NSAIDs-genes networking analysis demonstrated that the clinical effect of NSAIDs on COVID-19 was directly related to 26 genes. The results of KEGG pathway enrichment analysis of 26 genes suggested that 26 signaling pathways were associated with the occurrence and development of the COVID-19 symptoms. The correlations of 26 signaling pathways with COVID-19 symptoms were succinctly discussed as follows. PPAR (Peroxisome Proliferator-Activated Receptor) signaling pathway: A report shows that PPARγ (Peroxisome Proliferator-Activated Receptor-gamma), PPARα (Peroxisome Proliferator-Activated Receptor-alpha), and PPARβ/δ (Peroxisome Proliferator-Activated Receptor-beta/delta) agonists have anti-inflammatory and immunomodulatory functions 16. MAPK (Mitogen-Activated Protein Kinase) signaling pathway: The mechanisms of p38 MAPK inactivation might be a significant therapy against the SARS infected cells 17. Additionally, MAPK stimulates cytokine production such as IL-10 (Interleukin 10), TNF- α (Tumor Necrosis Factor-Alpha), IL-4 (Interleukin 4), and IFN- γ (Interferon gamma) 18. This report shows a coincidence with our suggested strategy in this study. ErbB (Erythroblastic Leukemia Viral Oncogene Homolog) signaling pathway: ErbB signaling reduces the proinflammatory activation in cardiac cells 19. RAS (Renin Angiotensin System) signaling pathway: Inactivation of RAS can reduce tissue damage in COVID-19 patients. In addition, ACE (Angiotensin Converting Enzyme) antagonists block the response of RAS system 20. cGMP-PKG (Cyclic GMP-Protein Kinase G) signaling pathway: The activation of cGMP-PKG signaling inhibits inflammatory response in the prostate, and also decreases CCL5 (C-C Motif Chemokine Ligand 5) release in CD8 + T cells (Cluster of Differentiation 8 T cells) 21. cAMP (Cyclic Adenosine Monophosphate) signaling pathway: The elevation of cAMP leads to diverse cellular effects, such as airway smooth muscle relaxation, repressed effects on cellular inflammation, and immune responses 22. NF-κB (Nuclear Factor kappa-light-chain-enhancer of activated B cells) signaling pathway: Activation of the NF-κB signaling pathway gives rise to the inflammation induced by the SARS-CoV infection. In contrast, NF-κB inhibitors are the potential antivirals against SARS-CoV, and can also contribute to other pathogenic human coronaviruses 23. FOXO (Forkhead box protein O1) signaling pathway: Decrease of FOXO3 (Forkhead box protein O3) in T cells inhibits apoptosis, enhances multifunction of CD8 cells, and elevates viral control 24. Sphingolipid signaling pathway: Sphingolipids play a vital role to protect lung from damages, and the control of sphingolipid signaling pathways may give a good therapeutic efficacy 25. Wnt (Wingless/Integrated) signaling pathway: Wnt signaling involves with the prime inflammatory pathways like intestinal inflammation. Also, elucidating the mutual modes of Wnt ligands and cytokines manifest new treatment strategies for chronic colitis and other inflammatory diseases 26. VEGF (Vascular Endothelial Growth Factor) signaling pathway: A report suggested that VEGFA (Vascular Endothelial Growth Factor A) is inhibited by the activation of ACE2 (Angiotensin-Converting Enzyme 2). However, ACE2 is downregulated by the attack of COVID-19. Subsequently, activation of VEGFA elevates vascular permeability and severity of endothelial damage 27. TLR (Toll-like receptor) signaling pathway: Toll-like receptors (TLRs) play a pivotal role in the innate immune system and contribute to defend host cells by recognizing PAMPs (Pathogen-Associated Molecular Patterns) induced by various microbes 28. The activation of TLRs triggers an array of response resulting into expression of different cytokines and chemokines, phagocytosis, and even apoptotic case activation to induce programmed cell death 29. NOD-like receptor (NLR) signaling pathway: Nod-like receptors (NLRs) have been revealed as the major microbial signals that take part in the universal immune responses to infection, and also contribute to the prevention of infections 30. RIG-I-like receptor (RLR) signaling pathway: RIG-I-like receptors (RLRs) play a vital role in pathogen sensor of RNA virus infection, which enhances the antiviral immunity by sensing foreign RNA 31. IL-17 (Interleukin-17) signaling pathway: IL-17 receptor inhibitors are widely used to ameliorate the inflammatory acuteness to date. Furthermore, it is a potential target to suppress severe inflammation induced by COVID-19 32. Fc epsilon RI signaling pathway: Fc epsilon RI interconnecting causes mast cell degranulation and synthesis of proinflammatory mediators 33. TNF (Tumor Necrosis Factor) signaling pathway: TNF deficit is associated with dysfunctional secretion of inflammatory cytokine, leading to lung pathology and death during respiratory poxvirus infection, and thus TNF is very significant element for regulating inflammation 34. Neurotrophin signaling pathway: COVID-19 causes severe brain damage and destruction of central nervous system derived from neurotrophin (Huang and Reichardt 2001). Insulin signaling pathway: Obesity-oriented insulin resistance is associated with the induction of proinflammatory macrophage, leads to inflammation of adipose tissue 37. GnRH (Gonadotropin-Releasing Hormone) signaling pathway: Disrupted BBB (Blood Brain Barrier) by viral infection, lymphocytes (B and T cells), monocytes, and granulocytes can penetrate in the brain parenchyma which induce inflammation, resulting in dysregulation of GnRH neurons. Additionally, the inflammation of GnRH neurons inhibits GnRH transport through proinflammatory cytokines by impairing the cytoskeleton 38. Prolactin signaling pathway: HIV (Human Immunodeficiency Virus) patients have greater prolactin quantity compared to others. Besides, prolactin is regarded as a cytokine to react in immune system 39,40. Adipocytokine signaling pathway: Adipocytokines stimulate inflammation and disrupting immune response which cause tissue damage. Adipocytokines might also induce proinflammation in RA (Rheumatoid Arthritis) patients and thus lead to the development of bone damage 41. Oxytocin signaling pathway: Oxytocin interrupts the production of proinflammatory cytokines by inactivating of the eIF-2α–ATF4 (Eukaryotic Initiation Factor -2 alpha- Activating Transcription Factor 4) pathway 42. Relaxin signaling pathway: Relaxin inhibitors are good therapeutic targets to suppress inflammation caused by airway dysfunction 43. AGE-RAGE (Advanced Glycation End product -Receptor of Advanced Glycation End product) signaling pathway in diabetic complications: The binding of AGE to its receptor RAGE can trigger the cytokine production, thus, can cause tissue damages, while the blockage of AGE-RAGE can effectively curtail the inflammation 44. Epithelial cell signaling in Helicobacter pylori infection: Helicobacter pylori interrupts T and B cell signaling to set immune system. It is apparent that COVID-19 patients with Helicobacter pylori might be vulnerable to inflammatory responses 45.
