Abstract

   Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes
   coronavirus disease 2019 (COVID-19), an epidemic disease characterized
   by rapid infection and a high death toll. The clinical diagnosis of
   patients with COVID-19 has risen sharply, especially in Western
   countries. Globally, an effective treatment for COVID-19 is still
   limited. Vitamin A (VA) exhibits pharmacological activity in the
   management of pneumonia. Thus, we reason that VA may potentially serve
   as an anti-SARS-CoV-2 regimen. In this study, bioinformatics analysis
   and computation assays using a network pharmacology method were
   conducted to explore and uncover the therapeutic targets and mechanisms
   of VA for treating COVID-19. We identified candidate targets,
   pharmacological functions, and therapeutic pathways of VA against
   SARS-CoV-2. Bioinformatics findings indicate that the mechanisms of
   action of VA against SARS-CoV-2 include enrichment of immunoreaction,
   inhibition of inflammatory reaction, and biological processes related
   to reactive oxygen species. Furthermore, seven core targets of VA
   against COVID-19, including MAPK1, IL10, EGFR, ICAM1, MAPK14, CAT, and
   PRKCB were identified. With this bioinformatics-based report, we
   reveal, for the first time, the anti-SARS-CoV-2 functions and
   mechanisms of VA and suggest that VA may act as a potent treatment
   option for COVID-19, a deadly global epidemic.

   Keywords: vitamin A, SARS-CoV-2, network pharmacology, target,
   mechanism

INTRODUCTION

   Coronavirus disease 2019 (COVID-19), a deadly disease induced by the
   novel coronavirus severe acute respiratory syndrome coronavirus 2
   (SARS-CoV-2), was first discovered in China, and is accompanied by a
   high degree of morbidity and mortality [[35]1]. The virus has spread
   across the world and infected very large populations, especially in the
   United States [[36]2]. Clinical features of COVID-19 may include
   pyrexia, asthenia, dyspnea, as well as acute respiratory distress
   syndrome, septic shock, and coagulation dysfunction in critically ill
   patients [[37]3]. It is believed that, due to its high fatality rate,
   SARS-CoV-2 may strongly bind to angiotensin-converting enzyme 2 (ACE2),
   which is a key factor in the pathological pathway of SARS-CoV infection
   in the host [[38]4]. The latest research findings have indicated that
   the affinity of SARS-CoV-2 to ACE2 may be 10 to 20 times greater than
   that of SARS-CoV [[39]5]. In the SARS-CoV-2-infected population, it has
   been reported that people aged over 65 years have the highest rate of
   death [[40]6]. Clinically, an effective and accurate diagnosis of
   SARS-CoV-2 has been achieved. However, the medical pharmacotherapy to
   treat COVID-19 is still insufficient [[41]7]. Accordingly, it is
   necessary to explore and develop bioactive compounds to treat COVID-19.
   Vitamins are considered as a food supplement and have been reported to
   play an important role in the immune system [[42]8]. Vitamin A (VA) has
   potent physiological functions, such as promoting growth and
   reproduction and maintaining bone, epithelial tissue, vision, and
   normal secretion of mucosal epithelium. VA and its derivatives can
   prevent precancerous lesions [[43]9]. An increasing number of reports
   indicate that VA is necessary to maintain immune function and that it
   is responsible for immune cell differentiation and proliferation
   [[44]10]. It has been reported that VA can improve the ability of
   immune cells to produce antibodies and induce T lymphocytes to release
   functional lymphokines through modulation of target genes via nuclear
   receptors [[45]11]. Additionally, VA is extremely important for
   maintaining sufficient levels of natural “killer cells” (showing
   antiviral activity) in circulating blood [[46]12]. VA adjuvant therapy
   may enhance body immune function by increasing IgM and IgG levels and
   activating T lymphocytes [[47]13]. More importantly, vitamin A was
   reported to play a significant role against pneumonia. It is evidenced
   that low VA content is linked to neonatal pneumonia [[48]14]. Clinical
   data show that VA deficiency is implicated in fatal mycoplasma-induced
   pneumonia in children [[49]15]. In addition, VA supplementation
   contributes to the reduction of clinical complications and shortening
   of in-hospital time for children with pneumonia [[50]16]. All these
   pieces of evidence suggest that vitamin A may be an optional treatment
   for COVID-19; however, to date there has been no investigation of VA
   against SARS-CoV-2, especially its pharmacological mechanism. In this
   report, we aimed to determine and identify the curative effect of VA
   for treating COVID-19 and to utilize a network pharmacology approach to
   uncover the mechanisms underlying the therapeutic role of VA.

