key: cord-0946900-53l48vbd
authors: Kong, Qi; Wu, Yue; Gu, Yu; Lv, Qi; Qi, Feifei; Gong, Shuran; Chen, Xiuping
title: Analysis of the molecular mechanism of Pudilan (PDL) treatment for COVID-19 by network pharmacology tools
date: 2020-05-30
journal: Biomed Pharmacother
DOI: 10.1016/j.biopha.2020.110316
sha: d14d114d813c5a5508db1b81da6471ca3401cdca
doc_id: 946900
cord_uid: 53l48vbd

BACKGROUND: Pudilan (PDL), a four-herb prescription with the traditional function of heat-clearing and detoxifying, has been clinically used as an anti-SARS-CoV-2 infectory agent in China for decades. PDL might also have therapeutic potentials for COVID-19 while the underlying mechanisms remain to be clarified. METHODS: We used network pharmacology analysis and selected 68 co-targeted genes/proteins as targets of both PDL and COVID-19. These co-targeted genes/proteins were predicted by SwissDock Server for their high-precision docking simulation, and analyzed by STRING for proteins to protein interaction (PPI), pathway and GO (gene ontology) enrichment. The therapeutic effect for PDL treatment on COVID-19 was validated by the TCMATCOV (TCM Anti COVID-19) platform. RESULTS: PDL might prevent the entrance of SARS-CoV-2 entry into cells by blocking the angiotensin-converting enzyme 2 (ACE2). It might inhibit the cytokine storm by affecting C-reactive protein (CRP), interferon-γ (IFN-γ), interleukin- 6 (IL-6), interleukin- 10 (IL-10), tumor necrosis factor (TNF), epidermal growth factor receptor (EGFR), C-C motif chemokine ligand 5 (CCL5), transforming growth factor-β1 (TGFβ1), and other proteins. PDL might moderate the immune system to shorten the course of the disease, delay disease progression, and reduce the mortality rate. CONCLUSION: PDL might have a therapeutic effect on COVID-19 through three aspects, including the moderate immune system, anti-inflammation, and anti-virus entry into cells.

included [1] . More than 85% of SARS-CoV-2 infected patients had received TCM treatment in China [2] . TCM, a traditional medical system, has more than two thousand years of clinical practice.

Compared with modern medicine, the herb-based TCM shows several advantages, including significant curative effects, few side-effects, and low cost. Clinical practice showed that early intervention by TCM is a practical medical way to improve the cure rate, shorten the disease course, delay the disease progression, and reduce the mortality rate [3, 4] . However, the underlying mechanisms remain unclear mainly due to the complicated ingredients of TCM. The proposed mechanisms include blocking the SARS-CoV-2 infection, balance the physiological activity, regulation of the immune response, inhibition of the inflammatory storm, and promoting patient recovery [3] . PDL was recorded in the Chinese Pharmacopoeia (2015 Edition) and has been recommended as a preferred drug for the prevention and treatment of H1N1 and hand, foot, and mouth disease (HFMD). PDL is also useful in the treatment of COVID-19 and is recommended for SARS-CoV-2 infection in children [5] . Our experimental studies using hACE2 mice and Vero E6 cells revealed that PDL oral liquid has a therapeutic effect against SARS-CoV-2 by anti-virus, anti-inflammatory, and moderate immunity [6] .

To explore the molecular mechanism for PDL against COVID-19, we tried to integrate the bioinformatics and network pharmacology tools to predict the target genes and proteins and to analyze the interactions between PDL ingredients with the targeted genes.

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The query four herbs of PDL were first transferred into a list of compositive ingredients/ingredients based on the formula-herb-ingredient association data collected and integrated by the TCMID (Traditional Chinese Medicine Integrated Database) database (http://www.megabionet.org/tcmid) [7] .

For each ingredient, candidate targets were predicted based on the target prediction method of BATMAN-TCM (Bioinformatics Analysis Tool for Molecular mechANism of Traditional Chinese Medicine, http://bionet.ncpsb.org/batman-tcm), which is a bioinformatics tool used for analyzing the molecular mechanism of TCMs by predicting the potential targets of the ingredients of TCMs, and then performing functional analyses on these targets including known ingredient-target interactions, protein interaction networks, and KEGG pathway data [8] .

