Network pharmacology reveals physiological manifestations of viperid envenomation and role of nitric oxide in their treatment

Network pharmacology reveals physiological manifestations of viperid envenomation and role of nitric oxide in their treatment | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Network pharmacology reveals physiological manifestations of viperid envenomation and role of nitric oxide in their treatment Madhu Sindhu, Umesh Thakur, Shiwani Latwal, Diksha Muwal, Anju Rao This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4512510/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Snakebite envenomations pose significant global health challenges with limited effective treatments available. The complex physiological manifestations induced by snake venoms, such as hypoxia, vasoconstriction, and pain, have not fully deciphered at the genetic level. This study employs network pharmacology combined with gene expression analysis to uncover the molecular mechanisms underlying these interventions, and to explore nitric oxide as potential therapeutic target for snakebites. We used NCBI and GeneCards databases to collect the gene expression profile and therapeutic targets for snake bites. We identified that upregulation of genes like HIF1A and HIF3A, and downregulation of EGFA indicate responses to venom induced hypoxia. Change in expression of phospholipases and KNG1 suggests alteration in mechanisms involved in vasoconstriction. The increase in expression of cytokines and PTGS2 potentially linked to inflammation and pain induction. We identified 100 nitric oxide-related genes in mouse including 20 key genes directly involved in these responses to envenomation. The protein-protein interaction analysis through Cytoscape indicates that nitric oxide could play pivotal role in neutralizing venom effects. We identified MAFK as master regulator in nitric oxide associated genes set. Our observations highlight a previously unrecognized patterns of gene expression linked to hypoxia, vasoconstriction, and pain, and lays the groundwork for innovative approaches for treating snakebites. Biological sciences/Drug discovery Biological sciences/Physiology Biological sciences/Systems biology Health sciences/Risk factors Health sciences/Signs and symptoms Snakebite Network pharmacology Nitric oxide Hypoxia Vasoconstriction Pain Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Snake bite is a neglected tropical disease. According to World Health Organization (WHO) data, more than 5 million people suffer from snake bites and results in about 81,000 to 138,000 deaths each year. Survivors often face long term disabilities, both physical and psychological. [ 1 ] A recent data indicates that deaths due to snake bites in India is around 58,000, comprising more than half of global fatalities, with vipers being the primary reason for majority of these deaths. [ 2 ] Viperid snake venom is highly complex and essentially proteinaceous in nature. Peptides, phospholipases, venom metalloproteinases, and serine proteinases contribute to toxicity of venom. Vipers are notorious for causing severe bites with both local and systemic effects, including neurotoxicity, hemotoxicity, coagulopathies, inflammation, and multiple organ failure. [ 3 ] Incidences of snake bites are disproportionately high in agricultural workers and children in rural communities, who often lack timely access to health care facilities. Antibody-based antivenom is a primary treatment for these bites, which often leads to anaphylactic reactions. [ 4 ] Moreover, maintenance of steady supply of antivenom in rural and remote areas is difficult due to production complexity, stringent storage conditions and shorter shelf life. [ 5 ] Considering the challenges associated with production and distribution of antivenom, it is important to explore alternative treatments for snake bites. Network pharmacology is a rapidly developing area in the field of drug discovery and development. [ 6 ] It integrates computational methods, high-throughput omics, and advanced pharmacological analysis, and can identify the interactions between compounds and their biological targets. [ 7 ] Traditional medicines have developed a vast array of treatments based on extensive clinical research and often target multiple pathways. To uncover the basis of traditional medicines, network pharmacology involves viewing entire biological system involved in the disease as a therapeutic target, to understand the mechanism of multitarget drugs. [ 8 ] In this context, a documented formulation from Jind district of Haryana, India, can offer valuable pharmacological insights which include kalmi shora or potassium nitrate as one of the ingredients. Potassium nitrate could be explored in the treatment of snakebite using network pharmacology. The rationale of using potassium nitrate in the treatment of snakebites is supported by its physiological properties. Potassium ions are known for their role in maintaining intracellular osmotic balance and cell membrane potential. Potassium ions decrease nerve excitation and pain by disturbing the synapse between nerve endings. [ 9 ] Nitrates are rapidly absorbed in proximal and small intestine and evenly distributed among body organs. Nitrates undergo reduction to nitrites by bacteria in oral cavity and nitrites can be further reduced to nitric oxides (NO). NO is a critical signaling molecule with wide ranging effects in physiological systems. NO is known for its role in vascular biology, immune system function, respiratory function, hemostasis, pain modulation. [ 10 , 11 , 12 ] Thus, metabolized nitrate in the form of NO was selected to study its impact on venom induced manifestations. Snakebite envenomation can cause severe and lasting systemic and neurological damage. [ 13 ] NO involvement in these manifestations can help to treat some critical impacts of snake venom. Neurotoxin in snake venom can cause oxygen deprivation results in hypoxic brain injury, manifesting critical conditions such as Lance- Adams syndrome. [ 14 ] Snake venom also causes immediate swelling and vasoconstriction leading to tissue necrosis by immediate affect due to cellular damage and vascular injuries. [ 15 ] This can trigger inflammatory reactions along with systemic injuries can cause or intensify severe pain, that can be local or remote. Pain in snake bites is heavily influenced by biological reactions of venom compounds. [ 16 ] These manifestations underline the importance of studying the effect of snake venom at genetic level. Genetic profiling of responses to snake venom can help in understanding physiological manifestations of venom, variations in susceptibility and outcomes among different populations. This is critical for understanding the physiology of snakebite envenomation. [ 17 ] Integrating the critical examination of genetic expression in response to snakebite envenomation with therapeutic potential of potassium nitrate can provide us a comprehensive strategy for understanding the physiology of snake bites. By utilizing the gene expression data of viperid envenomation from NCBI-GEO database, we analyzed the effects of snake venom on crucial genes associated with hypoxia, vasoconstriction, and pain. Upon intersecting the targets of nitric oxide with physiological manifestations, we aim to enhance effective treatment methodologies. By integrating this data, we can propose new treatments based on targeted effects of venom by increasing precision of treatment. The flow chat of study has shown in Fig. 1 . Materials and methods Acquisition of gene expression profile of venom-treated tissue and target genes of hypoxia, vasoconstriction, pain agnosia, and nitric oxide The gene expression profile of venom treated tissue was obtained from Gene Expression Omnibus (GEO), a public repository of NCBI using keyword ‘snake venom,’ and the data was available under heading ‘A complex pattern of gene expression in tissue affected by viperid snake envenoming: the emerging role of autophagy related genes’ ( https://www.ncbi.nlm.nih.gov/ ). The therapeutic targets for hypoxia, vasoconstriction, pain agnosia, and nitric oxide were sourced from the GeneCards database and selected on basis of relevance scores ( https://www.genecards.org/ ). This approach allowed us to identify the pattern of gene expression in the tissue affected by viperid venom and to enlist the genes involved in these physiological conditions. Screening of common targets To identify the overlapping elements with mouse gene expression profile, genes involved in hypoxia, vasoconstriction, pain agnosia, and NO were crossed with genes expressed in tissue affected by venom. We then compiled the genes of hypoxia, vasoconstriction, and pain agnosia, while removing redundant values. Finally, the obtained gene set was intersected with genes associated with nitric oxide. This intersection was important in identifying different genes combinations for different physiological manifestations, offering insights for pharmacological targets of NO. We used Venny 2.1, a free interactive tool for comparing lists and creating Venn diagrams ( https://csbg.cnb.csic.es/BioinfoGP/venny.html ). This tool enabled us to screen and visualize the gene sets of hypoxia, vasoconstriction, and pain agnosia in mouse tissue, and therapeutic targets of NO. Acquisition of protein-protein interaction (PPI) network data To construct the regulatory network of intersecting genes associated with hypoxia, vasoconstriction, pain agnosia, and targets of NO in these physiological manifestations in mice, we used the STIRNG (v12.0; Search Tool for the Retrieval of Interacting Genes/Proteins; https://string-db.org/ ) online database. The interaction score was set at medium confidence > 0.400 and the species was set to the ‘ Homo sapiens.’ Later, the important nodes were visualized by setting maximum FDR value < = 0.05 to reduce the redundancy and selectively highlight the functional enrichments in our networks. Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis The enrichment analysis of therapeutic targets of NO was done using ShinyGO, a web-based application ( http://bioinformatics.sdstate.edu/go76/ ). GO and KEGG pathway enrichment analysis was performed to identify the significantly enriched biological processes and pathways in overlapping genes. For analysis, maximum FDR cut off was set below 0.05, processes and pathways below this value were considered significant. The top 10 results of GO modules, biological processes (BP), cellular components (CC), and molecular function (MF) and KEGG pathway enrichments were represented using bar graphs. The degree of enrichment was presented using fold enrichment and -log10 transformed p values. PPI network analysis Later, the PPI network of NO-associated genes was imported to Cytoscape software (v3.10.2; https://cytoscape.org ). The hub genes from our network were identified by applying closeness, degree, edge percolated component (EPC) and maximum neighborhood component (MNC) algorithms in CytoHubba plugin (v0.1; https://apps.cytoscape.org/apps/cytoHubba ). Top 10 scoring genes in each category were selected and removed duplicates to identify the hub genes. For subcluster analysis, we used MCODE plugin (Molecular Complex Detection, v2.0.3; http://apps.cytoscape.org/apps/mcode ) and degree cutoff, maximum depth, k-core, and node score cutoff were set at 2, 100, 2, and 0.2 respectively. Finally, iRegulon plugin (v1.3; http://apps.cytoscape.org/apps/iregulon ) was used to detect master regulons based on motif enrichment in NO-associated gene set. The iRegulon predictions based on analysing transcription factor (TF) motifs and ChIP-Seq data to predict TFs regulating the given set of genes [ 18 ]. Key parameters included minimum identity between orthologous gene and false discovery rate (FDR), set at 0.05 and 0.001 respectively. The top 10 transcription factors were selected based on normalized enrichment score. Gene expression profile analysis The expression profiles of the targets of hypoxia, vasoconstriction, and pain agnosia and of identified hub genes were represented by generating heat maps. The heat maps were created using SRPLOT, an online tool for graphical and statistical analysis of biological data ( https://www.bioinformatics.com.cn/srplot ). For generating heat maps, complete clustering was selected along with bidirectional orientation, and distance method was configured at Euclidean distance. The heat maps were made using Log2 of transcripts counts of set of significant genes in each physiological condition and of hub genes. Results and Discussion Indian system of medicine has frequently used salts in the compositions for the treatment of different ailments. One such formulation is documented from Jind district of Haryana, India, that is used in the treatment of snakebites and aphid stings, and it includes potassium nitrate as one of the ingredients. Motivated by this ingredient, our study explores the potential of nitrates in the treatment of snakebites. Nitric oxide (NO) is metabolic product of dietary nitrate, and peak plasma levels are achieved within 2–3 hours. [19.] Nitric oxide plays a pivotal role in physiological processes such as hypoxia, vasoconstriction, and pain agnosia- the common complications of snakebites. [ 20 , 21 , 22 ] Such intersections lead us to study the network pharmacology of NO in relation to snakebite treatment. Considering the challenges of snake bites where professional medical help is not immediately available, properties of NO derived from nitrates offers a promising alternative. The ease of administration, and the speed at which NO becomes bioavailable make it promising candidate for emergency treatment in remote or resource limited areas. [ 23 ] Acquisition of gene expression profile Our study utilized the NCBI-GEO database, it provided supplementary file of gene expression profiles in mouse skeletal muscles at 1 hours, 6 hours, and 24 hours after injecting venom of Bothrops asper and Daboia russelii . For controls, mice were injected with phosphate-buffer saline (PBS). This data involved the mapped mouse genes to their human equivalents. We obtained a list of 760 genes expressed in the tissue affected by viperid venom. The expression profile under the influence of venom