Network pharmacology is indeed a growing and interdisciplinary field that has gained significant attention in the area of drug discovery and development. It combines computational biology, systems biology, and omics technologies to study the interactions between drugs and biological systems at a network level. The concept of network pharmacology recognizes that many drugs exert their therapeutic effects by acting on multiple targets and influencing various biological pathways simultaneously. This differs from the traditional "one drug, one target" paradigm. (Zimmermann et al., 2007). Understanding the complex pathways and targets affected by a drug is crucial for comprehending its mechanism of action and predicting its efficacy in different diseases. The use of drugs in combination is another important aspect addressed by network pharmacology. Combinations of drugs can have synergistic effects, targeting multiple disease-associated targets simultaneously. This approach has the potential to be more effective in treating complex diseases compared to single-target drugs. By studying the interactions and synergies between different drugs and their targets, network pharmacology aims to identify optimal drug combinations that maximize therapeutic outcomes. In recent years, network pharmacology has been applied to investigate the therapeutic benefits of herbal medicines and traditional Chinese medicines in various diseases (Luo et al., 2020). These studies have provided insights into the mechanisms of action of these natural products and their potential use in treating conditions such as DN, diabetic peripheral neuropathy and diabetic peripheral neuropathic pain. By mapping the interactions between the bioactive components of these medicines and the molecular targets in disease pathways, network pharmacology offers a systematic and holistic approach to understanding their therapeutic effects. A network pharmacology study carried out by Wang et al., 2022 evaluated the mechanism and active component of Radix Salviae, Salvianolic acid B for treatment of diabetic peripheral neuropathy and it was observed that that it downregulated the expressions of p-P38MAPK, inflammatory cytokines, and apoptosis targets, which are upregulated by hyperglycemia leading to beneficial effect in DPN. Another network pharmacology study identified the targets of Sappan Lignum (SL) and its potential mechanism in the treatment of DPN (Kang et al., 2021). Yet another research carried out by Lin et al., 2021 focused on identifying the key active components of Yi-Qi-Huo-Xue-Tong-Luo formula (YQHXTLF) which may potentially have therapeutic effects for DPN and its mechanism of action involves interacting with specific proteins such as TP53, MAPK1, JUN, and STAT3. By targeting these proteins, YQHXTLF has the ability to regulate various processes in the body, including inflammatory response, apoptosis (programmed cell death), and cell proliferation. The multitarget and multipathway action of YQHXTLF suggests that it can impact multiple biological pathways simultaneously, leading to a comprehensive therapeutic effect. This finding opens up new possibilities for understanding the pharmacological basis and mechanism of action of herbs in the treatment of DPN. Overall, network pharmacology holds great promise in accelerating the drug discovery process, facilitating the development of combination therapies, and exploring the therapeutic potential of natural products. By integrating computational modeling, network analysis, and experimental validation, this field can contribute to the development of more effective and personalized treatments for various complex diseases.
In the present network pharmacology approach was used to elucidate the targets of Vit.K2MK7 that could be beneficial in treatment of DN. Although there are clinical studies which prove the role of Vit.K2MK7 in the treatment of DN, there is dearth of knowledge regarding the molecular targets and pathways which are concurrent in its role. Since decades, the most well known physiological function of Vit. K specifically in humans is its involvement in the γ-carboxylation of numerous proteins. It acts as a crucial coenzyme for the posttranslational γ-carboxylation of glutamic acid residues on the luminal side of the rough endoplasmic reticulum. Vit. K is primarily indicated for the prevention of hemorrhagic disease in newborns and as an antidote for vitamin K antagonist overdose or poisoning (Raspini et al., 2015; Schulte et al., 2014). Oral administration is typically preferred in most cases. Vit. K plays a crucial role in blood coagulation by supporting the synthesis of clotting factors. However, the effects of Vit. K extend beyond coagulation to other areas of health. Vit. K is involved in the production of 18 to 19 Gla proteins, which have been found to have effects