, and CXCL8. The tissue- and gender-specific regulation of ACE2 by p53 was reported in pigs. The absence of the TP53 binding site increases the activity of ACE2 promoter in porcine kidney cells.³³ An earlier study reported that p53 is one of the important targets of traditional Chinese herbal compound prescription, Xuanfei Baidu Tang, in the treatment of COVID-19.³⁴ TNF-α and IFN-γ can cause a lethal cytokine shock and inflammatory cell death in mice with COVID-19.³⁵ The enrichment analyses also revealed that the potential therapeutic mechanisms of polydatin against COVID-19 involve various pathways and processes, including the IL-17 signaling pathway, T-cell activation, cytokines and inflammatory response, lipopolysaccharide-mediated signaling pathway, folate metabolism, and FOXO signaling pathway, as well as SARS-CoV-2 innate immunity evasion and cell-specific immune response. Studies have also confirmed that polydatin has the function of regulating inflammatory mediators and immune response.^36,37 These results suggest that polydatin may act against COVID-19 through these targets and mechanisms, indicating that the functions related to immune and inflammatory responses are largely associated with the activities of polydatin against COVID-19. The activation of G6PD is related to increased oxidative stress and inflammation in acute pulmonary injury. Polydatin can be used for patients in severe stages of COVID-19 as a potential G6PD inhibitor.⁴ Polydatin can moderate acute clinical, chronic subclinical inflammation, and pneumonia or lung inflammation processes through the TLR4-MyD88-NF-κB pathway.³⁸ It has also been reported that glucose and folate are depleted in cells infected with SARS-CoV-2, and viral replication is exquisitely sensitive to inhibitors of folate and one-carbon metabolism.³⁹ Decreased levels of serum folate are common among inpatients with COVID-19.⁴⁰ Multiple strategies enable SARS-CoV-2 to evade the innate immunity; these include low cytosine–phosphate–guanosine levels in the genome and suppression of IFN-α/β production at the earliest stage of infection.^41,42

According to a classic theory of traditional Chinese medicine, the lung and large intestine are “interior–exteriorly” related. It has been demonstrated that the frequency of gastrointestinal syndromes is high in patients with COVID-19.⁴³ Clinical data revealed diarrhea in a small number of patients with COVID-19, and SARS-CoV-2 was detectable in their stool samples.^44,45 Furthermore, gastrointestinal infection can lead to the progression of COVID-19 and the gut microbiota may influence the expression level of colonic ACE2.⁴³ As ACE2 is typically highly expressed in the colonocytes and is involved in intestinal inflammation, the candidate targets identified in the present study were reported to correlate with ACE2 expression in colonic endothelial cells.²⁰ Our enrichment analyses indicate that the common targets between polydatin and those correlated with ACE2 expression in colonic epithelial cells potentially function in cAMP, Ras, and Rap1 signaling pathways, as well as in immune mechanisms, such as C-type lectin receptor and T cell receptor signaling pathway. Potential mechanisms include digestive mechanisms, such as adherens junction, bile secretion, pancreatic secretion, and colorectal cancer, as well as infectious diseases, such as tuberculosis, pertussis, shigellosis, and cytomegalovirus infection. Intestinal mucosal epithelial cells maintain their barrier function by forming complex protein networks, including adherens junctions, tight junctions, and gap junctions. Polydatin may alleviate mucosal oxidative stress and immunological damage and may improve the intestinal epithelium barrier.^46,47

SARS-CoV-2 Mpro (also known as 3C-like protease) is an important enzyme for the replication of the coronavirus and is responsible for processing the polypeptide into functional proteins.⁴⁸ Furthermore, SARS-CoV-2 RdRp can also play a primary role in coronavirus replication and transcriptional machinery.⁴⁹ Human ACE2 is a cellular receptor for both SARS-CoV and SARS-CoV-2.²⁶ Accordingly, these three proteins have been reported to be potential targets for the development of antiviral drugs. Our molecular docking simulations suggest the potential of polydatin binding to the target proteins directly related to COVID-19, including SARS-CoV-2 Mpro, RdRp, and human ACE2, thereby, playing either a regulatory or therapeutic role. The potential binding affinity was the highest between polydatin and SARS-CoV-2 Mpro. A previous report based on molecular docking analyses suggests that the main bioactive compounds of Shufeng Jiedu capsules, including quercetin, wogonin, and polydatin, can bind directly to SARS-CoV-2 Mpro. However, the computational results in this report have not been verified experimentally.⁵⁰