of B. asper was used in this study to analyze the pattern of gene expression in the tissue. We pinpointed gene targets associated with the key complications of snakebites: hypoxia, vasoconstriction, and pain agnosia, and the pathways involving nitric oxide in these complications. Acquisition of targets of physiological manifestations and NO For target screening from gene expression profile, the targets of physiological complications and NO were meticulously sorted and selected based on relevance scores from the GeneCards database. We obtained 52, 44, 77, and 395 targets for hypoxia, vasoconstriction, pain agnosia and nitric oxide respectively from GeneCards. Upon intersection with mouse genes, a total of 11, 5, 19, and 100 genes corresponding to hypoxia, vasoconstriction, pain agnosia and nitric oxide were found overlapping. Four genes namely, PLCB2, PLCB3, PLCG1, and PLCG2 were added to the intersection list of vasoconstriction and 9 genes were analyzed by then. From the aggregated gene set of hypoxia, vasoconstriction, and pain agnosia, we obtained a set of 33 unique genes by removing duplicates. Lastly, by intersecting the combined values (33) and genes associated with nitric oxide (100) pathway, a total of 20 genes were found common and regarded as targets of NO in physiological manifestation of viperid envenomation (Fig. 2 . A- D). Protein-protein interaction network The protein-protein interaction network of intersecting genes associated with hypoxia, vasoconstriction, pain agnosia, and therapeutic targets of nitric oxide in mice was created using STRING. The interactions were visualized by selecting functions and pathways relevant for study from functional enrichments segment in the tool. This selective visualization allowed us to see the part of network that contributed to specific enrichments, and how these enrichments are interconnected through shared proteins (Table 1 – 3 ). The PPI networks are represented by highlighting nodes (Fig. 3 . A- C). Table 1 Highlighted functional enrichments in PPI network of hypoxia associated genes GO Term FDR Functional Enrichment Connected Nodes GO:0001666 5.91e-07 Response to hypoxia EPAS1, EP300, HIF1A, HIF3A, HMOX1, MTOR, TNF GO:0043619 2.63e-05 Regulation of transcription from DNA polymerase II promoter in response to oxidative stress EPAS1, HIF1A, HMOX1 GO:0051000 0.0049 Positive regulation of nitric oxide synthase activity HIF1A, TNF GO:0051403 0.0245 Stress- activated MAPK cascade TNF, MAPK1 Table 2 Highlighted functional enrichments in PPI network of vasoconstriction associated genes GO Term FDR Functional Enrichment Connected Nodes GO:0004435 2.70e-06 Phosphatidylinositol phospholipase C activity PLCB2, PLCB3, PLCG1, PLCG2 GO:0042311 0.0013 Vasodilation KNG1, NOS3, SOD1 GO:0051924 0.0022 Regulation of calcium ion transport NOS3, PTGS2, PLCG1, PLCG2 GO:0019229 0.0454 Regulation of vasoconstriction MMP2, PTGS2 Table 3 Highlighted functional enrichments in PPI network of pain agnosia associated genes GO term FDR Functional enrichment Connected Nodes GO:0000165 4.14E-09 MAPK cascade BRAF, CTNNB1, EGFR, IL1B, TGFB1, KRAS, NF1, TNF GO:0060559 0.00024 Positive regulation of calcidiol 1-monooxygense activity IL1B, TNF GO:0046328 0.00024 Regulation of JNK cascade APP, EGFR, IL1B, TNF, GO:1900017 0.0051 Positive regulation of cytokine production involved in inflammatory response IL6, TNF GO:0045202 0.0184 Synapse APP, AKT1, BRAF, CTNNB1, MECP2, PNOC, NF1 GO:0031394 0.00074 Positive regulation of prostaglandin biosynthetic process PTGS2, IL1B GO:0033280 0.0065 Response to vitamin D PTGS2, TGFB1 GO:0001666 0.00014 Response to hypoxia PTGS2, TGFB1, NF1, TNF, MECP2 Expression profile analysis of genes associated with physiological manifestations and generation of heat maps The analysis of expression pattern of genes associated with hypoxia facilitates identification of key genes involved in the cellular adaptation to hypoxia, these include VEGFA, CTNNB1, AP300, MTOR, EPAS1, TNF, MAPK1, HIF1A, HIF3A, HMOX1, and IL6 (Fig. 4 . (A)). The upregulated genes as compared to controls included, HIF1A, HIF3A, HMOX1, and IL6. The levels of both HIF1A and HIF3A has shown sustained upregulation, it suggests that cells are constantly facing hypoxia post venom injection. [ 24 ] Hypoxia increases the levels of HIF1A by inhibiting its degradation mediated by prolyl hydroxylase enzymes and activate transcription of genes that facilitate cellular adaptations to hypoxia. [ 25 ] Furthermore, HMOX1, is massively upregulated in response to venom and suggests that cells are facing severe oxidative stress and inflammation. Hypoxia is a contributing factor to the upregulation of HMOX1, by binding of HIF-1 complex to hypoxia response element (HRE) in its promoter region. [ 26 , 27 ] HIF1A and HMOX1 are known to increase VEGF expression but a drop in VEGFA levels has been observed, it implies that venom possesses specific toxins having antiangiogenic properties [ 28 ]. Our findings are consistent with the effect of B. moojeni metalloproteinases that decreased VEGF levels along with reduction in the production of NO. Reduced VEGFA levels post snakebites could be the markers of Bothrops sp. envenomation. [ 29 ] In the expression pattern of genes associated with vasoconstriction, we identified 9 genes associated with vasoconstriction, these include KNG1, MMP2, NOS3, PLCB2, PLCB3, PLCG1, PLCG2, PTGS2, and SOD1 (Fig. 4 . (B)). The expression profile reflects a complex interplay between vasodilative and vasoconstrictive responses to snake venom. Genes for phospholipases, PLCB2 and PLCG2 had shown sustained upregulation that can lead to increase in calcium ion signaling and has not returned to control levels which can affect muscle tone. Phospholipases are known for the release of calcium ions from intracellular stores and can lead to prolonged stress in the form of vasoconstriction. [ 30 ] A significant reduction in KNG1, a precursor to bradykinin, a potent vasodilator, has been observed. This reduced expression suggests lowered capacity of vasodilation. [ 31 ] NOS3 has shown a slight upregulation whereas NOS1 levels are reduced as compared to basal levels, this can lead to reduced blood flow and increased systemic vascular resistance. [ 32 ] In the expression pattern of genes associated with pain agnosia, a total of 19 genes related to pain perception were identified in gene expression profile (Fig. 4 . (C)). The upregulated genes in our gene set included APP, BRAF, TGFB1, PTGS2, IL1B, and IL6. The gene expression data shows robust inflammatory response after injection characterized by significant upregulation in pro inflammatory cytokines, IL1B and IL6 and decrease in anti-inflammatory cytokine IL10 [ 33 ]. The upregulation of IL1B and IL6 can directly sensitize the nociceptors which can amplify pain. [ 34 ]. IL1B and TNF are known to engage in NF-κB and MAPK pathways known for production of pro-inflammatory mediators than can further intensify pain perception. [ 35 ] The upregulation of PTGS2 or COX-2 imply the increased production of prostaglandins from arachidonic acid can cause acute or chronic pain by sensitizing nerve endings. [ 36 ] To counter the pain, vitamin D is known to suppress the expression of COX-2 to reduce prostaglandins and enhance the expression of TGFB1 to moderate the inflammatory response [ 37 , 38 ] But sustained reduction in CYP27B1 expression has been observed that can halt production of active form of vitamin D, could lead to intensified pain due to less controlled inflammation. [ 38 ] Prolonged deficiency of vitamin D can lead to weakened immunity and chronic inflammation. [ 39 ] Some genes have shown downregulation as compared to controls, few of them include CRP, MEMCP2, and PNOC. Identification of targets, biological processes, and pathways associated with NO in physiological manifestations We identified 20 targets of nitric oxide by combining the intersection genes of hypoxia, vasoconstriction, and pain agnosia. Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was performed on the 20 therapeutic targets of NO using ShinyGO. Top 10 enriched pathways and processes in GO and KEGG analyses were presented (Fig. 5 . A- D). GO and KEGG pathway analysis, provided a broader perspective of how NO can serve as crucial mediator in the biological responses to snake bite. Nitric oxide synthase has shown the highest enrichment, indicating that the genes involved in our datasets have roles in regulating nitric oxide synthase crucial for controlling production of nitric oxide. NO can increase the cellular ability to cope with oxidative stress and hypoxia, aligning with observed KEGG categories such as HIF-1 signaling pathway. NO increases the stability of HIF1A under hypoxic conditions, making cells more resilient to oxygen fluctuations, oxidative stress, and promoting angiogenesis. [ 40 ] NO is a marker molecule that promote angiogenesis, NO can activate AKT1, promoting endothelial cell survival, proliferation, and NOS production. This can create feedback loop to maintain endothelial function and vasodilation important for ensuring enough oxygen supply to tissue via blood stream. Interaction between AKT1, HIF1A, and NO can lead to improved vasculature and angiogenesis. [ 41 ] The GO analysis reveals that biological processes were enriched in responses to reactive oxygen species, chemical and oxidative stress, blood vessel development, vasculature, and tube development, etc. Enriched cellular components includes platelet alpha granule lumen, neuron projection cytoplasm, plasma membrane components such as membrane raft and microdomain. Intersection genes were highly enriched in molecular functions involving nitric oxide synthase regulator activity, growth factor receptor binding, cytokine receptor binding and phosphatase binding. The KEGG analysis reveals many signaling pathways, these include HIF-1 signaling pathway, AGE-RAGE signaling pathway in diabetic complications, and relaxin signaling pathway, and various disease pathways including those related to cancer and viral infections. The GO and KEGG analyses suggest that the target genes are related to oxidative stress, signal transduction, immune response, angiogenesis, and nitric oxide synthase regulator activity. Nitric-oxide synthase regulator activity shows the highest fold enrichment suggesting strong involvement of nitric oxide regulation in the gene expression profile under the influence of venom. PPI network analysis using CytoHubba, MCODE, and iRegulon plugins in Cytoscape The PPI network of therapeutic targets of NO was constructed using STRING database and imported to Cytoscape software. Top 10 genes namely APP, TNF, AKT1, IL1B, EGFR, PTGS2, MMP2, TGFB1, IL6 and HIF1A were identified as hub genes using closeness, degree, EPC, and MNC algorithms in CytoHubba plugin. The identified hub genes were presented in a network diagram shows the relationship among several hub genes and heat map displays the expression pattern of the genes shown in network diagram under influence of venom over time (1 hour, 6 hours, and 24 hours, and control) Fig. 6 . (A- B). KEGG pathways such as AGE-RAGE signaling in diabetic complications, provide insight into role of hub genes like AKT1, IL1B, and IL6 in these processes. NO can inhibit NF-κB activation, thereby reducing expression of proinflammatory cytokines. [ 42 ] Regarding IL-6, NO can enhance or suppress its expression. This dual nature reflects its complex nature in inflammatory responses. [ 43 ] To monitor the inflammatory response, NO can act as antioxidant or pro-oxidant, depending upon concentration and cellular requirement. This can also help in maintaining redox homeostasis. [ 44 ] Another cytokine TGFB1 is crucial for cellular proliferation, and extracellular matrix production. It has shown upregulation that can lead to tissue fibrosis and can be controlled by NO via Smad proteins. [ 45 ] The expression of MMP2 has decreased, indicates a reduced capacity to degrade ECM and subsequent fibrosis. NO can activate MMPs through the NO-cGMP pathway. Thus, NO can prevent excessive fibrosis by promoting effective tissue repair. [ 46 ] After hub gene identification, the MCODE plugin in Cytoscape was used to predict the modules in the PPI network of therapeutic targets of NO (Fig. 7 . (A)). The MCODE clustered the densely connected regions into single module comprised of 18 genes and 131 edges. The obtained module defined NOS3 as seed node, whereas genes PIK3CA and COL18A1 were found unclustered (Fig. 7 ). The central positioning of NOS3 in the module indicates that the cluster analysis started from this protein and it is potentially the major regulator in this network. Thus, we hypothesize that focusing on nitric oxide synthesis and its targets are of utmost importance in the understanding the pathophysiology of snakebite induced complications. Lastly, the iRegulon plugin in the Cytoscape was used to predict the transcription factors that may regulate the NO associated genes in these physiological conditions. The normalized enrichment scores (NES) were calculated and used for ranking purposes. The top 10 predicted TFs based on NES scores included MAFK, HDAC2, STAT5A, CEBPG, EGR1, GATA5, FOS, IRX4, FOXJ2, and BACH1 as shown in (Table 4 ). Among the identified transcription factors, MAFK stood first with highest NES (Fig. 7 . (B)). The results showed that MAFK is a key transcription factor regulating the 16 of 20 genes of PPI network and can play a defining role in regulating the expression pattern of genes related to NO pathway. As we witnessed the enrichment of pathways related to oxidative stress, MAFK can play a regulatory role to finely tune the cellular response to oxidative stress by preventing under- or over- activation of stress responses through ARE pathway [ 47 ]. A recent study demonstrates a complex role of MAFK in inflammation by regulating and modulating NF-κB activity to bind DNA, thereby promoting transcription of pro-inflammatory genes. [ 48 , 49 ] This regulation is important if acute immune responses are required, but as we observed immune response is already heightened after venom injection, if remains unchecked than this interaction can be detrimental leading to chronic inflammation rather beneficial. Thus, understanding the functioning of MAFK in the cases of snake