on bone and vascular calcification. Research has been conducted to investigate the potential benefits of increased intake or supplementation of Vit. K on conditions such as osteoporosis, fractures, and cardiovascular diseases (Simes et al., 2020; Palermo et al., 2017; Raspini et al., 2015). There have been suggestions of a possible relationship between Vit. K and cancer, as well as its role in cognitive function and brain physiology (Alisi et al., 2019). It is important to note that while there is ongoing research in these areas, the evidence regarding the specific effects of Vit. K on these conditions is still being studied and further research is needed to establish definitive conclusions with diabetic neuropathy being the matter of interest area here. We first obtained all the targets of Vit. K2MK7 from ChEMBL, Prediction Charite and SEA Search Server. A comprehensive network of molecules are present in databases that have drug like properties and biological activities that integrate genomic data, contribute to understanding interaction of proteins and ligands and helps in identifying new drugs and obtaining the targets for the drug molecules. Secondly, all the genes associated with DN were collected from CTD, DisGeNET and GeneCards and were then mapped to the target genes of Vit.K2MK7. We retrieved 69 targets in total out of 153 targets of Vit. K2MK7 that had a share in DN like diabetic peripheral neuropathic pain, DPN, diabetic neuralgia and diabetic neuropathies (Fig. 1). We constructed therapeutic moiety-target interaction network in cytoscape (Fig. 2a) and top 10 genes were identified by cytohubba plugin which were SCN3A, PLCG1, DPP8, SLC6A5, TRPV2, TRPM8, TBXA2R, KLF5, PDGFRA and FPR2 (Fig. 2b). 56 interacting proteins of the targets were identified from STRING database and subsequently a protein-protein interaction network was created using the cytoscape database. The top 10 genes identified by cytohubba plugin were NFKBIA, NFKB1, PSMA2, PSMA4, PSMC4, PSMD8, PSMA3, PSMD7, PSMB7 and PSMB2. An extensive knowledge of predicted and known protein-protein interaction networks is present in STRING database. Apart from the top 10 genes, 1 important genes is HIF1α. A recent research study conducted by Rojas et al., 2018 demonstrated that in peripheral sensory neurons, HIF1α acts like an upstream modulator of ROS by suppressing hyperglycemia-induced nerve damage and thereby reducing ROS levels. Furthermore it induced expression of VEGF which may promote peripheral nerve survival and overall be responsible for its protective action. Our data suggested that HIF1α stabilization may be thus a new strategy target for limiting sensory loss which is supposedly a debilitating late complication of diabetes. Our findings revealed that the 69 overlapping target genes participated in diabetic neuropathies (Table 1). Pathway analysis in Reactome revealed the key involvement of genes in 673 pathways among which some were immune mediated pathways such as interleukin signaling, cytokine signaling in immune system, nervous system development, signal transduction, diseases of signal transduction by growth factor receptor, MAPK1/MAPK3 signaling, metabolism and regulation of NF-κβ signaling (Fig. 4a and 4b). Various inflammatory mediators like interleukins, viz. IL-6, IL-35, IL-1β, IL-3 contribute to interleukin signaling pathway whereas production of pro-inflammatory cytokines viz. NF-κβ and TNFα have been known to participate in nerve fibre inflammation and damage implicated in pathophysiology of DPN (Cox et al., 2017; Jiang et al., 2019; Hangping et al., 2020; Chopra et al., 2010). Oxidative stress is implicated in pathophysiology of DPN. The overproduction of reactive oxygen species (ROS) and the deregulation of antioxidant defense systems in DPN can distort the redox balance and promote oxidative damage. This oxidative damage contributes to nerve dysfunction and plays a significant role in the development and progression of DPN (Lin et al.,2023). The reactome database points out a key pathway related to nuclear events mediated by Nuclear factor erythroid 2-related factor 2 (NFE2L2). It is also called as Nrf2 and it serves to be a crucial transcription factor involved in NRF2-KEAP1 pathway promoting mitochondrial biogenesis, antioxidant machinery and protection of oxidative stress (Zu et al., 2020). The regulation of detoxification enzymes, such as nucleotide adenosine diphosphate hydrogenase (NADPH), haem oxygenase-1 (HO-1) and quinone oxidoreductase-1 (NQO1), which aid in resisting oxidative stress and protecting cells is initiated by the Nrf2-ARE signaling pathway (Leng et al., 2020). This claim is further substantiated by recent research study which proved that metformin activated an antioxidant pathway involving nuclear factor erythroid 2-related factor (Nrf2) that binds to the antioxidant