Generally, SARS-CoV-2 invades in the lungs and throat, induces excessive inflammation, which causes the secretion of cytokines, resulting in severe complications like acute respiratory failure, pneumonia, and acute liver injury (Reyes and Peniche 2019; Nile et al. 2020). Researchers suggested that RAS is a potential route for SARS-CoV-2 induced cellular infection which may be linked to the imbalance of RAS. It was discovered that ACE-2 is the functional receptor for the SARS-CoV-2 to trigger infection in the lung alveolar epithelial cells. The internalization of virus leads to downregulate the ACE-2 on host cell surface that could cause the elevation and demotion of the angiotensin-II (AII) and angiotensin 1-7 (A1-7) respectively. Such an imbalance between these angiotensins may induce deleterious effects in the lung and heart. Thus, the SARS-CoV-2 affects humans through this mechanism 49–52. Therefore, blockade of the RAS may restore the RAS balance by reducing the deleterious effects associated with angiotensin-II 53. Recent evidence showed that RAS inhibitors might be a promising target for relieving acute-severe pneumonia caused by the COVID-19 54.
Interestingly, our study identified that the three genes (MAPK 8, MAPK 10, and BAD) are mainly associated with the RAS signaling pathway. MAPK 8 and MAPK 10 are members of the MAPK family which are the key mediators of the inflammation, vasoconstriction, and thrombosis. Besides, overwhelming heart and lung injury in COVID-19 infection might be due to the overactivation of MAPK 55. Therefore, inactivation of these genes can also be a viable strategy for relieving COVID-19 induced organ injury. In addition, disposal of inflammatory cells by promoting the cell death can be an innovative approach to control excessive inflammation. In this regard, inhibition of the anti-apoptotic Bcl-2 gene can also be a potential target to lessen inflammation 56,57. Our findings also explored that MAPK8, MAPK10 and BAD genes are related to three, twelve, and two NSAIDs, respectively. During the molecular docking analysis, 6MNA, rofecoxib, and indomethacin revealed promising binding affinity along with highest docking score against MAPK8, MAPK10 and BAD genes, respectively, which indicated that the three (6MNA, Rofecoxib, and Indomethacin) NSAIDs are very potential among all others, may possibly block the RAS signaling pathway by inactivating its associated genes (MAPK8, MAPK10 and BAD), and subsequently suppress SARS-CoV-2 induced cytokine storm.
Among various NSAIDs, indomethacin is a current drug of interest to the clinicians. Primary care physicians (New York) reported that indomethacin had been prescribed to a large number of COVID-19 patients and observed quick recovery from cough, pain, and other symptoms. Such improvements and well-being benefits were not evident in the case of ibuprofen and hydroxychloroquine implementation (Vaduganathan et al. 2020b; Little 2020). Importantly, many researchers previously reported varying degrees of antiviral activity of indomethacin against herpesvirus 60, pseudorabies virus 61, cytomegalovirus 62, hepatitis B virus 63, vesicular stomatitis virus 64, rotavirus 65, and canine coronavirus 11. In contrast, 6MNA (active metabolite of nabumetone) and Rofecoxib are also the potential anti-inflammatory drugs, but studies disclosed that they are less potent compared to the indomethacin 66,67. Hence, such compelling outcomes indicate that indomethacin can be considered to use alone or in combination for antiviral therapy which may assist in combating human coronavirus (SARS-CoV-2).
In summary, NSAIDs-genes network suggested that the therapeutic effect of NSAIDs on COVID-19 was associated with 26 signaling pathways. This study suggests that 6MNA, rofecoxib, and indomethacin are the most potent NSAIDs against COVID-19. The basis of this research is an understanding of how these NSAIDs (which stimulates anti-inflammatory processes against COVID-19) work on COVID-19 patients. That scientific evidence informs the selection of NSAIDs, in turn, provides for clinical design against COVID-19. Our research suggests that the inhibition of BAD-Indomethacin with other two hub genes MAPK8-6MNA, MAPK10-Rofecoxib might play cumulative actions by inactivating the RAS signaling pathway against COVID-19. Most recently, efficacy of indomethacin against COVID-19 has been approved clinically. Our study presents that indomethacin is a potent therapeutic candidate to relieve COVID-19 symptoms, which is in line with the many previous studies. However, further clinical trial on indomethacin should be warranted in COVID-19 patients in order to slow down the progression of SARS-CoV-2 and mitigating the severity as well.