bites is important as it can aggravate the pathological condition. Table 4 The top 10 iRegulon predicted transcription factors (ranked by NES) related to 20 N0-associated genes. S. No. Transcription factor NES Targets Number Motifs/Tracks 1. MAFK 7.592 16 59 2. HDAC2 5.938 4 2 3. STAT5A 5.608 11 10 4. CEBPG 5.596 4 7 5. EGR1 4.955 9 1 6. GATA5 4.832 12 18 7. FOS 4.694 11 4 8. IRX4 4.558 10 8 9. FOXJ2 4.558 12 17 10. BACH1 4.460 3 1 Focusing on enrichment in nitric oxide synthase regulator activity and presence of NOS3 as seed node in MCODE, suggests an external supplementation of nitric oxide can have significant therapeutic implications in the treatment of snake bites and more particularly in these resulting complications. MAFK is identified as a key transcription factor in our study, therefore understanding its role could provide insights into its potential as a therapeutic target in the cases of snake bites. Conclusion Our research began by considering the metabolic product of an ingredient in a formulation used to treat snake bites in Jind, a district in Haryana, India. We identified the gene combinations and changes in their expression pattern leading to complications such as hypoxia, vasoconstriction, and pain agnosia post viperid venom injection. We focused on the interplay of nitric oxide associated genes in these complications that opens new avenues for exploring the therapeutic potential of nitric oxide, a metabolic product of potassium nitrate. Such studies are crucial for developing therapeutic interventions based on understanding of the molecular basis of snake venom pathology. Like the parent formulation, potassium nitrate is stable under normal storage conditions. Further investigations are required into such formulations and their ingredients, which are reported to be life-saving. To achieve the World Health Organization's target to reduce mortality by half by 2030, effective, stable, and affordable alternatives to present day antivenoms are required. These alternatives are crucial for saving the lives of field workers and children who are more susceptible to snake bites. Therefore, publication of this study could guide researchers to consider the traditional formulations as potential source for developing new, stable, and effective antivenom treatments. Abbreviations ARE - Antioxidant Response Element BP - Biological Process CC - Cellular Component MF - Molecular Function CTNNB1- Catenin Beta 1. DNA- Deoxyribonucleic Acid EGFA- Epidermal Growth Factor Alpha EPAS1- Endothelial PAS Domain Protein 1 EPC- Edge Percolated Component MNC - Maximum Neighborhood Component FDR - False Discovery Rate GO - Gene Ontology HIF1A/HIF3A - Hypoxia-Inducible Factor 1-alpha/3-alpha KEGG - Kyoto Encyclopedia of Genes and Genomes KNG1 - Kininogen 1 MAFK - Small MAF transcription factor K. MAPK1 - Mitogen-Activated Protein Kinase 1 MCODE - Molecular Complex Detection MTOR - Mechanistic Target of Rapamycin NCBI - National Center for Biotechnology Information NCBI GEO - Gene Expression Omnibus NO - Nitric Oxide NOS3 - Nitric Oxide Synthase 3 PLCB2, PLCB3, PLCG1, PLCG2 - Phospholipase C Beta 2, Beta 3, Gamma 1, Gamma 2 PTGS2 - Prostaglandin-Endoperoxide Synthase 2, also known as COX-2 SOD1 - Superoxide Dismutase 1 STRING - Search Tool for the Retrieval of Interacting Genes/Proteins. TF - Transcription Factor TNF - Tumor Necrosis Factor VEGFA - Vascular Endothelial Growth Factor A WHO - World Health Organization Declarations Data Availability Data in this study can be obtained from the corresponding author upon reasonable request. Contribution M.S. conceptualized, designed, performed, and wrote the present study, U.T. edited the manuscript, S.L., D.M., A.R. critically revised the article. Corresponding author Correspondence to Madhu Sindhu. Funding No funding is available. Acknowledgement Authors would like to express our gratitude to NCBI-GEO (https://www.ncbi.nlm.nih.gov/geo/) for providing valuable gene expression profile for data analysis. Competing interests The authors declare no potential conflicts of interest with respect to research, authorship, and/or publication of this article. References Pach, S. et al. Paediatric snakebite envenoming: the world's most neglected 'Neglected Tropical Disease'? Archives of disease in childhood . 105 (12), 1135–1139; https://doi.org/10.1136/archdischild-2020-319417 (2020). Gopalakrishnan, M., Saurabh, S., Sagar, P., Bammigatti, C. & Dutta, T. K. A simple mortality risk prediction score for viper envenoming in India (VENOMS): A model development and validation study. PLOS Negl. Trop. Dis. 16 (2), e0010183; https://doi.org/10.1371/journal.pntd.0010183 (2022). Pinto, L. J. et al. Case Report: Hemothorax in Envenomation by the Viperid Snake Bothrops asper . Am. J. Trop. Med. Hyg. 100 (3), 714–716; https://doi.org/10.4269/ajtmh.18-0826 (2019). Kalita, B., Mackessy, S. P. & Mukherjee, A. K. Proteomic analysis reveals geographic variation in venom composition of Russell’s Viper in the Indian subcontinent: implications for clinical manifestations post-envenomation and antivenom treatment. Expert Rev. Proteomics. 15 (10), 837–849; https://doi.org/10.1080/14789450.2018.1528150 (2018). Xie, C. et al. Neutralizing effects of small molecule inhibitors and metal chelators on coagulopathic Viperinae snake venom toxins. Biomedicines. 8 (9), 297; 10.3390/biomedicines8090297 (2020). Noor, F., Ashfaq, U. A., Albutti, A., Alwashmi, A. S. & Aljasir, M. A. Network Pharmacology Approach for Medicinal Plants: Review and Assessment. Pharmaceuticals. 15 (5), 572; https://doi.org/10.3390/ph15050572 (2022). Zeng, L. et al. Network Pharmacological Study on the Mechanism of Cynanchum paniculatum (Xuchangqing) in the Treatment of Bungarus multicinctus Bites. Biomed. Res. Int. 3887072; https://doi.org/10.1155/2022/3887072 (2022). Sun, S. et al. Exploring the Mechanism of Cytisine in Treating Respiratory Depression following Venomous Snake Bites Based on Network Pharmacology and Molecular Docking. Am. J. Transl. Res. 16 (4), 1442–1453; https://doi.org/10.62347/MCPD8618 (2024). Tsantoulas, C. & McMahon, S. B. Opening Paths to Novel Analgesics: The Role of Potassium Channels in Chronic Pain. Trends Neurosci. 37 (3), 146–158; https://doi.org/10.1016/j.tins.2013.12.002 (2014). Moncada, S., Palmer, R. M. & Higgs, E. A. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol. Rev. 43 (2), 109–142 (1991). Ignarro, L. J. Nitric oxide: a unique endogenous signaling molecule in vascular biology. Biosci. Rep. 19 (2), 51–71 (1999). Petrizzo, R., Mohr, J., Mantione, K. & Goldstein, L. The Role of Endogenous Morphine and Nitric Oxide in Pain Management. Pract. Pain Manag. 14 (9), (2014). Resiere, D., Mehdaoui, H. & Neviere, R. Inflammation and Oxidative Stress in Snakebite Envenomation: A Brief Descriptive Review and Clinical Implications. Toxins 14 (11), 802; https://doi.org/10.3390/toxins14110802 (2022). Ghosh, R., Maity, A., Biswas, U., Das, S. & Benito-León, J. Lance-Adams Syndrome: An Unusual Complication of Snakebite Envenomation. Toxicon 209, 50–55; https://doi.org/10.1016/j.toxicon.2022.02.008 , (2022). Bozkurt, M., Kulahci, Y., Zor, F. & Kapi, E. The Management of Pit Viper Envenomation of the Hand. Hand 3 (4), 324 – 33; https://doi.org/10.1007/s11552-008-9114-2 (2008). Mehta, S. R. & Sashindran, V. K. Clinical Features and Management of Snake Bite. Med. J. Armed Forces India. 58 (3), 247–249; https://doi.org/10.1016/S0377-1237(02)80140-X (2002). Zdenek, C. N., et al. Children and Snakebite: Snake Venom Effects on Adult and Paediatric Plasma. Toxins 15 (2), 158; https://doi.org/10.3390/toxins15020158 (2023). Janky, R. et al. iRegulon: from a gene list to a gene regulatory network using large motif and track collections. PLoS computational biology. 10 (7), e1003731; https://doi.org/10.1371/journal.pcbi.1003731 (2014). Larsen, F. J. Ekblom, B., Sahlin, K., Lundberg, J. O. & Weitzberg, E. Effects of dietary nitrate on blood pressure in healthy volunteers. N. Engl. J. Med. 355 (26), 2792–2793; https://doi.org/10.1056/NEJMc062800 (2006). Umbrello, M., Dyson, A., Feelisch, M. & Singer, M. The key role of nitric oxide in hypoxia: hypoxic vasodilation and energy supply-demand matching. Antioxid Redox Signal. 19 (14), 1690–1710; https://doi.org/10.1089/ars.2012.4979 (2013). Kourembanas, S., McQuillan, L. P., Leung, G. K. & Faller, D. V. Nitric oxide regulates the expression of vasoconstrictors and growth factors by vascular endothelium under both normoxia and hypoxia. J. Clin. Invest. 92 (1), 99–104; https://doi.org/10.1172/JCI116604 (1993). Anbar, M. et al. Role of nitric oxide in the physiopathology of pain. J. Pain Symptom Manage. 14 (4), 225–254 (1997). Lundberg, J. O., Weitzberg, E. & Gladwin, M. T. The nitrate-nitrite-nitric oxide pathway in physiology and therapeutics. Nat. Rev. Drug Discov. 7 (2), 156–167; https://doi.org/10.1038/nrd2466b (2008). Belaiba, R. S. et al. Hypoxia up-regulates hypoxia-inducible factor-1alpha transcription by involving phosphatidylinositol 3-kinase and nuclear factor kappaB in pulmonary artery smooth muscle cells. Molecular biology of the cell, 18 (12), 4691–4697; https://doi.org/10.1091/mbc.e07-04-0391 (2007). Semenza G. L. Hypoxia-inducible factors in physiology and medicine. Cell, 148 (3), 399–408; https://doi.org/10.1016/j.cell.2012.01.021 (2012). Chen, S., Wang, X., Nisar, M. F., Lin, M. & Zhong, J. L. Heme Oxygenases: Cellular Multifunctional and Protective Molecules against UV-Induced Oxidative Stress. Oxid. Med. Cell. Longev, 208, 5416728; https://doi.org/10.1155/2019/5416728 (2019). Dunn, L. L. et al. Hmox1 (Heme Oxygenase-1) Protects Against Ischemia-Mediated Injury via Stabilization of HIF-1α (Hypoxia-Inducible Factor-1α). Arteriosclerosis, thrombosis, and vascular biology , 41 (1), 317–330; https://doi.org/10.1161/ATVBAHA.120.315393 (2021). Shweiki, D., Itin, A., Soffer, D. & Keshet, E. Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis. Nature. 359 (6398), 843–845; https://doi.org/10.1038/359843a0 (1992). Oliveira, V. Q. et al. Antiangiogenic properties of BthMP, a P-I metalloproteinase from Bothrops moojeni snake venom by VEGF pathway in endothelial cells. Biochemical and biophysical research communications. 706, 149748; https://doi.org/10.1016/j.bbrc.2024.14974824 (2024). Wilson, D. P., Susnjar, M., Kiss, E., Sutherland, C. & Walsh, M. P. Thromboxane A2-induced contraction of rat caudal arterial smooth muscle involves activation of Ca2 + entry and Ca2 + sensitization: Rho-associated kinase-mediated phosphorylation of MYPT1 at Thr-855, but not Thr-697. The Biochemical journal, 389 (3), 763–774; https://doi.org/10.1042/BJ20050237 (2005). Kaplan, A. P. & Joseph, K. Pathogenic mechanisms of bradykinin mediated diseases: dysregulation of an innate inflammatory pathway. Advances in immunology. 121, 41–89; https://doi.org/10.1016/B978-0-12-800100-4.00002-7 (2014). Joyner, M. J. & Dietz, N. M. Nitric oxide and vasodilation in human limbs. Journal of Applied Physiology, 83 (6), 1785–1796; doi: 10.1152/jappl.1997.83.6.1785 (1997). Resiere, D. et al. Inflammation and Oxidative Stress in Snakebite Envenomation: A Brief Descriptive Review and Clinical Implications. Toxins. 14 (11), 802; https://doi.org/10.3390/toxins14110802 (2022). Ren, K. & Torres, R. Role of interleukin-1β during pain and inflammation. Brain Res. Rev. 60 (1), 57–64; https://doi.org/10.1016/j.brainresrev.2008.12.020 (2009). Lawrence, T. & Fong, C. The role of the NF-κB pathway in the pathogenesis of inflammation and cancer. Nat. Rev. Immunol. 10, 361–378; https://doi.org/10.1038/nri2742 (2010). Ricciotti, E. & FitzGerald, G. A. Prostaglandins and inflammation. Arterioscler. Thromb. Vasc. Biol. 31 (5), 986–1000; https://doi.org/10.1161/ATVBAHA.110.207449 (2011). Feldman, D. et al. Vitamin D inhibition of the prostaglandin pathway as therapy for prostate cancer. Nutrition Reviews. 65 (8 Pt 2), S113-S115; https://doi.org/10.1111/j.1753-4887.2007.tb00335.x (2007). Shipton, E. A. & Shipton, E. E. Vitamin D and Pain: Vitamin D and Its Role in the Aetiology and Maintenance of Chronic Pain States and Associated Comorbidities. Pain Res. Treat . 904967; https://doi.org/10.1155/2015/904967 (2015). Habib, A. M., Nagi, K., Thillaiappan, N. B., Sukumaran, V. & Akhtar, S. Vitamin D and Its Potential Interplay with Pain Signaling Pathways. Frontiers in immunology. 11, 820; https://doi.org/10.3389/fimmu.2020.00820 (2020). Hendrickson, M. & Poyton, R. Crosstalk between nitric oxide and hypoxia-inducible factor signaling pathways: an update. Res. Rep. Biochem. 5, 147–161; https://doi.org/10.2147/RRBC.S58280 (2015). Dimmeler, S. et al. Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation. Nature 399 (6736), 601–605; https://doi.org/10.1038/21224 (1999). Jue, D. M., Jeon, K. I. & Jeong, J. Y. Nuclear factor kappaB (NF-kappaB) pathway as a therapeutic target in rheumatoid arthritis. Journal of Korean medical science, 14 (3), 231–238; https://doi.org/10.3346/jkms.1999.14.3 (1999). Siednienko, J., Nowak, J., Moynagh, P. N. & Gorczyca, W. A. (2011). Nitric oxide affects IL-6 expression in human peripheral blood mononuclear cells involving cGMP-dependent modulation of NF-κB activity. Cytokine , 54 (3), 282–288; https://doi.org/10.1016/j.cyto.2011.02.015 (2011). Patel, R. P., Levonen, A., Crawford, J. H. & Darley-Usmar, V. M. Mechanisms of the pro- and anti-oxidant actions of nitric oxide in atherosclerosis. Cardiovascular research. 47 (3), 465–474; https://doi.org/10.1016/s0008-6363(00)00086-9 (2000). Saura, M. et al. Nitric oxide regulates transforming growth factor-beta signaling in endothelial cells. Circulation research. 97 (11), 1115–1123; https://doi.org/10.1161/01.RES.0000191538.76771.66 (2005). Man Chow, B. S. et al. Relaxin Signals through a RXFP1-pERK-nNOS-NO-cGMP-Dependent Pathway to Up-Regulate Matrix Metalloproteinases: The Additional Involvement of iNOS. PLOS ONE. 7 (8), e42714; https://doi.org/10.1371/journal.pone.0042714 (2012). Katsuoka, F. et al. Genetic Evidence that Small Maf Proteins Are Essential for the Activation of Antioxidant Response Element-Dependent Genes. Molecular and Cellular Biology, 25 (18), 8044–8051; https://doi.org/10.1128/MCB.25.18.8044-8051 (2005). Nguyen, T., Huang, H. C. & Pickett, C. B. Transcriptional regulation of the antioxidant response element. Activation by Nrf2 and repression by MafK. The Journal of biological chemistry. 