RESULTS

Identification of SARS-CoV-2- and vitamin A-associated genes

   In order to determine the SARS-CoV-2-associated and VA-pharmacological
   action genes, we conducted a series of bioinformatic analyses. We
   assayed and identified 393 SARS-CoV-2-associated genes from the
   Genecard and OMIM datasets. In addition, 122 VA-pharmacological action
   genes were identified following data correction using the UniProt tool
   ([51]Figure 1). When we compared the target (VA-associated and
   SARS-CoV-2-associated) genes, 15 VA-associated targets against
   SARS-CoV-2 were identified, and the common genes were subjected to
   target-function-protein interaction network analysis ([52]Figure 1). By
   setting the median degree of freedom to 2.923, the maximum degree of
   freedom to 7, and the core target screening condition ranged to 3–7),
   seven core targets of VA against SARS-CoV-2 were identified, namely
   MAPK1, IL10, EGFR, ICAM1, MAPK14, CAT, and PRKCB ([53]Figure 2).

Figure 1.

   [54]Figure 1
   [55]Open in a new tab

   Identification of vitamin A and SARS-CoV-2 associated genes. Venn
   diagram showing the intersection targets of vitamin A against
   SARS-CoV-2 with an identified PPI network.

Figure 2.

   Figure 2
   [56]Open in a new tab

   Gene network of the seven core targets of vitamin A against SARS-CoV-2

Biological role of vitamin A against SARS-CoV-2

   The core targets were subjected to gene ontology (GO) biological
   process and KEGG pathway enrichment analysis to understand the possible
   biological role of VA and the signaling pathway against SARS-CoV-2. The
   GO results highlighted that VA mediated a number of biological
   processes related to cellular response to the virus, immunity, cytokine
   production and secretion, and inflammatory response ([57]Figure 3A and
   [58]Supplementary Table 1). Immune responses are mediated by different
   classes of immune cells, such as neutrophils and lymphocytes, through
   the immune response-activating cell surface receptor signaling pathway.
   The VA-mediated immune response is also regulated by immunoglobulin
   production ([59]Figure 3A and [60]Supplementary Table 1). In addition,
   VA played a role in both acute and chronic inflammatory responses
   against SARS-CoV-2 ([61]Figure 3A and [62]Supplementary Table 1).

Figure 3.

   [63]Figure 3
   [64]Open in a new tab

   Gene ontology analysis of the seven core targets of vitamin A against
   SARS-CoV-2. (A) Bubble diagram showing the vitamin A-mediated
   biological process against SARS-CoV-2. (B) All core biotargets of
   vitamin A against SARS-CoV-2 were linked to the top 10 most enriched GO
   terms in Circro diagrams.

Vitamin A-mediated signaling pathway and network against SARS-CoV-2

   We conducted a KEGG pathway analysis to further understand the possible
   mechanism underlying the anti-SARS-CoV-2 role of vitamin A. Our results
   highlighted that vitamin A might regulate a series of signaling
   pathways related to viral infections, such as human cytomegalovirus,
   influenza A, Kaposi sarcoma-associated herpes virus, human
   immunodeficiency virus 1, hepatitis C, Epstein-Barr, and human
   papilloma virus ([65]Figure 4 and Online Resource 2). In addition, we
   also found that vitamin A was involved in many important cell signaling
   pathways such as FoxO, VEGF, TNF, Ras, nuclear factor kappa B (NF-κB),
   phospholipase D, mTOR, and JAK-STAT ([66]Figure 4 and [67]Supplementary
   Table 2). More importantly, we found the involvement of vitamin A in
   immune responses such as T cell receptor signaling pathway, leukocyte
   transendothelial migration, natural killer cell mediated cytotoxicity,
   Fc epsilon RI signaling pathway, B cell receptor signaling pathway, Th1
   and Th2 cell differentiation, Fc gamma R-mediated phagocytosis, IL-17
   signaling pathway, Toll-like receptor signaling pathway, Th17 cell
   differentiation, and human T-cell leukemia virus 1 infection
   ([68]Figure 4 and [69]Supplementary Table 2). Vitamin A regulates
   cytokine production via NOD-like receptor signaling pathway and
   chemokine signaling pathway ([70]Figure 4 and [71]Supplementary Table
   2). These findings suggest that vitamin A could be a potential
   treatment for COVID-19. Finally, we used Cytoscape software to
   construct the gene network diagram of VA-target-GO-KEGG-SARS-CoV-2
   ([72]Figure 5).

Figure 4.