GeneCards (https://www.genecards.org) provides gene-centric information that is automatically mined and integrated from myriad data sources, resulting in the webbased card for COVID-19 disease targeted genes by searching the "novel coronavirus" in GeneCards and obtained a list of COVID-19-targeted genes [9] .

With STRING (https://string-db.org), we analyzed the co-targeted proteins that are encoded by COVID-19-associated genes that interact with PDL ingredient-targeted genes to explore their relationship within a PPI network, GO, and Reactome pathway analysis [10] . WebGestalt (http://www.webgestalt.org) was used as the enrichment method for COVID-19 and PDL co-targeted GSEA [11] . The Reactome Knowledgebase (https://reactome.org) provides molecular details of pathways and reactions in human biology. We used Reactome to draw two pathways that COVID-19 and PDL co-targeted gene set enriched [12] . With pathway builder tool 2.0, we simulated the possible ways for PDL treatment on COVID-19.

TCM Anti COVID-19 (http://tcmatcov.bbtcml.com, TCMATCOV) was a platform to predict the efficacy of the anti-coronavirus pneumonia effect of TCM. TCMATCOV is based on J o u r n a l P r e -p r o o f the interaction network imitating the disease network of COVID-19 [13] . TCMATCOV utilizes a quantitative evaluation algorithm to analyze disease network disturbance after multitarget drug attacks to predict potential drug effects. Based on the TCMATCOV platform, PDL was calculated and predicted to have a high disturbance score and to account for a high proportion of the classic anti-COVID-19 prescriptions used by clinicians.

The steps used in the entire analysis performed in this study are shown in Fig. 1 . COVID-19 disease targeted genes/proteins were mined by GeneCards. The PDL ingredients were identified targeted by TCMID and their targeted genes/proteins and pathways were identified by BATMAN-TCM. These co-targeted genes/proteins were enriched by STRING, WebGestalt, and predicted by SwissDock, and TCMATCOV.

All analyses were performed with the default values for each of the tools used.

Continuous variables were commonly described as the median and range. The cutoff of the FDR value was set as 0.01. Only the predicted candidate target proteins with scores >= 20 are presented in the query results of BATMAN-TCM. All reported P values are two-tailed, and P<0.01 was considered statistically significant.

The PDL ingredients were identified targeted by TCMID and their targeted genes/proteins and pathways were identified by BATMAN-TCM. PDL includes four kinds of herbs, which contain 181 ingredients. Among them, 67 ingredients have no structural information, and thus their targets could not be predicted. Finally, 114 ingredients were predicted to interact with 1281 targeted genes, and 64 ingredients had potential targets with scores larger than 20 (Supplementary Table S1 ). The results of the PDL ingredients targeted gene-disease enrichment analysis in TTD (Therapeutic Target Database) indicate that PDL might treat some respiratory system disease including asthma, chronic obstructive J o u r n a l P r e -p r o o f pulmonary disease (COPD), obstructive airway disease, and cough, which are closely related to COVID-19 (Table 1 , P<0.01, Enrich ratio˃1.5).

COVID-19 disease targeted genes/proteins were mined by GeneCards. We searched for "Novel Coronavirus" in GeneCards and obtained 350 COVID-19 related genes with targeted scores (Supplementary Table S2 ). Several TCM herb prescriptions, including Lianhuaqingwen (LHQW), and Shufengjiedu (SFJD) were reported to be useful for the treatment of COVID-19, similar to PDL. We compared their targeted genes and the data are shown in Fig. 2A . The 68 co-targeted genes that were among both the PDL targeted genes and the COVID-19 disease-associated genes are shown in the Venn diagram of Table 2 showed the top 10 target prediction results for COVID-19 disease-associated genes interaction with PDL ingredients with predicted scores. Among which, ACE2 is the receptor for SARS-CoV-2 entry into cells. TNF, SPIDR, IFN-γ, IL-6, TP53, CRP, EGFR, and CCL5 proteins play important roles in the pathogenic process of COVID-19. The result may explain the efficacy of PDL oral liquid therapy in COVID-19 patients.