275 (20), 15466–15473; https://doi.org/10.1074/jbc.M000361200 (2000). Hwang, Y. J. et al. MafK positively regulates NF-κB activity by enhancing CBP-mediated p65 acetylation. Scientific reports. 3, 3242; https://doi.org/10.1038/srep03242 (2013). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4512510","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":314199637,"identity":"b07653e8-7673-4572-8f46-6c23acd70b4c","order_by":0,"name":"Madhu Sindhu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9ElEQVRIiWNgGAWjYHACMyCWYGC4wcD4IKECyGZmbiBaC7PBhzMgLYxEaWEAaWGTnNkGYhHQott+eNtjnj8WeXy3ewykeefVRvO3A7X8qNiG24ozaeXGvG0SxZJ3zhgY8247njvjMGMDY8+Z27i1HMgxk+ZtkEjccCPHIJl327HcBqAWZsY2PFrOvzGT5vkD0XKYd86x3PkEtdwA2sLDBtZi2DizoSZ3A2Etz8ok57ZJJM68kVbM8OHYgdyNQC0H8frlfPI2iTd/6hL7biRv/5FQU5c77/zhgw9+VODWgg4Og8kDRKsHgjpSFI+CUTAKRsEIAQBOLWF3f+T/pQAAAABJRU5ErkJggg==","orcid":"","institution":"Panjab University","correspondingAuthor":true,"prefix":"","firstName":"Madhu","middleName":"","lastName":"Sindhu","suffix":""},{"id":314199638,"identity":"fd1f821f-788a-4cb1-8151-f1916f4130fd","order_by":1,"name":"Umesh Thakur","email":"","orcid":"","institution":"Panjab University","correspondingAuthor":false,"prefix":"","firstName":"Umesh","middleName":"","lastName":"Thakur","suffix":""},{"id":314199639,"identity":"c3a57dc6-8789-439c-a59c-ce47e0728da7","order_by":2,"name":"Shiwani Latwal","email":"","orcid":"","institution":"Panjab University","correspondingAuthor":false,"prefix":"","firstName":"Shiwani","middleName":"","lastName":"Latwal","suffix":""},{"id":314199640,"identity":"07a8a14e-364c-46fb-8450-b69148768035","order_by":3,"name":"Diksha Muwal","email":"","orcid":"","institution":"Panjab University","correspondingAuthor":false,"prefix":"","firstName":"Diksha","middleName":"","lastName":"Muwal","suffix":""},{"id":314199641,"identity":"63ad7bf2-355c-4cbe-ab18-306849010550","order_by":4,"name":"Anju Rao","email":"","orcid":"","institution":"Panjab University","correspondingAuthor":false,"prefix":"","firstName":"Anju","middleName":"","lastName":"Rao","suffix":""}],"badges":[],"createdAt":"2024-06-01 07:40:45","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4512510/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4512510/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":58390607,"identity":"2c1d34f5-0914-473f-bdcb-3b77571e9037","added_by":"auto","created_at":"2024-06-14 20:15:34","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":85855,"visible":true,"origin":"","legend":"\u003cp\u003eThe flow chart of network pharmacology to investigate the physiological manifestations of viperid envenomation and identification of targets of NO in their treatment\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4512510/v1/c3c80960920e546d86cf02c0.jpeg"},{"id":58390605,"identity":"cc8e7d65-22d1-427c-947a-2292b0603a39","added_by":"auto","created_at":"2024-06-14 20:15:34","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":141409,"visible":true,"origin":"","legend":"\u003cp\u003eIdentification of targets of physiological manifestations of viperid venom (A.) Hypoxia (B.) Vasoconstriction (C) Pain agnosia. (D) Venn diagram showing 20 intersection genes identified as targets of NO in physiological manifestations\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4512510/v1/ddf6b6b758efbcba971cd003.jpeg"},{"id":58390790,"identity":"4fd81236-d2a9-4692-a0cb-a7639a5aab4c","added_by":"auto","created_at":"2024-06-14 20:23:34","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":220787,"visible":true,"origin":"","legend":"\u003cp\u003eProtein-protein interaction network (A) A network diagram of hypoxia associated genes (B) A network diagram of vasoconstriction associated genes (C) A network diagram of pain agnosia associated genes\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4512510/v1/9cc19c1b84574344a741809b.jpeg"},{"id":58390611,"identity":"172c9436-206c-48ff-b63d-4e5fa9e76d6c","added_by":"auto","created_at":"2024-06-14 20:15:35","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":159653,"visible":true,"origin":"","legend":"\u003cp\u003eHeat maps of intersection targets of physiological alterations in muscle tissue injected with PBS (control) and B. asper venom (A) Hypoxia associated genes (B) Vasoconstriction associated genes (C) Pain agnosia associated genes. Each row represent change in gene expression over time after injecting venom. Heat maps was presented using Log2 of transcript counts of identified genes. SRPLOT was used to generate heat maps and to perform hierarchical clustering. Complete linkage and Euclidean distance methods were selected.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4512510/v1/0c6174c64a31089ef9a5604a.jpeg"},{"id":58390609,"identity":"54ded35d-fc3f-4515-98bf-87e72ac4a0cf","added_by":"auto","created_at":"2024-06-14 20:15:34","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":223852,"visible":true,"origin":"","legend":"\u003cp\u003eGO enrichment analysis (A) Biological process (B) Cellular component (CC) (C) Molecular function (MF). (D) KEGG enrichment analysis of 20 identified targets of NO in combined gene list.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4512510/v1/832cb232a2386ab5bab95155.jpeg"},{"id":58390610,"identity":"3dcf4ea9-8ad5-4944-857d-a801bece578b","added_by":"auto","created_at":"2024-06-14 20:15:35","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":206391,"visible":true,"origin":"","legend":"\u003cp\u003e(A). Top 10 identified hub genes (in red central wheel) from intersection targets of NO in physiological alterations in muscle tissue injected with PBS as control and B. asper venom. (B) Heat maps of hub genes, presented using Log2 of transcript counts of identified hub genes. SRPLOT was used to generate heat maps and to perform hierarchical clustering. Complete linkage and Euclidean distance methods were selected.\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4512510/v1/6932679b87791dabcad4f9ba.jpeg"},{"id":58390791,"identity":"d6d8f133-a698-43fc-9f0d-4a377cc0c1a6","added_by":"auto","created_at":"2024-06-14 20:23:35","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":260389,"visible":true,"origin":"","legend":"\u003cp\u003ePPI network of 20 NO associated genes through cytoscape (A) MCODE module : The top module containing NOS3 as seed (Dark red colored in center), 18 clustered nodes (salmon colored; clockwise as per MCODE score starting from AKT1), and 2 unclustered nodes (green in color) and 131 edges. (B) The interaction between the transcription factor MAFK (red octagon) and its core targets (ellipse) was analyzed and constructed using iRegulon plugin in Cytoscape software\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4512510/v1/3138388b602252f428d3dcf7.jpeg"},{"id":74996075,"identity":"9a5a3655-f240-4504-a0b7-eeed5b4973d1","added_by":"auto","created_at":"2025-01-29 08:46:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2089366,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4512510/v1/936277aa-789d-46e5-99ba-e68e8fe2f10b.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Network pharmacology reveals physiological manifestations of viperid envenomation and role of nitric oxide in their treatment","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSnake bite is a neglected tropical disease. According to World Health Organization (WHO) data, more than 5\u0026nbsp;million people suffer from snake bites and results in about 81,000 to 138,000 deaths each year. Survivors often face long term disabilities, both physical and psychological. [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] A recent data indicates that deaths due to snake bites in India is around 58,000, comprising more than half of global fatalities, with vipers being the primary reason for majority of these deaths. [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] Viperid snake venom is highly complex and essentially proteinaceous in nature. Peptides, phospholipases, venom metalloproteinases, and serine proteinases contribute to toxicity of venom. Vipers are notorious for causing severe bites with both local and systemic effects, including neurotoxicity, hemotoxicity, coagulopathies, inflammation, and multiple organ failure. [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] Incidences of snake bites are disproportionately high in agricultural workers and children in rural communities, who often lack timely access to health care facilities. Antibody-based antivenom is a primary treatment for these bites, which often leads to anaphylactic reactions. [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] Moreover, maintenance of steady supply of antivenom in rural and remote areas is difficult due to production complexity, stringent storage conditions and shorter shelf life. [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] Considering the challenges associated with production and distribution of antivenom, it is important to explore alternative treatments for snake bites.\u003c/p\u003e \u003cp\u003eNetwork pharmacology is a rapidly developing area in the field of drug discovery and development. [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] It integrates computational methods, high-throughput omics, and advanced pharmacological analysis, and can identify the interactions between compounds and their biological targets. [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] Traditional medicines have developed a vast array of treatments based on extensive clinical research and often target multiple pathways. To uncover the basis of traditional medicines, network pharmacology involves viewing entire biological system involved in the disease as a therapeutic target, to understand the mechanism of multitarget drugs. [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eIn this context, a documented formulation from Jind district of Haryana, India, can offer valuable pharmacological insights which include kalmi shora or potassium nitrate as one of the ingredients. Potassium nitrate could be explored in the treatment of snakebite using network pharmacology. The rationale of using potassium nitrate in the treatment of snakebites is supported by its physiological properties. Potassium ions are known for their role in maintaining intracellular osmotic balance and cell membrane potential. Potassium ions decrease nerve excitation and pain by disturbing the synapse between nerve endings. [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] Nitrates are rapidly absorbed in proximal and small intestine and evenly distributed among body organs. Nitrates undergo reduction to nitrites by bacteria in oral cavity and nitrites can be further reduced to nitric oxides (NO). NO is a critical signaling molecule with wide ranging effects in physiological systems. NO is known for its role in vascular biology, immune system function, respiratory function, hemostasis, pain modulation. [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] Thus, metabolized nitrate in the form of NO was selected to study its impact on venom induced manifestations.\u003c/p\u003e \u003cp\u003eSnakebite envenomation can cause severe and lasting systemic and neurological damage. [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] NO involvement in these manifestations can help to treat some critical impacts of snake venom. Neurotoxin in snake venom can cause oxygen deprivation results in hypoxic brain injury, manifesting critical conditions such as Lance- Adams syndrome. [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] Snake venom also causes immediate swelling and vasoconstriction leading to tissue necrosis by immediate affect due to cellular damage and vascular injuries. [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] This can trigger inflammatory reactions along with systemic injuries can cause or intensify severe pain, that can be local or remote. Pain in snake bites is heavily influenced by biological reactions of venom compounds. [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] These manifestations underline the importance of studying the effect of snake venom at genetic level. Genetic profiling of responses to snake venom can help in understanding physiological manifestations of venom, variations in susceptibility and outcomes among different populations. This is critical for understanding the physiology of snakebite envenomation. [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eIntegrating the critical examination of genetic expression in response to snakebite envenomation with therapeutic potential of potassium nitrate can provide us a comprehensive strategy for understanding the physiology of snake bites. By utilizing the gene expression data of viperid envenomation from NCBI-GEO database, we analyzed the effects of snake venom on crucial genes associated with hypoxia, vasoconstriction, and pain. Upon intersecting the targets of nitric oxide with physiological manifestations, we aim to enhance effective treatment methodologies. By integrating this data, we can propose new treatments based on targeted effects of venom by increasing precision of treatment. The flow chat of study has shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e \u003cb\u003eAcquisition of gene expression profile of venom-treated tissue and target genes of hypoxia, vasoconstriction, pain agnosia, and nitric oxide\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe gene expression profile of venom treated tissue was obtained from Gene Expression Omnibus (GEO), a public repository of NCBI using keyword \u0026lsquo;snake venom,\u0026rsquo; and the data was available under heading \u0026lsquo;A complex pattern of gene expression in tissue affected by viperid snake envenoming: the emerging role of autophagy related genes\u0026rsquo; \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e(\u003c/span\u003e\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The therapeutic targets for hypoxia, vasoconstriction, pain agnosia, and nitric oxide were sourced from the GeneCards database and selected on basis of relevance scores (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.genecards.org/\u003c/span\u003e\u003cspan address=\"https://www.genecards.