   [73]Figure 4
   [74]Open in a new tab

   Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of the
   seven core targets of vitamin A against SARS-CoV-2. (A) Bubble diagram
   showing the vitamin A-mediated cell signaling pathways against
   SARS-CoV-2. (B) Identified core biotargets of vitamin A against
   SARS-CoV-2 were associated with the 10 most enriched KEGG terms in
   Circro diagrams.

Figure 5.

   [75]Figure 5
   [76]Open in a new tab

   Interaction network of the target-KEGG pathways of VA against
   SARS-CoV-2. The middle part represented the anti-SARS-CoV-2 targets of
   vitamin A, and the enriched top 20 biological functions and KEGG
   pathways.

DISCUSSION

   Recently, the prevalence and mortality of SARS-CoV-2 has increased
   sharply worldwide, especially in developed countries in 2020 [[77]17].
   However, there are few existing drug treatments for COVID-19, and some
   of the immunotherapy treatments and antiviral drugs used in clinical
   practice have shown limited therapeutic effectiveness [[78]18]. Thus,
   further investigation and development of potential therapeutic agents
   is warranted. In this bioinformatics report, we aimed to identify and
   reveal the anti-SARS-CoV-2 targets and molecular mechanisms of VA
   through an emerging approach to network pharmacology. VA may exert
   potent, beneficial pharmacological activity for the treatment of
   COVID-19 via associated cytoprotection, anti-viral and
   anti-inflammatory effects, and immunoregulation. By using the network
   pharmacology strategy to uncover the molecular functions, the
   anti-SARS-CoV-2 effects of VA could be shown to be modulated by some
   key molecules and corresponding genes, including MAPK1, IL10, EGFR,
   ICAM1, MAPK14, CAT, and PRKCB. In our previous report, we demonstrated
   that vitamin C could also modulate a cluster of core targets against
   SARS-CoV-2 [[79]19]. When we compared the findings, we found that two
   core targets, MAPK1 and EGFR, were found in the effect of both vitamin
   A and vitamin C. MAPK1, a functional protein kinase, is a key
   connection in the switch from extracellular irritation to intracellular
   signaling. Changes in the signaling pathway are evidenced in complex
   diseases, including cancers [[80]20]. It has been reported that
   anti-pneumonia action exerted by VA is related to specific suppression
   of the MAPK signaling pathway, including MAPK1 activity [[81]21]. EGFR,
   a tyrosine kinase receptor, plays important roles in modulating cell
   proliferation, division, differentiation, survival, and oncogenesis
   [[82]22]. It has been reported that EGFR-mutated patients are likely to
   have hospital-acquired pneumonia [[83]23]. IL-10, a pivotal
   anti-inflammatory cytokine, can effectively control inflammatory Th
   cells and immunopathology and secure cellular homeostasis [[84]24].
   Some reports have shown that inactivation of IL-10 is linked to an
   increased risk of developing pneumonia [[85]25–[86]26]. ICAM-1, a
   transmembrane glycoprotein receptor, can recruit inflammatory cells and
   cytokines to target tissue [[87]27]. Reportedly, blood ICAM-1 content
   may function as a potent biomarker of patients with pneumonia [[88]28],
   including pediatric pneumonia [[89]29]. Catalase, a well-known
   antioxidant enzyme, acts as an oxidative catalyst for some biological
   functions [[90]30]. Biologically, catalase is found to have potential
   therapeutic effectiveness against influenza-induced pneumonia [[91]31].
   PRKCB, an important regulator of B cells, can regulate metabolic and
   mitochondrial reprogramming responsible for B cell fate [[92]32]. Some
   evidence indicates that PRKCB overexpression is associated with the
   development of pneumonia via activation of the NF-κB pathway [[93]33].
   Collectively, these predictive peptides may be used as potential
   markers for detecting SARS-CoV-2 and may serve as pharmacological
   targets against SARS-CoV. In further bioinformatics and computational
   assays, the therapeutic mechanisms of VA for managing COVID-19 could be
   conjunctively actualized through collective regulation of the FoxO
   signaling pathway, GnRH signaling pathway, PD-L1 expression, and PD-1
   checkpoint pathway. Accordingly, VA demonstrates several
   pharmacological mechanisms against SARS-CoV, namely, cytoprotective
   action, anti-viral activity, anti-inflammatory effects, and
   immunity-based immunomodulation. The anti-coronavirus benefits may be
   the dual efficacy of a nutrient agent and bioactive compound to treat
   complex disease by synergistically modulating all presumptive
   multi-targets and multi-pathways. Adjuvant supplementation of VA may
   enhance the therapeutic efficacy of current clinical anti-viral agents
   and immunotherapy to treat potentially fatal COVID-19. However, the
   current findings should be further validated clinically.