The expression of the ACE2 receptor has been observed in multiple tissues including the lung, heart, kidney, endothelium, and intestine.^51,52 ACE2 is one of the crucial enzymes in the renin–angiotensin system (RAS), which can regulate blood pressure, isohydria, inflammation, and vascular resistance. The SARS-CoV-2 spike protein is able to directly bind with the ACE2 receptor of the host cell surface, leading to virus entry and ultimately multiple organ damage.⁵³ Considering the importance of ACE2 and the analysis results of our network pharmacology and molecular docking analyses, we carried out experimental verification of our findings. The experimental results demonstrated that polydatin reduced the infection of the SARS-CoV-2 spike pseudovirus in HEK293T-ACE2 cells, which overexpress ACE2. We suggest that polydatin may retard the entry of SARS-CoV-2 into cells by competitively binding to human ACE2 (Figure 10). Molecular docking simulations in another report suggest that polydatin can bind to the SARS-CoV-2 Spike and ACE2 proteins. These simulations were confirmed by biochemical assays using the purified ACE2 protein and biotinylated SARS-CoV-2 Spike S1 protein.⁵⁴ This report corroborates our findings. We performed different experimental assays, using the SARS-CoV-2 spike pseudovirus and HEK293T-ACE2 cells, to better simulate the process of virus infection of human cells. However, owing to the limitations of the current research conditions employed, the mechanisms underlying the action of polydatin in the treatment of COVID-19 are worthy of further research.

Figure 10.

Potential mechanism of action of polydatin against COVID-19. Polydatin has the potential to reduce the entry of SARS-CoV-2 into cells by competitively binding to human ACE2.

Conclusions

In this study, we explored the potential therapeutic mechanisms of polydatin action against COVID-19 using a combined approach of network pharmacology, molecular docking, and experimental verification. Polydatin may exhibit a functional role in the lungs and intestine, which are mainly involved in regulating immune functions and inflammatory responses. Polydatin may also exhibit potential regulatory or therapeutic effects by binding to the target proteins that are directly related to COVID-19, such as SARS-CoV-2 Mpro, RdRp, and human ACE2. Our experimental results show that polydatin can decrease the infection of the SARS-CoV-2 spike pseudovirus in HEK293T-ACE2 cells. Hence, polydatin may retard the entry of the virus into cells by competitively binding to human ACE2. These results suggest the potential of developing polydatin as a treatment for patients with COVID-19.

Supplemental Material

sj-doc-1-npx-10.1177_1934578X221095352 - Supplemental material for Combined Network Pharmacology, Molecular Docking, and Experimental Verification Approach to Investigate the Potential Mechanisms of Polydatin Against COVID-19

Supplemental material, sj-doc-1-npx-10.1177_1934578X221095352 for Combined Network Pharmacology, Molecular Docking, and Experimental Verification Approach to Investigate the Potential Mechanisms of Polydatin Against COVID-19 by Meng Wang, Kaijian Qin and Xiaofeng Zhai in Natural Product Communications

Footnotes

Acknowledgments

The authors would particularly like to acknowledge the team members for their wonderful collaboration and patient support.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

This work was supported by Shanghai major clinical research project on traditional Chinese medicine [grant number ZY(2018-2020)-CCCX-4003] and Three-year Action Plan for the Development of TCM in Hongkou District, Shanghai [grant number HGY-KY-2018-01].

Authors Contributions

Meng Wang: Conception of the study, analysis and interpretation of the data, and drafting the article. Kaijian Qin: Acquisition of data and revising the article critically. Xiaofeng Zhai: Design of the study and final approval of the version to be submitted.

Data Availability

The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

Ethical Approval

Not applicable, because this article does not contain any studies with human or animal subjects.

Informed Consent

Not applicable, because this article does not contain any studies with human or animal subjects.

Trial Registration

Not applicable, because this article does not contain any clinical trials.

ORCID iD

Meng Wang

Supplemental Material

Supplemental material for this article is available online.

References

Ang

Lee H

Choi J

, et al. Herbal medicine and pattern identification for treating COVID-19: a rapid review of guidelines[J]. Integr Med Res. 2020;9(2):100407. doi:10.1016/j.imr.2020.100407

Wang

Liu

. Handbook of diagnosis and treatment of novel coronavirus pneumonia in TCM. 1st ed[G]. China Press of Traditional Chinese Medicine; 2020.