response element (ARE) to improve mitochondrial function and reduce oxidative stress and thereby protect spinal cord from damage (Demaré et al., 2021). The major enriched pathways identified using Funrich database include signaling events mediated by VEGFR1 and VEGFR2, VEGF and VEGFR signaling network, mTOR signaling pathway, Insulin Pathway, IL3-mediated signaling events and TCR signaling (Fig. 3a and 3b). Neuronal insulin signaling may play a significant role in DN (Zochodne 2014; 2015). The Diabetes Control and Complications Trial (DCCT) is a strong evidence linking poor glucose control and DN (Diabetes Control Complications Trial Research Group, 1995a; Grote and Wright., 2016). The results of this trial demonstrated that patients who achieved intensive glycemic control through multiple daily insulin injections or an external pump had a 64% reduction in neuropathy over a 5-year period compared to patients on conventional therapy, which involved fewer daily insulin injections with mixed rapid and intermediate-acting insulin. VEGFR1 and VEGFR2 are receptors of Vascular Endothelial Growth Factor (VEGF) which is implicated in DPN. VEGF is a potent growth factor known for its role in promoting angiogenesis, the formation of new blood vessels. The vascular alterations observed in peripheral nerves after mechanical injury or in metabolic disorders are well-documented. In a research study carried out by Samii et al., 1999, the chronic hyperglycemia associated with type I diabetes likely contributes to the sensory neuropathy observed in the rat model. The sensory neuropathy is characterized by impaired sensory function in the peripheral nerves. The up-regulation of VEGF in Schwann cells and neurons suggests that VEGF may play a crucial role in the regeneration of nervous tissue in response to the functional alterations caused by sensory neuropathy. Although further research might be needed to elucidate the precise mechanisms by which VEGF influences nerve regeneration and to explore potential therapeutic interventions based on these findings. The analysis thus reveals the involvement of Vit. K2MK7’s targets in these pathways.
The analysis of GO (MF, CC, TF and BP) revealed that the 69 genes participated in cell communication, protein modification, protein metabolism, signal transduction, nucleoside, nucleotide and nucleic acid metabolism, energy pathways, protein tyrosine/ serine/ threonine phosphatase activity, tyrosine kinase activity and ion channel activity, catalytic activity, transcription regulator activity and G-Protein Coupled Receptor activity while others consisted of innate immune response, transport metabolism, lipid metabolism and DNA repair (Fig. 9a, 9b). The genes were localized in different cellular components such as plasma membrane, integral to plasma membrane, cytoplasm, endoplasmic reticulum, nucleus, mitochondrion, exosomes, etc (Fig. 7a and 7b). Further, among the total of 210 transcription factors (TF) found, the significant ones were PPARG or PPAR-γ, SP4, KLF7, EGR1, SP1, NFYA and HNF4A. PPAR-γ is another key transcription factor that is involved in numerous cellular functions like energy metabolism, regulation of mitochondrial function, regulation of anti-oxidant defence mechanisms and oxidation of fatty acids (Fig. 8a and 8b). In a recent research study carried out by Santos et al., 2022 it was seen that pioglitazone, a PPAR-γ agonist exhibited greater anti-hyperalgesic responses to pioglitazone in females as compared to males in mouse models of chemical-induced nociception, postsurgical pain, neuropathic pain, and DPN. Our analysis, too, identified PPAR-γ as the key transcription factor via gene ontology (Fig. 8a). Thus, the GO enrichment data further strengthens the role of Vit. K2MK7 in treating DPN possibly through pathways of inflammation, gene transcription, oxidative stress and mitochondrial pathways. The overlaid doughnut and bar plots of biological pathways revealed that the 69 genes participated in pathways of insulin signaling, signaling events mediated by VEGFR1 and VEGFR2, VEGF and VEGFR signaling network, glypican pathway, Insulin-like Growth Factor (IGF) activity by Insulin-like Growth Factor Binding Proteins (IGFBPs), atypical NF-kappaβ pathway, signaling by interleukins, canonical NF-κβ pathway, VEGF binds to VEGFR leading to receptor dimerization and insulin receptor signaling cascade. further suggesting their potential role in DPN pathogenesis (Fig. 6a,6b).
Very meagre knowledge is available in public domain about the mechanistic targets of Vit.K2MK7 and our present study will surely unlock the possibilities for conducting more preclinical studies and subsequently clinical studies for unveiling the mechanisms which would eventually pave way to cross potential barriers in circumventing the failure of current therapies used for the management of DN.