Uncovering the pharmacological mechanism of strychnos nux-uomical in the treatment of peripheral neuropathy based on a network pharmacology approach

Background: Strychnos nux-uomical (Ma Qianzi in pinyin), a classic chinese herb, has been extensively used to treat peripheral neuropathy in clinical practice in china for many years. However, the pharmacological mechanisms of strychnos nux-uomical remain vague. The aim of this study was to decrypt the underlying mechanisms of strychnos nux-uomical in the treatment of peripheral neuropathy using a systems harmacology approach. Methods: Using pharmacokinetic parameters [oral bioavailability (OB) ≥ 30% and drug likeness (DL) ≥ 0.18] of traditional chinese medicine system pharmacology platform (TCMSP) to screen the active ingredient of strychnos nux-uomical, and then integrating target prediction human gene database to predict and screen its target genes for the treatment of strychnos nux-uomical, network topology attribute analysis software cytoscape 3.6.0 was utilized to construct the active components-peripheral neuropath targets network. String database and cytoscape software were used to draw the protein interaction network. Finally gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analyses by panther database. Results: A total of 13 potential active ingredients were screened based on their OB and DL parameters, and 72 core targets were related to disease target. The results of pathway enrichment analysis indicated that putative targets of strychnos nux-uomical mostly participated in various pathways associated with cellular process, response to stimulus, biological regulation, metabolic process, localization. The putative targets of strychnos nux-uomical (endothelial growth factor, tumor necrosis factor-α, interleukin-10, interleukin-6, interleukin 1β and so on) were recognized as active factors involved in the main biological functions of treatment, which implied that these were involved in the underlying mechanisms of strychnos nux-uomical on peripheral neuropathy. Conclusions: In this study, a network pharmacology-based strategy integrating target prediction, network construction, GO and KEGG pathway enrichment analyses, was adopted to predict the targets of strychnos The potential targets and molecular mechanisms underlying the effects of strychnos nux-uomical against peripheral neuropathy mediated by chemokine and cytokine signaling pathway, p53 pathway feedback loops 2, CCKR signaling map, ras pathway, angiogenesis, interleukin signaling pathway, gonadotropin-releasing hormone receptor pathway, VEGF signaling pathway, apoptosis signaling pathway, T cell activation in the treatment of peripheral neuropathy were systematically investigated by network pharmacology. This research provides a scientic basis for further elucidating the mechanism underlying the effects of Strychnos nux-uomical in the treatment of Peripheral neuropathy and suggests that the network pharmacology approach can be an effective tool to reveal the mechanisms of TCM. However, to improve the reliability of the results, further experimental experiments are needed to validate these results. and cholecystokinin receptor; VEGF: Vascular endothelial growth factor; GnRH: gonadotropin-releasing hormone; IGF: insulin like growth factor; NGF: nerve growth factor; BDNF: brain derived neurotrophic factor; FGF2: broblast

that putative targets of strychnos nux-uomical mostly participated in various pathways associated with cellular process, response to stimulus, biological regulation, metabolic process, localization. The putative targets of strychnos nux-uomical (endothelial growth factor, tumor necrosis factor-α, interleukin-10, interleukin-6, interleukin 1β and so on) were recognized as active factors involved in the main biological functions of treatment, which implied that these were involved in the underlying mechanisms of strychnos nux-uomical on peripheral neuropathy.
Conclusions: In this study, a network pharmacology-based strategy integrating target prediction, network construction, GO and KEGG pathway enrichment analyses, was adopted to predict the targets of strychnos nux-uomical relevant to the treatment of peripheral neuropathy and to further explore the involved pharmacological mechanisms.