   In conclusion, the bioinformatics and computational findings from this
   study highlight the role of vitamin A in anti-viral, anti-inflammatory,
   and immunomodulatory effects via different biological processes and
   cell signaling pathways, as revealed by network pharmacology analysis.
   More importantly, VA may be used clinically for the treatment of
   COVID-19, as evidenced by the identified biological processes—which
   indicate pharmacological functions—and the signaling pathways, which
   suggest therapeutic mechanisms ([94]Figure 6).

Figure 6.

   [95]Figure 6
   [96]Open in a new tab

   A schematic diagram to summarize the workflow of the study.

MATERIALS AND METHODS

Identification of candidate genes activated by VA

   By using online datasets, in particular TCMSP, Drugbank, SuperPred,
   Swiss Target Prediction, ChemMapper, and BATMAN TCM, all target genes
   activated by VA were obtained. The genes activated by VA were collected
   for target correction from reviewed (Swiss-Prot) and Uniprot databases.
   The Genecard and OMIM datasets were employed to harvest SARS-CoV-2
   genes. All the shortlisted genes and targets of VA and SARS-CoV-2 were
   subjected to intersection analysis via an online platform with
   graphical output
   ([97]http://bioinformatics.psb.ugent.be/webtools/Venn/) in the form of
   Venn diagrams were plotted to display the relational VA-activated genes
   and targets of VA against SARS-CoV-2 [[98]34, [99]35].

Gene Ontology (GO) and KEGG pathway enrichment analyses

   The R-language package ClusterProfiler, ReactomePA, org.Hs.eg.Db, and
   GOplot were applied in the assay and visualization of the relational
   targets. GO analysis was conducted with the use of org.Hs.eg.Db, with a
   p-value cutoff of 0.05 in enriched output of bubble charts and
   Circos-circle charts. The Pathview package in R-language was used to
   merge the relational targets of enriched KEGG pathways by drawing
   pathway diagrams [[100]36, [101]37].

Construction of network visualization in core targets

   The software Cytoscape (v3.7.1) was used to plot the
   component-target-pathway network and the GO biological process and KEGG
   pathway-based visual graphics of VA against SARS-CoV-2 [[102]38,
   [103]39].

Establishment of the biological process and KEGG molecular pathway of VA

   To compare the anti-SARS-CoV-2 effects of VA, bioinformatics data of
   top biological functions and KEGG-enriched pathways were applied for
   pairwise comparison, including the value of -log10 (p-adjust) as the
   heat-map parameter, and the heat-map was drawn using HemI 1.0 software
   [[104]19, [105]40].

Construction of construction of protein-protein interaction (PPI) network
graphics of core targets

   The relational targets of VA against SARS-CoV were employed as inputs
   to the software STRING for plotting target-to-target network
   interactions and target interaction PPI network diagram. The
   NetworkAnalyzer setting in Cytoscape software was applied to analyze
   topology parameters, such as median degrees of freedom and maximum
   degrees of freedom in the network. The optimal targets were collected
   based on degree values (DV). The upper limit of the filtering range was
   the maximum DV in the topology data, and the lower limit was twice the
   median of freedom, as previously reported [[106]41, [107]42].

Supplementary Material

   Supplementary Table 1
   [108]aging-12-103888-s001..xlsx^ (76.9KB, xlsx)
   Supplementary Table 2
   [109]aging-12-103888-s002..pdf^ (272.4KB, pdf)

Abbreviations

   CAT
          Catalase

   EGFR
          Epidermal growth factor receptor

   ICAM1
          Intercellular Adhesion Molecule 1

   IL10
          Interleukin 10

   KEGG
          Kyoto Encyclopedia of Genes and Genomes

   MAPK1
          Mitogen-Activated Protein Kinase 1

   MAPK14
          Mitogen-activated protein kinase 14

   OMIM
          Online Mendelian Inheritance in Man

   PRKCB
          Protein kinase C beta type

   TCMSP
          The traditional Chinese medicine systems pharmacology database
          and analysis platform

Footnotes

   CONFLICTS OF INTEREST: The authors declare that they have no conflicts
   of interest.

   FUNDING: This study was supported by the National Natural Science
   Foundation of China (No. 81560134) and the National Natural Science
   Foundation of Guangxi (No. 2019GXNSFBA185015; 2018GXNSFAA281153). Keng
   Po Lai was supported by Hong Kong SAR, Macao SAR, and the Taiwan
   Province Talent Young Scientist Program of Guangxi.

REFERENCES