Using STRING, we analyzed the interactions of 68 proteins that are COVID-19associated genes interaction with PDL ingredient-targeted genes, and the multiple proteins to protein interaction (PPI) enrichment were obvious (P<1.0e -16 ) (Fig. 2B) .

Separate interaction scores are available as well as part of the underlying evidence. The interaction scores from STRING represent the expression of approximate confidence that the association is true given all the available evidence. Table 3 ). The major pathology of COVID-19 is viral pneumonia with pulmonary edema and patchy inflammatory cellular infiltration. The above biological processes or activities may infer in the pathogenic of COVID-19 and these pathological changes may be treated by PDL.

With TCMATCOV, Fig. 2C showed the network of PDL ingredient-drug target-DEGs consists of ingredient-target relations (from BATMAN-TCM, confidence score ≥ 20), and drug target-disease protein relations (protein-protein interaction from the string, confidence score = 0.4). Fig. 2D is the enlarged part of the TCMATCOV network from 

Using STRING, we also analyzed the PDL ingredient-targeted Reactome pathways enrichment. The results indicated that the pathways were enriched in cytokine signaling in the immune system, signaling by interleukins, the immune system, interleukin-4, and interleukin-13 signaling, signal transduction, and interleukin-10 signaling among other pathways (Table 5) 

To make a GSEA pathway enrichment, we used WebGestalt as the enrichment tool with COVID-19 and PDL co-targeted genes with scores for GSEA enrichment. The GSEA enrichment results are shown in Fig. 4A -B and the gene set enrichment plots with P values and enrichment scores were listed in Fig. 4C . As the results showed, the 68 PDL-COVID-19 co-targeted genes were enriched. Ten positively related categories were identified, including tuberculosis, human cytomegalovirus infection, C-type lectin receptor signaling pathway, and Influenza A. Four negatively related categories were also identified, including cholinergic synapse, inflammatory mediator regulation of TRP channels, cAMP signaling pathway, and metabolic pathways.

CRP, IL-6, IL-10, and TNF-α were remarkably higher in severe cases than in moderate cases of COVID-19 [14] . We selected 6 more potential PDL and COVID-19 co-targeted proteins with ingredients for molecular docking using the SwissDock server. The data show these PDL ingredients are well docking with PDL and COVID-19 co-targeted proteins ( Fig. 5A-F) . Among them, IL-6 is an important factor elevated during the pathology of COVID-19 with a cytokine storm [15] . The percentage of IFN-γ producing CD4 + T cells and CD8 + T cells was increased in severe patients of COVID-19 [16] .

Among the PDL ingredients, quinazolinone, and oxysophocarpine may be useful in the treatment of COVID-19. These results can prove that PDL ingredients work with COVID-19 targeted proteins in molecular docking simulation. The results may serve as the validation of the activity of the single substance components of the herb mixture.

Our previous study analyzed the importance of ACE2 and TMPRSS2 in the susceptibility of SARS-CoV-2 infection [17] .Other reports also supposed that integrins [18] and CD147 [19] might be the potential receptors of SARS-CoV-2, and integrins were targeted J o u r n a l P r e -p r o o f as the COVID-19 targeted genes, but they were not predicted in PDL-COVID-19 cotargeted genes. Therefore, PDL might have not effect on integrins and CD147.

PDL, a famous TCM formula recorded in Chinese Pharmacopeia, is widely prescribed for the treatment of acute and chronic inflammation. The reported side effects of PDL include gastrointestinal symptoms and allergic reactions. PDL oral liquid alleviates LPS-induced respiratory injury by decreasing nitroxidative stress and blocking toll-like receptor 4 (TLR4) activation along with nuclear factor kappa B (NF-κB) phosphorylation in mice [20, 21] , and reduces the levels of pro-inflammatory mediators including IL-10, TNF-α, and NF-κB in serum [22] .