Background
The term peripheral neuropathy refers to any disorder of the peripheral nervous system including single and multiple (asymmetric) mononeuropathy, and symmetrical involvement of many nerves (polyneuropathy) [1] . Peripheral neuropathies result when damage occurs to peripheral nerves due to a systemic disease, toxicant exposure or due to direct nerve injury [2] , result in loss of motor, sensory and autonomic functions in the denervated segments of the body, thus having strong impact in the quality of life of affected patients. Neuropathic pain is the most common clinical manifestation of peripheral neuropathy resulting from diabetes, chronic alcohol intake, certain cancers, vitamin B de ciency, infections, nerve-related diseases, toxins, and certain drugs [3] . Peripheral neuropathy occurs as a component of several common and many rare diseases [1] . Acquired neuropathies such as diabetic peripheral neuropathy, chemotherapy induced peripheral neuropathy and trauma/injury induced neuropathies occur as a result of enhanced oxidative stress and in ammation in peripheral neurons.
There are still no therapeutic agents available that can mitigate neuropathies in patients, and thus, more research is needed to identify molecular factors that can be effectively targeted.
Traditional chinese medicine (TCM) is featured as multiple targets and complex mechanisms of action and widely used in asian countries, especially China [4] , characterized by its satisfying therapeutic effects and minor side effects. Network pharmacology is an innovative method to study the mechanisms of drugs at the systemic level [5] , especially multi-target drugs. It encompasses bioinformatics, network analysis, and experimental approaches and integrates multiple sources of information [6] . Therefore, network approaches can accurately discriminate potential drug-target interactions [7] .
The strychnine tree (Strychnos nux-vomica L) (S.nux-vomica) belonging to family loganiaceae has been a very promising medication for certain disorders,used in TCM indications [8] : protracted arthritis, rheumatoid arthralgia, numbness and paralysis, sequela of poliomyelitis, traumatic injury, boils and sores. Disease groups treated in TCM: nervous system ailments, locomotor, immune system ailments. However, the pharmacological mechanisms of S.nux-vomica are still unknown.
The current review is aimed at identifying the potential targets and molecular mechanisms underlying the effects of S.nux-vomica associated with various peripheral neuropathy by the network pharmacology strategy. First, we predicted the molecular targets of S.nux-vomica through chemical similarity analysis and pharmacophore model screening. Pathological targets were identi ed using various bioinformatics platforms. We then performed enrichment analysis according to GO and KEGG terms and constructed a drug-target-pathway network.

Disease targets database building
We collected peripheral neuropathy targets from the human gene database is a searchable, integrative database that provides comprehensive, user-friendly information on all annotated and predicted human genes, including genomic, transcriptomic, proteomic, genetic, clinical and functional information [9] . "Peripheral neuropathy" as keywords to screen, there are 1231 targets, relevance score > 7 were selected associated with peripheral neuropathy were screened through Gene-Cards as shown in Fig. 1. Finally, we matched the prediction of the targets of strychnos nux-uomical active ingredients and the retrieval of the related targets of peripheral neuropathy and chose the overlapping targets as the related targets of strychnos nux-uomical for the treatment of peripheral neuropathy. The targets were then processed by String to build a protein-protein interaction (PPI) network to analyze the interactions among these targets and screen the hub targets based on topology [10] .

Network construction method
To collect the ingredients in strychnos nux-uomical, we used the traditional chinese medicine system pharmacology database [11] , a specialized pharmacological platform for TCM, to screen the active ingredients in strychnos nux-uomical by absorption, distribution, metabolism and excretion screening. In this process, the OB threshold was set as 30% and those ingredients with OB ≥ 30% were selected as the active ingredients for the next step [12] . The DL level of the compounds was set as 0.18, as this is the selection criterion for "drug-like" compounds in traditional chinese herbs [13] . In this study, those ingredients with the number of related targes > 3 OB ≥ 30% and DL ≥ 0.18 were selected [14] .
Active ingredients and related targets screening in strychnos nux-uomical, were then imported into cytoscape v3.2.1, an open software package project for visualizing, integrating, modeling and analyzing the interaction networks to generate compound-target network [15] .
The aim of our study used twice median degree values, was to screen the strychnos nux-uomical active ingredients and the related targets of peripheral neuropathy using cytoscape software merge tool to build a drug-target interaction network [16] .
To identify the hub nodes and essential proteins in the PPI network, the topological parameters of the node degree were calculated by Network Analyzer, and the three centralities (betweenness, closeness, and subgraph) were determined by the CytoNCA [17] plugin to screen the core target of strychnos nux-uomical, used twice median degree values [16] .

Enrichment Analyses
The core targets associated with peripheral neuropathy treatment by strychnos nux-uomical were extracted from KEGG database and GO enrichment analysis, KEGG terms by panther database [18] and constructed a drug-target-pathway network.