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). This approach allowed us to identify the pattern of gene expression in the tissue affected by viperid venom and to enlist the genes involved in these physiological conditions.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eScreening of common targets\u003c/h2\u003e \u003cp\u003eTo identify the overlapping elements with mouse gene expression profile, genes involved in hypoxia, vasoconstriction, pain agnosia, and NO were crossed with genes expressed in tissue affected by venom. We then compiled the genes of hypoxia, vasoconstriction, and pain agnosia, while removing redundant values. Finally, the obtained gene set was intersected with genes associated with nitric oxide. This intersection was important in identifying different genes combinations for different physiological manifestations, offering insights for pharmacological targets of NO. We used Venny 2.1, a free interactive tool for comparing lists and creating Venn diagrams (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://csbg.cnb.csic.es/BioinfoGP/venny.html\u003c/span\u003e\u003cspan address=\"https://csbg.cnb.csic.es/BioinfoGP/venny.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). This tool enabled us to screen and visualize the gene sets of hypoxia, vasoconstriction, and pain agnosia in mouse tissue, and therapeutic targets of NO.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eAcquisition of protein-protein interaction (PPI) network data\u003c/h2\u003e \u003cp\u003eTo construct the regulatory network of intersecting genes associated with hypoxia, vasoconstriction, pain agnosia, and targets of NO in these physiological manifestations in mice, we used the STIRNG (v12.0; Search Tool for the Retrieval of Interacting Genes/Proteins; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://string-db.org/\u003c/span\u003e\u003cspan address=\"https://string-db.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) online database. The interaction score was set at medium confidence\u0026thinsp;\u0026gt;\u0026thinsp;0.400 and the species was set to the \u0026lsquo;\u003cem\u003eHomo sapiens.\u0026rsquo;\u003c/em\u003e Later, the important nodes were visualized by setting maximum FDR value\u0026thinsp;\u0026lt;\u0026thinsp;=\u0026thinsp;0.05 to reduce the redundancy and selectively highlight the functional enrichments in our networks.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eGene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis\u003c/h2\u003e \u003cp\u003eThe enrichment analysis of therapeutic targets of NO was done using ShinyGO, a web-based application (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bioinformatics.sdstate.edu/go76/\u003c/span\u003e\u003cspan address=\"http://bioinformatics.sdstate.edu/go76/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). GO and KEGG pathway enrichment analysis was performed to identify the significantly enriched biological processes and pathways in overlapping genes. For analysis, maximum FDR cut off was set below 0.05, processes and pathways below this value were considered significant. The top 10 results of GO modules, biological processes (BP), cellular components (CC), and molecular function (MF) and KEGG pathway enrichments were represented using bar graphs. The degree of enrichment was presented using fold enrichment and -log10 transformed p values.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003ePPI network analysis\u003c/h2\u003e \u003cp\u003eLater, the PPI network of NO-associated genes was imported to Cytoscape software (v3.10.2; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cytoscape.org\u003c/span\u003e\u003cspan address=\"https://cytoscape.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The hub genes from our network were identified by applying closeness, degree, edge percolated component (EPC) and maximum neighborhood component (MNC) algorithms in CytoHubba plugin (v0.1; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://apps.cytoscape.org/apps/cytoHubba\u003c/span\u003e\u003cspan address=\"https://apps.cytoscape.org/apps/cytoHubba\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Top 10 scoring genes in each category were selected and removed duplicates to identify the hub genes. For subcluster analysis, we used MCODE plugin (Molecular Complex Detection, v2.0.3; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://apps.cytoscape.org/apps/mcode\u003c/span\u003e\u003cspan address=\"http://apps.cytoscape.org/apps/mcode\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and degree cutoff, maximum depth, k-core, and node score cutoff were set at 2, 100, 2, and 0.2 respectively. Finally, iRegulon plugin (v1.3; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://apps.cytoscape.org/apps/iregulon\u003c/span\u003e\u003cspan address=\"http://apps.cytoscape.org/apps/iregulon\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to detect master regulons based on motif enrichment in NO-associated gene set. The iRegulon predictions based on analysing transcription factor (TF) motifs and ChIP-Seq data to predict TFs regulating the given set of genes [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Key parameters included minimum identity between orthologous gene and false discovery rate (FDR), set at 0.05 and 0.001 respectively. The top 10 transcription factors were selected based on normalized enrichment score.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eGene expression profile analysis\u003c/h2\u003e \u003cp\u003eThe expression profiles of the targets of hypoxia, vasoconstriction, and pain agnosia and of identified hub genes were represented by generating heat maps. The heat maps were created using SRPLOT, an online tool for graphical and statistical analysis of biological data (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.bioinformatics.com.cn/srplot\u003c/span\u003e\u003cspan address=\"https://www.bioinformatics.com.cn/srplot\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). For generating heat maps, complete clustering was selected along with bidirectional orientation, and distance method was configured at Euclidean distance. The heat maps were made using Log2 of transcripts counts of set of significant genes in each physiological condition and of hub genes.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003eIndian system of medicine has frequently used salts in the compositions for the treatment of different ailments. One such formulation is documented from Jind district of Haryana, India, that is used in the treatment of snakebites and aphid stings, and it includes potassium nitrate as one of the ingredients. Motivated by this ingredient, our study explores the potential of nitrates in the treatment of snakebites. Nitric oxide (NO) is metabolic product of dietary nitrate, and peak plasma levels are achieved within 2\u0026ndash;3 hours. [19.] Nitric oxide plays a pivotal role in physiological processes such as hypoxia, vasoconstriction, and pain agnosia- the common complications of snakebites. [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] Such intersections lead us to study the network pharmacology of NO in relation to snakebite treatment. Considering the challenges of snake bites where professional medical help is not immediately available, properties of NO derived from nitrates offers a promising alternative. The ease of administration, and the speed at which NO becomes bioavailable make it promising candidate for emergency treatment in remote or resource limited areas. [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eAcquisition of gene expression profile\u003c/p\u003e \u003cp\u003eOur study utilized the NCBI-GEO database, it provided supplementary file of gene expression profiles in mouse skeletal muscles at 1 hours, 6 hours, and 24 hours after injecting venom of \u003cem\u003eBothrops asper\u003c/em\u003e and \u003cem\u003eDaboia russelii\u003c/em\u003e. For controls, mice were injected with phosphate-buffer saline (PBS). This data involved the mapped mouse genes to their human equivalents. We obtained a list of 760 genes expressed in the tissue affected by viperid venom. The expression profile under the influence of venom of \u003cem\u003eB. asper\u003c/em\u003e was used in this study to analyze the pattern of gene expression in the tissue. We pinpointed gene targets associated with the key complications of snakebites: hypoxia, vasoconstriction, and pain agnosia, and the pathways involving nitric oxide in these complications.\u003c/p\u003e \u003cp\u003eAcquisition of targets of physiological manifestations and NO\u003c/p\u003e \u003cp\u003eFor target screening from gene expression profile, the targets of physiological complications and NO were meticulously sorted and selected based on relevance scores from the GeneCards database. We obtained 52, 44, 77, and 395 targets for hypoxia, vasoconstriction, pain agnosia and nitric oxide respectively from GeneCards. Upon intersection with mouse genes, a total of 11, 5, 19, and 100 genes corresponding to hypoxia, vasoconstriction, pain agnosia and nitric oxide were found overlapping. Four genes namely, PLCB2, PLCB3, PLCG1, and PLCG2 were added to the intersection list of vasoconstriction and 9 genes were analyzed by then. From the aggregated gene set of hypoxia, vasoconstriction, and pain agnosia, we obtained a set of 33 unique genes by removing duplicates. Lastly, by intersecting the combined values (33) and genes associated with nitric oxide (100) pathway, a total of 20 genes were found common and regarded as targets of NO in physiological manifestation of viperid envenomation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. A- D).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eProtein-protein interaction network\u003c/p\u003e \u003cp\u003eThe protein-protein interaction network of intersecting genes associated with hypoxia, vasoconstriction, pain agnosia, and therapeutic targets of nitric oxide in mice was created using STRING. The interactions were visualized by selecting functions and pathways relevant for study from functional enrichments segment in the tool. This selective visualization allowed us to see the part of network that contributed to specific enrichments, and how these enrichments are interconnected through shared proteins (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The PPI networks are represented by highlighting nodes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. A- C).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eHighlighted functional enrichments in PPI network of hypoxia associated genes\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGO Term\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFDR\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFunctional Enrichment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eConnected Nodes\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGO:0001666\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.91e-07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eResponse to hypoxia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eEPAS1, EP300, HIF1A, HIF3A, HMOX1, MTOR, TNF\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGO:0043619\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.63e-05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRegulation of transcription from DNA polymerase II promoter in response to oxidative stress\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eEPAS1, HIF1A, HMOX1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGO:0051000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.0049\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePositive regulation of nitric oxide synthase activity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHIF1A, TNF\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGO:0051403\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.0245\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eStress- activated MAPK cascade\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTNF, MAPK1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eHighlighted functional enrichments in PPI network of vasoconstriction associated genes\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGO Term\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFDR\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFunctional Enrichment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eConnected Nodes\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGO:0004435\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.70e-06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePhosphatidylinositol phospholipase C activity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePLCB2, PLCB3, PLCG1, PLCG2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGO:0042311\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.0013\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eVasodilation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eKNG1, NOS3, SOD1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGO:0051924\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.0022\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRegulation of calcium ion transport\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNOS3, PTGS2, PLCG1, PLCG2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGO:0019229\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.0454\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRegulation of vasoconstriction\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMMP2, PTGS2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eHighlighted functional enrichments in PPI network of pain agnosia associated genes\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGO term\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFDR\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFunctional enrichment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eConnected Nodes\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGO:0000165\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.14E-09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMAPK cascade\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBRAF, CTNNB1, EGFR, IL1B, TGFB1, KRAS, NF1, TNF\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGO:0060559\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.00024\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePositive regulation of calcidiol 1-monooxygense activity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIL1B, TNF\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGO:0046328\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.00024\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRegulation of JNK cascade\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAPP, EGFR, IL1B, TNF,\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGO:1900017\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.0051\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePositive regulation of cytokine production involved in inflammatory response\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIL6, TNF\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGO:0045202\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.0184\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSynapse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAPP, AKT1, BRAF, CTNNB1, MECP2, PNOC, NF1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGO:0031394\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.00074\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePositive regulation of prostaglandin biosynthetic process\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePTGS2, IL1B\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGO:0033280\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.0065\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eResponse to vitamin D\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePTGS2, TGFB1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGO:0001666\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.00014\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eResponse to hypoxia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePTGS2, TGFB1, NF1, TNF, MECP2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eExpression profile analysis of genes associated with physiological manifestations and generation of heat maps\u003c/p\u003e \u003cp\u003eThe analysis of expression pattern of genes associated with hypoxia facilitates identification of key genes involved in the cellular adaptation to hypoxia, these include VEGFA, CTNNB1, AP300, MTOR, EPAS1, TNF, MAPK1, HIF1A, HIF3A, HMOX1, and IL6 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. (A)). The upregulated genes as compared to controls included, HIF1A, HIF3A, HMOX1, and IL6. The levels of both HIF1A and HIF3A has shown sustained upregulation, it suggests that cells are constantly facing hypoxia post venom injection. [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] Hypoxia increases the levels of HIF1A by inhibiting its degradation mediated by prolyl hydroxylase enzymes and activate transcription of genes that facilitate cellular adaptations to hypoxia. [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] Furthermore, HMOX1, is massively upregulated in response to venom and suggests that cells are facing severe oxidative stress and inflammation. Hypoxia is a contributing factor to the upregulation of HMOX1, by binding of HIF-1 complex to hypoxia response element (HRE) in its promoter region. [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] HIF1A and HMOX1 are known to increase VEGF expression but a drop in VEGFA levels has been observed, it implies that venom possesses specific toxins having antiangiogenic properties [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Our findings are consistent with the effect of \u003cem\u003eB. moojeni\u003c/em\u003e metalloproteinases that decreased VEGF levels along with reduction in the production of NO. Reduced VEGFA levels post snakebites could be the markers of \u003cem\u003eBothrops sp.\u003c/em\u003e envenomation. [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eIn the expression pattern of genes associated with vasoconstriction, we identified 9 genes associated with vasoconstriction, these include KNG1, MMP2, NOS3, PLCB2, PLCB3, PLCG1, PLCG2, PTGS2, and SOD1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. (B)). The expression profile reflects a complex interplay between vasodilative and vasoconstrictive responses to snake venom. Genes for phospholipases, PLCB2 and PLCG2 had shown sustained upregulation that can lead to increase in calcium ion signaling and has not returned to control levels which can affect muscle tone. Phospholipases are known for the release of calcium ions from intracellular stores and can lead to prolonged stress in the form of vasoconstriction. [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] A significant reduction in KNG1, a precursor to bradykinin, a potent vasodilator, has been observed. This reduced expression suggests lowered capacity of vasodilation. [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] NOS3 has shown a slight upregulation whereas NOS1 levels are reduced as compared to basal levels, this can lead to reduced blood flow and increased systemic vascular resistance. [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eIn the expression pattern of genes associated with pain agnosia, a total of 19 genes related to pain perception were identified in gene expression profile (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. (C)). The upregulated genes in our gene set included APP, BRAF, TGFB1, PTGS2, IL1B, and IL6. The gene expression data shows robust inflammatory response after injection characterized by significant upregulation in pro inflammatory cytokines, IL1B and IL6 and decrease in anti-inflammatory cytokine IL10 [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The upregulation of IL1B and IL6 can directly sensitize the nociceptors which can amplify pain. [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. IL1B and TNF are known to engage in NF-κB and MAPK pathways known for production of pro-inflammatory mediators than can further intensify pain perception. [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] The upregulation of PTGS2 or COX-2 imply the increased production of prostaglandins from arachidonic acid can cause acute or chronic pain by sensitizing nerve endings. [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] To counter the pain, vitamin D is known to suppress the expression of COX-2 to reduce prostaglandins and enhance the expression of TGFB1 to moderate the inflammatory response [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] But sustained reduction in CYP27B1 expression has been observed that can halt production of active form of vitamin D, could lead to intensified pain due to less controlled inflammation. [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] Prolonged deficiency of vitamin D can lead to weakened immunity and chronic inflammation. [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e] Some genes have shown downregulation as compared to controls, few of them include CRP, MEMCP2, and PNOC.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIdentification of targets, biological processes, and pathways associated with NO in physiological manifestations\u003c/p\u003e \u003cp\u003eWe identified 20 targets of nitric oxide by combining the intersection genes of hypoxia, vasoconstriction, and pain agnosia. Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was performed on the 20 therapeutic targets of NO using ShinyGO. Top 10 enriched pathways and processes in GO and KEGG analyses were presented (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. A- D). GO and KEGG pathway analysis, provided a broader perspective of how NO can serve as crucial mediator in the biological responses to snake bite. Nitric oxide synthase has shown the highest enrichment, indicating that the genes involved in our datasets have roles in regulating nitric oxide synthase crucial for controlling production of nitric oxide. NO can increase the cellular ability to cope with oxidative stress and hypoxia, aligning with observed KEGG categories such as HIF-1 signaling pathway. NO increases the stability of HIF1A under hypoxic conditions, making cells more resilient to oxygen fluctuations, oxidative stress, and promoting angiogenesis. [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e] NO is a marker molecule that promote angiogenesis, NO can activate AKT1, promoting endothelial cell survival, proliferation, and NOS production. This can create feedback loop to maintain endothelial function and vasodilation important for ensuring enough oxygen supply to tissue via blood stream. Interaction between AKT1, HIF1A, and NO can lead to improved vasculature and angiogenesis. [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eThe GO analysis reveals that biological processes were enriched in responses to reactive oxygen species, chemical and oxidative stress, blood vessel development, vasculature, and tube development, etc. Enriched cellular components includes platelet alpha granule lumen, neuron projection cytoplasm, plasma membrane components such as membrane raft and microdomain. Intersection genes were highly enriched in molecular functions involving nitric oxide synthase regulator activity, growth factor receptor binding, cytokine receptor binding and phosphatase binding. The KEGG analysis reveals many signaling pathways, these include HIF-1 signaling pathway, AGE-RAGE signaling pathway in diabetic complications, and relaxin signaling pathway, and various disease pathways including those related to cancer and viral infections. The GO and KEGG analyses suggest that the target genes are related to oxidative stress, signal transduction, immune response, angiogenesis, and nitric oxide synthase regulator activity. Nitric-oxide synthase regulator activity shows the highest fold enrichment suggesting strong involvement of nitric oxide regulation in the gene expression profile under the influence of venom.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePPI network analysis using CytoHubba, MCODE, and iRegulon plugins in Cytoscape\u003c/p\u003e \u003cp\u003eThe PPI network of therapeutic targets of NO was constructed using STRING database and imported to Cytoscape software. Top 10 genes namely APP, TNF, AKT1, IL1B, EGFR, PTGS2, MMP2, TGFB1, IL6 and HIF1A were identified as hub genes using closeness, degree, EPC, and MNC algorithms in CytoHubba plugin. The identified hub genes were presented in a network diagram shows the relationship among several hub genes and heat map displays the expression pattern of the genes shown in network diagram under influence of venom over time (1 hour, 6 hours, and 24 hours, and control) Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. (A- B). KEGG pathways such as AGE-RAGE signaling in diabetic complications, provide insight into role of hub genes like AKT1, IL1B, and IL6 in these processes. NO can inhibit NF-κB activation, thereby reducing expression of proinflammatory cytokines. [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] Regarding IL-6, NO can enhance or suppress its expression. This dual nature reflects its complex nature in inflammatory responses. [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] To monitor the inflammatory response, NO can act as antioxidant or pro-oxidant, depending upon concentration and cellular requirement. This can also help in maintaining redox homeostasis. [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e] Another cytokine TGFB1 is crucial for cellular proliferation, and extracellular matrix production. It has shown upregulation that can lead to tissue fibrosis and can be controlled by NO via Smad proteins. [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e] The expression of MMP2 has decreased, indicates a reduced capacity to degrade ECM and subsequent fibrosis. NO can activate MMPs through the NO-cGMP pathway. Thus, NO can prevent excessive fibrosis by promoting effective tissue repair. [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAfter hub gene identification, the MCODE plugin in Cytoscape was used to predict the modules in the PPI network of therapeutic targets of NO (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e. (A)). The MCODE clustered the densely connected regions into single module comprised of 18 genes and 131 edges. The obtained module defined NOS3 as seed node, whereas genes PIK3CA and COL18A1 were found unclustered (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). The central positioning of NOS3 in the module indicates that the cluster analysis started from this protein and it is potentially the major regulator in this network. Thus, we hypothesize that focusing on nitric oxide synthesis and its targets are of utmost importance in the understanding the pathophysiology of snakebite induced complications.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eLastly, the iRegulon plugin in the Cytoscape was used to predict the transcription factors that may regulate the NO associated genes in these physiological conditions. The normalized enrichment scores (NES) were calculated and used for ranking purposes. The top 10 predicted TFs based on NES scores included MAFK, HDAC2, STAT5A, CEBPG, EGR1, GATA5, FOS, IRX4, FOXJ2, and BACH1 as shown in (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Among the identified transcription factors, MAFK stood first with highest NES (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e. (B)). The results showed that MAFK is a key transcription factor regulating the 16 of 20 genes of PPI network and can play a defining role in regulating the expression pattern of genes related to NO pathway. As we witnessed the enrichment of pathways related to oxidative stress, MAFK can play a regulatory role to finely tune the cellular response to oxidative stress by preventing under- or over- activation of stress responses through ARE pathway [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. A recent study demonstrates a complex role of MAFK in inflammation by regulating and modulating NF-κB activity to bind DNA, thereby promoting transcription of pro-inflammatory genes. [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e] This regulation is important if acute immune responses are required, but as we observed immune response is already heightened after venom injection, if remains unchecked than this interaction can be detrimental leading to chronic inflammation rather beneficial. Thus, understanding the functioning of MAFK in the cases of snake bites is important as it can aggravate the pathological condition.