A joint research team from Shanghai Institute of Materia Medica of Chinese Academy of Sciences and Shanghai University of Science and Technology has found a number of traditional Chinese medicines that may have a therapeutic effect on the novel pneumonia [internet]. 2020 [cited February 25, 2022]. http://www.simm.cas.cn/web/xwzx/kydt/202001/t20200125_5646357.html

Doustimotlagh A

Eftekhari

. Glucose-6-phosphate dehydrogenase inhibitor for treatment of severe COVID-19: polydatin[J]. Clin Nutr ESPEN. 2021;43:197‐199. doi:10.1016/j.clnesp.2021.02.021

Jiang

, et al. Old dog, new tricks: polydatin as a multitarget agent for current diseases[J]. Phytother Res. 2022;36(1):214‐230. doi:10.1002/ptr.7306

Khuntia B

Sharma

Qazi

, et al. Ayurvedic medicinal plants against COVID-19: an in silico analysis[J]. Nat Prod Commun. 2021;16(11):1‐9. doi:10.1177/1934578X211056753

Isa M

Mustapha

Qazi

, et al. In silico molecular docking and molecular dynamic simulation of potential inhibitors of 3C-like main proteinase (3CLpro) from severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) using selected African medicinal plants[J]. Adv Tradit Med. 2022;22:107‐123. doi:10.1007/s13596-020-00523-w

Qazi

Das

Khuntia B

. In silico molecular docking and molecular dynamic simulation analysis of phytochemicals from Indian foods as potential inhibitors of SARS-CoV-2 RdRp and 3CLpro[J]. Nat Prod Commun. 2021;16(9):1‐12. doi:10.1177/1934578X211031707

Yadav M

Ahmad

Raza

, et al. Predictive modeling and therapeutic repurposing of natural compounds against the receptor-binding domain of SARS-CoV-2[J]. J Biomol Struct Dyn. 2022:1‐13.34974820

10.

Qazi

Sheikh

Raza

. In silico approach to understand the epigenetic mechanism of SARS-CoV-2 and its impact on the environment[J]. VirusDisease. 2021;32(2):286‐297. doi:10.1007/s13337-021-00655-w

11.

Hopkins A

. Network pharmacology: the next paradigm in drug discovery[J]. Nat Chem Biol. 2008;4(11):682‐690. doi:10.1038/nchembio.118

12.

Kim

Chen

Cheng

, et al. Pubchem 2019 update: improved access to chemical data[J]. Nucleic Acids Res. 2019;47(D1):D1102‐D1109. doi:10.1093/nar/gky1033

13.

Wang

, et al. TCMSP: a database of systems pharmacology for drug discovery from herbal medicines[J]. J Cheminform. 2014;6(1):13. doi:10.1186/1758-2946-6-13

14.

Zhang

Liu

, et al. ETCM: an encyclopaedia of traditional Chinese medicine[J]. Nucleic Acids Res. 2019;47(D1):D976‐D982. doi:10.1093/nar/gky987

15.

Davis A

Grondin C

Johnson R

, et al. Comparative toxicogenomics database (CTD): update 2021[J]. Nucleic Acids Res. 2021;49(D1):D1138‐D1143. doi:10.1093/nar/gkaa891

16.

The UniProt Consortium. Uniprot: a worldwide hub of protein knowledge[J]. Nucleic Acids Res. 2019;47(D1):D506‐D515. doi:10.1093/nar/gky1049

17.

Zhou

Pache

, et al. Metascape provides a biologist-oriented resource for the analysis of systems-level datasets[J]. Nat Commun. 2019;10(1):1523. doi:10.1038/s41467-019-09234-6

18.

Piñero

Ramírez-Anguita J

Saüch-Pitarch

, et al. The DisGeNET knowledge platform for disease genomics: 2019 update[J]. Nucleic Acids Res. 2019;48(D1):D845‐D855.

19.

Stelzer

Rosen

Plaschkes

, et al. The GeneCards suite: from gene data mining to disease genome sequence analyses[J]. Curr Protoc Bioinform. 2016;54(1):1‐30. doi:10.1002/cpbi.5

20.