Pudilan (PDL) is a four-herb prescription, among which Pu Gong Ying could alleviate inflammatory injury by inhibiting phosphorylation of NF-κB and TLR4/NF-κB signal pathway [23] . Ku Di Ding could inhibit the protein expression of iNOS, TNF-α, IL-6 and IL-1β in vitro and in vivo [24] . Ban Lan Gen could dose-dependently inhibited cleavage activity of the 3C-like protease (3CLpro) of SARS-coronavirus [25] . Baicalin is a bioactive flavone extracted from the Huang Qin was predicted to inhibit the activity of SARS-CoV-2 [26] . The study of the molecular mechanism for PDL and COVID-19 interactions has contributed extensively to the understanding of PDL therapeutic effect on COVID-19 including inflammatory cytokines.

Acute respiratory distress syndrome (ARDS) with cytokine storms might be the main cause of death due to COVID-19. Many inflammatory cytokines (IFN-α, IFN-γ, IL-1β, IL-6, IL-12, IL-18, IL-33, TNF-α, and TGFβ) and chemokines (CCL2, CCL3, CCL5, CXCL8, CXCL9, and CXCL10) were detected in COVID-19 patients [27] . Human coronaviruses (HCoVs) may modulate various cellular processes, such as apoptosis, innate immunity, mitogen-activated protein kinase (MAPK) pathway, and nuclear factor kappa B (NF-κB) pathway [28] . When the host immune system is exposed to viral pathogens, it reacts straightaway by triggering a diverse array of defense mechanisms to establish a more efficacious shield, as characterized by the increased production of type I interferons (IFNα and IFN-β) and other inflammatory cytokines. The cytokine family of interferons is dedicated to the conveyance of the presence of infection [29] . expression at the mRNA level [30] . A recent report indicated that these herbal products could markedly relieve major symptoms such as fever and cough and could promote the recovery. For example, Shen Fu injection inhibited the lung inflammation and decrease the levels of IL-1β, IL-6, and other cytokines [4] . Re Du Ning injection markedly reduced the levels of IL-1β, TNF-α, IL-8, and IL-10 in acute lung injury in a rat model [2] .

PDL was recommended in the treatment for COVID-19, due to its anti-inflammation effects, its capability to reduce fever and to clear the infection, especially in children [5, 31] . PDL also exhibited potent COVID-19 effects and produced good outcomes in the hACE2 mouse model and Vero cells with SARS-CoV-2 infection [6] .

In the network pharmacology analysis, 68 co-targeted genes/proteins were selected as targets of both PDL and COVID-19. PDL works efficiently to block SARS-CoV-2 entry into cells by blocking the ACE2 protein.

Sixty-eight genes were identified as COVID-19, PDL, LHQW, and SFJD co-targeted genes, including ACE2, TNF, IFN-γ, IL-6, TP53, CRP, EGFR, CCL5, IL-10, TGFβ1, BCL2, HSPA5, BAX, IL-1β, PIK3CA, and other genes. Many of these genes were inferred to be involved in the ARDS and cytokine storms. PDL may attenuate cytokine storms by affecting TNF, IFN-γ, IL-6, CRP, EGFR, CCL5, IL-10, TGFβ1, and other genes. These genes may be the hub genes involved in the effects of PDL, LHQW, and SFJD on COVID-19.

In conclusion, our study showed that PDL, a TCM formula, might be useful in the treatment of COVID-19 through regulating and targeting many cytokines and chemokines. PDL could balance the physiological activity, regulate the immune response, inhibit the inflammatory storm in animal and cell experiments. However, these potential targets predicted by bioinformatic and network pharmacology tools need further investigation to confirm. J o u r n a l P r e -p r o o f J o u r n a l P r e -p r o o f J o u r n a l P r e -p r o o f 

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We thank American Journal Experts (www.aje.com) for language standard editing.

The authors declare that there are no conflicts of interest.

Qi Kong designed the project and drafted the manuscript. Xiuping Chen attended to design the project, reviewed the manuscript, and provided comments and suggestions.Other authors were involved in data analysis and interpretation. Acknowledgment: These pictures were drawn based on the database of Reactome.J o u r n a l P r e -p r o o f

Description PDL and COVID- 19 J o u r n a l P r e -p r o o f