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe top 10 iRegulon predicted transcription factors (ranked by NES) related to 20 N0-associated genes.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS. No.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTranscription factor\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNES\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTargets Number\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMotifs/Tracks\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMAFK\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7.592\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e59\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHDAC2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5.938\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSTAT5A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5.608\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCEBPG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5.596\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEGR1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.955\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGATA5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.832\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFOS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.694\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIRX4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.558\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFOXJ2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.558\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBACH1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.460\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eFocusing on enrichment in nitric oxide synthase regulator activity and presence of NOS3 as seed node in MCODE, suggests an external supplementation of nitric oxide can have significant therapeutic implications in the treatment of snake bites and more particularly in these resulting complications. MAFK is identified as a key transcription factor in our study, therefore understanding its role could provide insights into its potential as a therapeutic target in the cases of snake bites.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOur research began by considering the metabolic product of an ingredient in a formulation used to treat snake bites in Jind, a district in Haryana, India. We identified the gene combinations and changes in their expression pattern leading to complications such as hypoxia, vasoconstriction, and pain agnosia post viperid venom injection. We focused on the interplay of nitric oxide associated genes in these complications that opens new avenues for exploring the therapeutic potential of nitric oxide, a metabolic product of potassium nitrate.\u003c/p\u003e \u003cp\u003eSuch studies are crucial for developing therapeutic interventions based on understanding of the molecular basis of snake venom pathology. Like the parent formulation, potassium nitrate is stable under normal storage conditions. Further investigations are required into such formulations and their ingredients, which are reported to be life-saving. To achieve the World Health Organization's target to reduce mortality by half by 2030, effective, stable, and affordable alternatives to present day antivenoms are required. These alternatives are crucial for saving the lives of field workers and children who are more susceptible to snake bites. Therefore, publication of this study could guide researchers to consider the traditional formulations as potential source for developing new, stable, and effective antivenom treatments.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003col\u003e\n \u003cli\u003eARE - Antioxidant Response Element\u003c/li\u003e\n \u003cli\u003eBP - Biological Process\u003c/li\u003e\n \u003cli\u003eCC - Cellular Component\u003c/li\u003e\n \u003cli\u003eMF - Molecular Function\u003c/li\u003e\n \u003cli\u003eCTNNB1- Catenin Beta 1.\u003c/li\u003e\n \u003cli\u003eDNA- Deoxyribonucleic Acid\u003c/li\u003e\n \u003cli\u003eEGFA- Epidermal Growth Factor Alpha\u003c/li\u003e\n \u003cli\u003eEPAS1- Endothelial PAS Domain Protein 1\u003c/li\u003e\n \u003cli\u003eEPC- Edge Percolated Component\u003c/li\u003e\n \u003cli\u003eMNC - Maximum Neighborhood Component\u003c/li\u003e\n \u003cli\u003eFDR - False Discovery Rate\u003c/li\u003e\n \u003cli\u003eGO - Gene Ontology\u003c/li\u003e\n \u003cli\u003eHIF1A/HIF3A - Hypoxia-Inducible Factor 1-alpha/3-alpha\u003c/li\u003e\n \u003cli\u003eKEGG - Kyoto Encyclopedia of Genes and Genomes\u003c/li\u003e\n \u003cli\u003eKNG1 - Kininogen 1\u003c/li\u003e\n \u003cli\u003eMAFK - Small MAF transcription factor K.\u003c/li\u003e\n \u003cli\u003eMAPK1 - Mitogen-Activated Protein Kinase 1\u003c/li\u003e\n \u003cli\u003eMCODE - Molecular Complex Detection\u003c/li\u003e\n \u003cli\u003eMTOR - Mechanistic Target of Rapamycin\u003c/li\u003e\n \u003cli\u003eNCBI - National Center for Biotechnology Information\u003c/li\u003e\n \u003cli\u003eNCBI GEO - Gene Expression Omnibus\u003c/li\u003e\n \u003cli\u003eNO - Nitric Oxide\u003c/li\u003e\n \u003cli\u003eNOS3 - Nitric Oxide Synthase 3\u003c/li\u003e\n \u003cli\u003ePLCB2, PLCB3, PLCG1, PLCG2 - Phospholipase C Beta 2, Beta 3, Gamma 1, Gamma 2\u003c/li\u003e\n \u003cli\u003ePTGS2 - Prostaglandin-Endoperoxide Synthase 2, also known as COX-2\u003c/li\u003e\n \u003cli\u003eSOD1 - Superoxide Dismutase 1\u003c/li\u003e\n \u003cli\u003eSTRING - Search Tool for the Retrieval of Interacting Genes/Proteins.\u003c/li\u003e\n \u003cli\u003eTF - Transcription Factor\u003c/li\u003e\n \u003cli\u003eTNF - Tumor Necrosis Factor\u003c/li\u003e\n \u003cli\u003eVEGFA - Vascular Endothelial Growth Factor A\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eWHO - World Health Organization\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData in this study can be obtained from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eM.S. conceptualized, designed, performed, and wrote the present study, U.T. edited the manuscript, S.L., D.M., A.R. critically revised the article.\u003c/p\u003e\n\u003cp\u003eCorresponding author\u003c/p\u003e\n\u003cp\u003eCorrespondence to Madhu Sindhu.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo funding is available.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors would like to express our gratitude to NCBI-GEO (https://www.ncbi.nlm.nih.gov/geo/) for providing valuable gene expression profile for data analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no potential conflicts of interest with respect to research, authorship, and/or publication of this article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePach, S. et al. Paediatric snakebite envenoming: the world's most neglected 'Neglected Tropical Disease'? \u003cem\u003eArchives of disease in childhood\u003c/em\u003e. 105 (12), 1135\u0026ndash;1139; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1136/archdischild-2020-319417\u003c/span\u003e\u003cspan address=\"10.1136/archdischild-2020-319417\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGopalakrishnan, M., Saurabh, S., Sagar, P., Bammigatti, C. \u0026amp; Dutta, T. K. A simple mortality risk prediction score for viper envenoming in India (VENOMS): A model development and validation study. PLOS Negl. Trop. Dis. 16 (2), e0010183; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1371/journal.pntd.0010183\u003c/span\u003e\u003cspan address=\"10.1371/journal.pntd.0010183\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePinto, L. J. et al. Case Report: Hemothorax in Envenomation by the Viperid Snake \u003cem\u003eBothrops asper\u003c/em\u003e. Am. J. Trop. Med. Hyg. 100 (3), 714\u0026ndash;716; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.4269/ajtmh.18-0826\u003c/span\u003e\u003cspan address=\"10.4269/ajtmh.18-0826\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKalita, B., Mackessy, S. P. \u0026amp; Mukherjee, A. K. Proteomic analysis reveals geographic variation in venom composition of Russell\u0026rsquo;s Viper in the Indian subcontinent: implications for clinical manifestations post-envenomation and antivenom treatment. Expert Rev. Proteomics. 15 (10), 837\u0026ndash;849; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/14789450.2018.1528150\u003c/span\u003e\u003cspan address=\"10.1080/14789450.2018.1528150\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXie, C. et al. Neutralizing effects of small molecule inhibitors and metal chelators on coagulopathic Viperinae snake venom toxins. Biomedicines. 8 (9), 297; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/biomedicines8090297\u003c/span\u003e\u003cspan address=\"10.3390/biomedicines8090297\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNoor, F., Ashfaq, U. A., Albutti, A., Alwashmi, A. S. \u0026amp; Aljasir, M. A. Network Pharmacology Approach for Medicinal Plants: Review and Assessment. Pharmaceuticals. 15 (5), 572; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/ph15050572\u003c/span\u003e\u003cspan address=\"10.3390/ph15050572\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZeng, L. et al. Network Pharmacological Study on the Mechanism of \u003cem\u003eCynanchum paniculatum\u003c/em\u003e (Xuchangqing) in the Treatment of \u003cem\u003eBungarus multicinctus\u003c/em\u003e Bites. Biomed. Res. Int. 3887072; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1155/2022/3887072\u003c/span\u003e\u003cspan address=\"10.1155/2022/3887072\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSun, S. et al. Exploring the Mechanism of Cytisine in Treating Respiratory Depression following Venomous Snake Bites Based on Network Pharmacology and Molecular Docking. Am. J. Transl. Res. 16 (4), 1442\u0026ndash;1453; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.62347/MCPD8618\u003c/span\u003e\u003cspan address=\"10.62347/MCPD8618\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTsantoulas, C. \u0026amp; McMahon, S. B. Opening Paths to Novel Analgesics: The Role of Potassium Channels in Chronic Pain. Trends Neurosci. 37 (3), 146\u0026ndash;158; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.tins.2013.12.002\u003c/span\u003e\u003cspan address=\"10.1016/j.tins.2013.12.002\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoncada, S., Palmer, R. M. \u0026amp; Higgs, E. A. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol. Rev. 43 (2), 109\u0026ndash;142 (1991).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIgnarro, L. J. Nitric oxide: a unique endogenous signaling molecule in vascular biology. Biosci. Rep. 19 (2), 51\u0026ndash;71 (1999).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePetrizzo, R., Mohr, J., Mantione, K. \u0026amp; Goldstein, L. The Role of Endogenous Morphine and Nitric Oxide in Pain Management. Pract. Pain Manag. 14 (9), (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eResiere, D., Mehdaoui, H. \u0026amp; Neviere, R. Inflammation and Oxidative Stress in Snakebite Envenomation: A Brief Descriptive Review and Clinical Implications. Toxins 14 (11), 802; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/toxins14110802\u003c/span\u003e\u003cspan address=\"10.3390/toxins14110802\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGhosh, R., Maity, A., Biswas, U., Das, S. \u0026amp; Benito-Le\u0026oacute;n, J. Lance-Adams Syndrome: An Unusual Complication of Snakebite Envenomation. Toxicon 209, 50\u0026ndash;55; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.toxicon.2022.02.008\u003c/span\u003e\u003cspan address=\"10.1016/j.toxicon.2022.02.008\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBozkurt, M., Kulahci, Y., Zor, F. \u0026amp; Kapi, E. The Management of Pit Viper Envenomation of the Hand. Hand 3 (4), 324\u0026thinsp;\u0026ndash;\u0026thinsp;33; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11552-008-9114-2\u003c/span\u003e\u003cspan address=\"10.1007/s11552-008-9114-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2008).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMehta, S. R. \u0026amp; Sashindran, V. K. Clinical Features and Management of Snake Bite. Med. J. Armed Forces India. 58 (3), 247\u0026ndash;249; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/S0377-1237(02)80140-X\u003c/span\u003e\u003cspan address=\"10.1016/S0377-1237(02)80140-X\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2002).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZdenek, C. N., et al. Children and Snakebite: Snake Venom Effects on Adult and Paediatric Plasma. Toxins 15 (2), 158; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/toxins15020158\u003c/span\u003e\u003cspan address=\"10.3390/toxins15020158\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJanky, R. et al. iRegulon: from a gene list to a gene regulatory network using large motif and track collections. PLoS computational biology. 10 (7), e1003731; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1371/journal.pcbi.1003731\u003c/span\u003e\u003cspan address=\"10.1371/journal.pcbi.1003731\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLarsen, F. J. Ekblom, B., Sahlin, K., Lundberg, J. O. \u0026amp; Weitzberg, E. Effects of dietary nitrate on blood pressure in healthy volunteers. N. Engl. J. Med. 355 (26), 2792\u0026ndash;2793; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1056/NEJMc062800\u003c/span\u003e\u003cspan address=\"10.1056/NEJMc062800\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2006).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUmbrello, M., Dyson, A., Feelisch, M. \u0026amp; Singer, M. The key role of nitric oxide in hypoxia: hypoxic vasodilation and energy supply-demand matching. Antioxid Redox Signal. 19 (14), 1690\u0026ndash;1710; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1089/ars.2012.4979\u003c/span\u003e\u003cspan address=\"10.1089/ars.2012.4979\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKourembanas, S., McQuillan, L. P., Leung, G. K. \u0026amp; Faller, D. V. Nitric oxide regulates the expression of vasoconstrictors and growth factors by vascular endothelium under both normoxia and hypoxia. J. Clin. Invest. 