Wang

Zhao

Liu

, et al. ACE2 Expression by colonic epithelial cells is associated with viral infection, immunity and energy metabolism [J]. medRxiv. 2020. https://doi.org/10.1101/2020.02.05.20020545

21.

Venny

Oliveros JC.

. An interactive tool for comparing lists with Venn’s diagrams; 2007–2015 [internet]. [cited May 25, 2021]. https://bioinfogp.cnb.csic.es/tools/venny/index.html.

22.

Szklarczyk

Gable A

Lyon

, et al. STRING V11: protein–protein association networks with increased coverage, supporting functional discovery in genome-wide experimental datasets[J]. Nucleic Acids Res. 2019;47(D1):D607‐D613. doi:10.1093/nar/gky1131

23.

Shannon

. Cytoscape: a software environment for integrated models of biomolecular interaction networks[J]. Genome Res. 2003;13(11):2498‐2504. doi:10.1101/gr.1239303

24.

Burley S

Bhikadiya

, et al. RCSB protein data bank: powerful new tools for exploring 3D structures of biological macromolecules for basic and applied research and education in fundamental biology, biomedicine, biotechnology, bioengineering and energy sciences[J]. Nucleic Acids Res. 2021;49(D1):D437‐D451. doi:10.1093/nar/gkaa1038

25.

Friesner

Banks

Murphy

, et al. Glide: a new approach for rapid, accurate docking and scoring. 1. Method and assessment of docking accuracy[J]. J Med Chem. 2004;47(7):1739‐1749. doi:10.1021/jm0306430

26.

Yan

Zhang

, et al. Structural basis for the recognition of SARS-CoV-2 by full-length human ACE2[J]. Science. 2020;367(6485):1444‐1448. doi:10.1126/science.abb2762

27.

Lin

Chen

, et al. Polygonum cuspidatum and its active components inhibit replication of the influenza virus through toll-like receptor 9-induced interferon beta expression[J]. PlosOne. 2015;10(2):e117602.

28.

Portsmore

Tran Nguyen

Beacham

, et al. Combined IL-6 and JAK/STAT inhibition therapy in COVID-19-related sHLH, potential game changer[J]. Br J Haematol. 2020;190(4):525‐528. doi:10.1111/bjh.16966

29.

Marino

Vargas

Skein

, et al. Metabolic and inflammatory health in SARS-CoV-2 and the potential role for habitual exercise in reducing disease severity[J]. Inflammation Res. 2022;71(1):27‐38. doi:10.1007/s00011-021-01517-3

30.

Khwatenge C

Pate

Miller L

, et al. Immunometabolic dysregulation at the intersection of obesity and COVID-19[J]. Front Immunol. 2021;12:732913. doi:10.3389/fimmu.2021.732913

31.

Jia

Yin

, et al. Two things about COVID-19 might need attention[J]. Preprint. 2020. https://doi.org/10.20944/preprints202002.0315.v1

32.

Shi

Wang

, et al. Pathological findings of COVID-19 associated with acute respiratory distress syndrome[J]. Lancet Respir Med. 2020;8(4):420‐422. doi:10.1016/S2213-2600(20)30076-X

33.

Zhang

Niu

Flisikowska

, et al. A tissue- and gender-specific regulation of the SARS-CoV-2 receptor ACE2 by p53 in pigs[J]. Biochem Biophys Res Commun. 2021;553:25‐29. doi:10.1016/j.bbrc.2021.03.068

34.

Wang

Zhang

Zhan

, et al. Screening out anti-inflammatory or anti-viral targets in Xuanfei baidu tang through a new technique of reverse finding target[J]. Bioorg Chem. 2021;116:105274. doi:10.1016/j.bioorg.2021.105274

35.

Karki

Sharma B

Tuladhar

, et al. Synergism of TNF-α and IFN-γ triggers inflammatory cell death, tissue damage, and mortality in SARS-CoV-2 infection and cytokine shock syndromes[J]. Cell. 2021;184(1):149‐168. doi:10.1016/j.cell.2020.11.025

36.

Yao

Wang

Liu

, et al. Polydatin ameliorates DSS-induced colitis in mice through inhibition of nuclear factor-kappaB activation[J]. Planta Med. 2011;77(05):421‐427. doi:10.1055/s-0030-1250462

37.

Jiang

Zhao

Deng

, et al. Polydatin ameliorates Staphylococcus aureus-induced mastitis in mice via inhibiting TLR2- mediated activation of the p38 MAPK/NF-κB pathway[J]. Acta Pharmacol Sin. 2017;38(2):211‐222. doi:10.1038/aps.2016.123

38.

Jiang

Guo

, et al. Protective effects of polydatin on lipopolysaccharide-induced acute lung injury through TLR4-MyD88-NF-κB pathway[J]. Int Immunopharmacol. 2015;29(2):370‐376. doi:10.1016/j.intimp.2015.10.027

39.

Zhang

Guo

Kim S

, et al. SARS-CoV-2 hijacks folate and one-carbon metabolism for viral replication[J]. Nat Commun. 2021;12(1):1676. doi:10.1038/s41467-021-21903-z

40.

Meisel

Efros

Bleier

, et al. Folate levels in patients hospitalized with coronavirus disease 2019[J]. Nutrients. 2021;13(3):812. doi:10.3390/nu13030812

41.

Bouayad

. Innate immune evasion by SARS-CoV-2: comparison with SARS-CoV[J]. Rev Med Virol. 2020;30(6):1‐9. doi:10.1002/rmv.2135

42.

Amor

Fernández Blanco

Baker

. Innate immunity during SARS-CoV-2: evasion strategies and activation trigger hypoxia and vascular damage[J]. Clin Exp Immunol. 2020;202(2):193‐209. doi:10.1111/cei.13523

43.

Yang

Huang

Fan

, et al. Gastrointestinal microenvironment and the gut-lung axis in the immune responses of severe COVID-19[J]. Front Mol Biosci. 2021;8:647508. doi:10.3389/fmolb.2021.647508

44.

Guan

, et al. Clinical characteristics of coronavirus disease 2019 in China[J]. N Engl J Med. 2020;382(18):1708‐1720. doi:10.1056/NEJMoa2002032

45.

Liang

Feng

Rao

, et al. Diarrhoea may be underestimated: a missing link in 2019 novel coronavirus[J]. Gut. 2020;69(6):1141‐1143. doi:10.1136/gutjnl-2020-320832

46.

Zhang

Chen

, et al. Protective effect of polydatin on jejunal mucosal integrity, redox status, inflammatory response, and mitochondrial function in intrauterine growth-retarded weanling piglets[J]. Oxid Med Cell Longevity. 2020;2020:1‐14.

47.

Chen

Yang

Wen

, et al. Polydatin has anti-inflammatory and antioxidant effects in LPS-induced macrophages and improves DSS-induced mice colitis[J]. Immun Inflamm Dis. 2021;9(3):959‐970. doi:10.1002/iid3.455

48.

Liu

Wang

. Potential inhibitors against 2019-nCoV coronavirus M protease from clinically approved medicines[J]. J Genet Genomics. 2020;47(2):119‐121. doi:10.1016/j.jgg.2020.02.001

49.

Gao

Yan

Huang

, et al. Structure of the RNA-dependent RNA polymerase from COVID-19 virus[J]. Science. 2020;368(6492):779‐782. doi:10.1126/science.abb7498

50.

Xia

Shi

, et al. Shufeng jiedu, a promising herbal therapy for moderate COVID-19: antiviral and anti-inflammatory properties, pathways of bioactive compounds, and a clinical real-world pragmatic study[J]. Phytomedicine. 2021;85:153390. doi:10.1016/j.phymed.2020.153390

51.

Crackower

Sarao

Oudit

, et al. Angiotensin-converting enzyme 2 is an essential regulator of heart function[J]. Nature. 2002;417(6891):822‐828. doi:10.1038/nature00786

52.

Hamming

Timens

Bulthuis

, et al. Tissue distribution of ACE2 protein, the functional receptor for SARS coronavirus. A first step in understanding SARS pathogenesis[J]. J Pathol. 2004;203(2):631‐637. doi:10.1002/path.1570

53.

Zhang

Penninger J

, et al. Angiotensin-converting enzyme 2 (ACE2) as a SARS-CoV-2 receptor: molecular mechanisms and potential therapeutic target[J]. Intensive Care Med. 2020;46(4):586‐590. doi:10.1007/s00134-020-05985-9

54.

Perrella

Coppola

Petrone

, et al. Interference of polydatin/resveratrol in the ACE2:spike recognition during COVID-19 infection. A focus on their potential mechanism of action through computational and biochemical assays[J]. Biomolecules. 2021;11(7):1048. doi:10.3390/biom11071048

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