92 (1), 99\u0026ndash;104; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1172/JCI116604\u003c/span\u003e\u003cspan address=\"10.1172/JCI116604\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (1993).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAnbar, M. et al. Role of nitric oxide in the physiopathology of pain. J. Pain Symptom Manage. 14 (4), 225\u0026ndash;254 (1997).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLundberg, J. O., Weitzberg, E. \u0026amp; Gladwin, M. T. The nitrate-nitrite-nitric oxide pathway in physiology and therapeutics. Nat. Rev. Drug Discov. 7 (2), 156\u0026ndash;167; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/nrd2466b\u003c/span\u003e\u003cspan address=\"10.1038/nrd2466b\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2008).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBelaiba, R. S. et al. Hypoxia up-regulates hypoxia-inducible factor-1alpha transcription by involving phosphatidylinositol 3-kinase and nuclear factor kappaB in pulmonary artery smooth muscle cells. Molecular biology of the cell, \u003cem\u003e18\u003c/em\u003e (12), 4691\u0026ndash;4697; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1091/mbc.e07-04-0391\u003c/span\u003e\u003cspan address=\"10.1091/mbc.e07-04-0391\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2007).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSemenza G. L. Hypoxia-inducible factors in physiology and medicine. Cell, 148 (3), 399\u0026ndash;408; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.cell.2012.01.021\u003c/span\u003e\u003cspan address=\"10.1016/j.cell.2012.01.021\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen, S., Wang, X., Nisar, M. F., Lin, M. \u0026amp; Zhong, J. L. Heme Oxygenases: Cellular Multifunctional and Protective Molecules against UV-Induced Oxidative Stress. Oxid. Med. Cell. Longev, 208, 5416728; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1155/2019/5416728\u003c/span\u003e\u003cspan address=\"10.1155/2019/5416728\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDunn, L. L. et al. Hmox1 (Heme Oxygenase-1) Protects Against Ischemia-Mediated Injury via Stabilization of HIF-1α (Hypoxia-Inducible Factor-1α). \u003cem\u003eArteriosclerosis, thrombosis, and vascular biology\u003c/em\u003e, 41 (1), 317\u0026ndash;330; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1161/ATVBAHA.120.315393\u003c/span\u003e\u003cspan address=\"10.1161/ATVBAHA.120.315393\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShweiki, D., Itin, A., Soffer, D. \u0026amp; Keshet, E. Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis. Nature. 359 (6398), 843\u0026ndash;845; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/359843a0\u003c/span\u003e\u003cspan address=\"10.1038/359843a0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (1992).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOliveira, V. Q. et al. Antiangiogenic properties of BthMP, a P-I metalloproteinase from \u003cem\u003eBothrops moojeni\u003c/em\u003e snake venom by VEGF pathway in endothelial cells. Biochemical and biophysical research communications. 706, 149748; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.bbrc.2024.14974824\u003c/span\u003e\u003cspan address=\"10.1016/j.bbrc.2024.14974824\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWilson, D. P., Susnjar, M., Kiss, E., Sutherland, C. \u0026amp; Walsh, M. P. Thromboxane A2-induced contraction of rat caudal arterial smooth muscle involves activation of Ca2\u0026thinsp;+\u0026thinsp;entry and Ca2\u0026thinsp;+\u0026thinsp;sensitization: Rho-associated kinase-mediated phosphorylation of MYPT1 at Thr-855, but not Thr-697. The Biochemical journal, 389 (3), 763\u0026ndash;774; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1042/BJ20050237\u003c/span\u003e\u003cspan address=\"10.1042/BJ20050237\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2005).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKaplan, A. P. \u0026amp; Joseph, K. Pathogenic mechanisms of bradykinin mediated diseases: dysregulation of an innate inflammatory pathway. Advances in immunology. 121, 41\u0026ndash;89; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/B978-0-12-800100-4.00002-7\u003c/span\u003e\u003cspan address=\"10.1016/B978-0-12-800100-4.00002-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJoyner, M. J. \u0026amp; Dietz, N. M. Nitric oxide and vasodilation in human limbs. Journal of Applied Physiology, 83 (6), 1785\u0026ndash;1796; doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1152/jappl.1997.83.6.1785\u003c/span\u003e\u003cspan address=\"10.1152/jappl.1997.83.6.1785\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (1997).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eResiere, D. et al. Inflammation and Oxidative Stress in Snakebite Envenomation: A Brief Descriptive Review and Clinical Implications. Toxins. 14 (11), 802; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/toxins14110802\u003c/span\u003e\u003cspan address=\"10.3390/toxins14110802\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRen, K. \u0026amp; Torres, R. Role of interleukin-1β during pain and inflammation. Brain Res. Rev. 60 (1), 57\u0026ndash;64; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.brainresrev.2008.12.020\u003c/span\u003e\u003cspan address=\"10.1016/j.brainresrev.2008.12.020\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2009).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLawrence, T. \u0026amp; Fong, C. The role of the NF-κB pathway in the pathogenesis of inflammation and cancer. Nat. Rev. Immunol. 10, 361\u0026ndash;378; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/nri2742\u003c/span\u003e\u003cspan address=\"10.1038/nri2742\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRicciotti, E. \u0026amp; FitzGerald, G. A. Prostaglandins and inflammation. Arterioscler. Thromb. Vasc. Biol. 31 (5), 986\u0026ndash;1000; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1161/ATVBAHA.110.207449\u003c/span\u003e\u003cspan address=\"10.1161/ATVBAHA.110.207449\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFeldman, D. et al. Vitamin D inhibition of the prostaglandin pathway as therapy for prostate cancer. Nutrition Reviews. 65 (8 Pt 2), S113-S115; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1753-4887.2007.tb00335.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1753-4887.2007.tb00335.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2007).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShipton, E. A. \u0026amp; Shipton, E. E. Vitamin D and Pain: Vitamin D and Its Role in the Aetiology and Maintenance of Chronic Pain States and Associated Comorbidities. \u003cem\u003ePain Res. Treat\u003c/em\u003e. 904967; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1155/2015/904967\u003c/span\u003e\u003cspan address=\"10.1155/2015/904967\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHabib, A. M., Nagi, K., Thillaiappan, N. B., Sukumaran, V. \u0026amp; Akhtar, S. Vitamin D and Its Potential Interplay with Pain Signaling Pathways. Frontiers in immunology. 11, 820; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fimmu.2020.00820\u003c/span\u003e\u003cspan address=\"10.3389/fimmu.2020.00820\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHendrickson, M. \u0026amp; Poyton, R. Crosstalk between nitric oxide and hypoxia-inducible factor signaling pathways: an update. Res. Rep. Biochem. 5, 147\u0026ndash;161; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2147/RRBC.S58280\u003c/span\u003e\u003cspan address=\"10.2147/RRBC.S58280\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDimmeler, S. et al. Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation. Nature 399 (6736), 601\u0026ndash;605; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/21224\u003c/span\u003e\u003cspan address=\"10.1038/21224\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (1999).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJue, D. M., Jeon, K. I. \u0026amp; Jeong, J. Y. Nuclear factor kappaB (NF-kappaB) pathway as a therapeutic target in rheumatoid arthritis. Journal of Korean medical science, 14 (3), 231\u0026ndash;238; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3346/jkms.1999.14.3\u003c/span\u003e\u003cspan address=\"10.3346/jkms.1999.14.3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (1999).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSiednienko, J., Nowak, J., Moynagh, P. N. \u0026amp; Gorczyca, W. A. (2011). Nitric oxide affects IL-6 expression in human peripheral blood mononuclear cells involving cGMP-dependent modulation of NF-κB activity. \u003cem\u003eCytokine\u003c/em\u003e, 54 (3), 282\u0026ndash;288; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.cyto.2011.02.015\u003c/span\u003e\u003cspan address=\"10.1016/j.cyto.2011.02.015\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePatel, R. P., Levonen, A., Crawford, J. H. \u0026amp; Darley-Usmar, V. M. Mechanisms of the pro- and anti-oxidant actions of nitric oxide in atherosclerosis. Cardiovascular research. 47 (3), 465\u0026ndash;474; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/s0008-6363(00)00086-9\u003c/span\u003e\u003cspan address=\"10.1016/s0008-6363(00)00086-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2000).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSaura, M. et al. Nitric oxide regulates transforming growth factor-beta signaling in endothelial cells. Circulation research. 97 (11), 1115\u0026ndash;1123; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1161/01.RES.0000191538.76771.66\u003c/span\u003e\u003cspan address=\"10.1161/01.RES.0000191538.76771.66\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2005).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMan Chow, B. S. et al. Relaxin Signals through a RXFP1-pERK-nNOS-NO-cGMP-Dependent Pathway to Up-Regulate Matrix Metalloproteinases: The Additional Involvement of iNOS. PLOS ONE. 7 (8), e42714; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1371/journal.pone.0042714\u003c/span\u003e\u003cspan address=\"10.1371/journal.pone.0042714\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKatsuoka, F. et al. Genetic Evidence that Small Maf Proteins Are Essential for the Activation of Antioxidant Response Element-Dependent Genes. Molecular and Cellular Biology, 25 (18), 8044\u0026ndash;8051; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/MCB.25.18.8044-8051\u003c/span\u003e\u003cspan address=\"10.1128/MCB.25.18.8044-8051\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2005).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNguyen, T., Huang, H. C. \u0026amp; Pickett, C. B. Transcriptional regulation of the antioxidant response element. Activation by Nrf2 and repression by MafK. The Journal of biological chemistry. 275 (20), 15466\u0026ndash;15473; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1074/jbc.M000361200\u003c/span\u003e\u003cspan address=\"10.1074/jbc.M000361200\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2000).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHwang, Y. J. et al. MafK positively regulates NF-κB activity by enhancing CBP-mediated p65 acetylation. Scientific reports. 3, 3242; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/srep03242\u003c/span\u003e\u003cspan address=\"10.1038/srep03242\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2013).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Snakebite, Network pharmacology, Nitric oxide, Hypoxia, Vasoconstriction, Pain","lastPublishedDoi":"10.21203/rs.3.rs-4512510/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4512510/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSnakebite envenomations pose significant global health challenges with limited effective treatments available. The complex physiological manifestations induced by snake venoms, such as hypoxia, vasoconstriction, and pain, have not fully deciphered at the genetic level. This study employs network pharmacology combined with gene expression analysis to uncover the molecular mechanisms underlying these interventions, and to explore nitric oxide as potential therapeutic target for snakebites. We used NCBI and GeneCards databases to collect the gene expression profile and therapeutic targets for snake bites. We identified that upregulation of genes like HIF1A and HIF3A, and downregulation of EGFA indicate responses to venom induced hypoxia. Change in expression of phospholipases and KNG1 suggests alteration in mechanisms involved in vasoconstriction. The increase in expression of cytokines and PTGS2 potentially linked to inflammation and pain induction. We identified 100 nitric oxide-related genes in mouse including 20 key genes directly involved in these responses to envenomation. The protein-protein interaction analysis through Cytoscape indicates that nitric oxide could play pivotal role in neutralizing venom effects. We identified MAFK as master regulator in nitric oxide associated genes set. Our observations highlight a previously unrecognized patterns of gene expression linked to hypoxia, vasoconstriction, and pain, and lays the groundwork for innovative approaches for treating snakebites.\u003c/p\u003e","manuscriptTitle":"Network pharmacology reveals physiological manifestations of viperid envenomation and role of nitric oxide in their treatment","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-14 20:15:30","doi":"10.21203/rs.3.rs-4512510/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"02d7a633-b5d4-4b2f-8149-37944ee8be6b","owner":[],"postedDate":"June 14th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":33231480,"name":"Biological sciences/Drug discovery"},{"id":33231481,"name":"Biological sciences/Physiology"},{"id":33231482,"name":"Biological sciences/Systems biology"},{"id":33231483,"name":"Health sciences/Risk factors"},{"id":33231484,"name":"Health sciences/Signs and symptoms"}],"tags":[],"updatedAt":"2025-01-29T08:38:48+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-14 20:15:30","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4512510","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4512510","identity":"rs-4512510","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}