Results
Active compounds and compound-target network networkin strychnos nux-uomical Retrieved from TCMSP, there were 13 active compounds were selected in strychnos nux-uomical.
According to the active ingredient screening thresholds of OB ≥ 30%, DL ≥ 0.18 and the number of related targes > 3, 13 active ingredients were selected as shown in Table 1, 56 related targets were listed in Fig. 2.  to generate the protein-protein interaction (PPI) network about Component-disease interactions (Fig. 3).
We used the CytoHubba plugin in Cytoscape software according to twice median degree values to screen node component-disease interaction core target network, after screening, 72 core targets were obtained, as shown in Fig. 3.

GO biological process and KEGG pathway enrichment analysis
By enrichment analysis of the core target of strychnos nux-uomical in the PPI network, biological processes enrichment analysis and KEGG pathway enrichment analysis results in peripheral neuropathy shown in Fig. 4 and Fig. 5. The top ve GO biological processes were mainly involved in cellular process, response to stimulus, biological regulation, metabolic process, localization. We selected 10 KEGG pathways for analysis.  Fig. 4. Based on these main aspects, a complex multi-path synergetic effect may be the cause of the effect of strychnos nux-uomical on peripheral neuropathy shown in Table 2.

Discussion
There is an urgent need to develop new treatment strategies because of the growing crisis resulting from peripheral neuropathy and the poor e cacy of the existing approved drugs. We discovered that strychnos nux-uomical signifcantly alleviated the symptoms of peripheral neuropathy and believe that its impact may be related to its potential function. Hence, a network pharmacology strategy was used to reveal the mechanism underlying the effects of strychnos nux-uomical. The basic pathological changes of peripheral neuropathy include enhanced oxidative stress, in ammation and mitochondrial dysfunction in peripheral neurons [19] .
Therapeutic targets and the signaling pathways in which they participate were explored by PPI network construction, and pathway enrichment analysis. Information from various online servers and databases was integrated. The results we obtained are very reliable, as they bene ted from multiple information sources and different target identi cation methods.
Indicated by the KEGG pathway enrichment analysis, we proposed that strychnos nux-uomical plays a therapeutic role in the treatment of Peripheral neuropathy that is mediated by In ammation mediated by chemokine and cytokine signaling pathway, p53 pathway feedback loops 2, CCKR signaling map, Ras Pathway, Angiogenesis, Interleukin signaling pathway, Gonadotropin-releasing hormone receptor pathway, VEGF signaling pathway, Apoptosis signaling pathway, T cell activation and so on.
In ammation mediated by chemokine and cytokine signaling pathway In ammation has been indicated as a powerful driver of peripheral neuropathy. chemotherapy-induced increase in peripheral pro-in ammatory cytokines and a strong correlation with peripheral neuropathy, such as interleukin-6 (IL-6), tumor necrosis factor-α (TNFα), interleukin-1β (IL1β), and interleukin-10 (IL-10) may play a role in pain [20] , IL1β [21] , which starts an immune response and leads to chronic in ammation.
This eventually results in apoptosis and bioenergetic dysfunction associated with DN. Glucose induced advanced glycation end products (AGE) and its further association with receptors for AGE also involved in activation of NF-κB heterodimer and thus releases proin ammatory mediators such as IL-6, TNF-α, cyclooxygenase-2 release and thus produces neuro in ammation [22] .
p53 pathway feedback loops 2 The p53 circuit communicates with the Wnt-beta-catenin, IGF-1-AKT, Rb-E2F, p38 MAP kinase, cyclin-cdk, p14/19 ARF pathways and the cyclin PP2A, and p73 gene products [23] . One of the most intriguing methods of activating the p53 protein in a cell results from the mutational inactivation of some tumor suppressor genes including retinoblastoma, and adenomatous polyposis coli or the mutational activation of some oncogenes such as ras and myc.
Cytochrome c interacts with APAF-1 (a p53-regulated gene) to initiate a protease cascade, leading to the activation of caspase 9 and then caspase-3 followed by apoptosis. This is the intrinsic apoptotic pathway that is initiated by a number of stress signals that activate the p53 pathway. In addition to the intrinsic pathway, p53 regulates a series of genes that initiate the extrinsic apoptotic pathway (Fas ligand, killer Dr receptor), resulting in the caspase-8 and − 3 activities and apoptosis [23] .

Ras Pathway
Ras homolog family (Rho, belong to Ras superfamily)/Rho-associated coiled-coil kinase (ROCK) signaling pathway widely exists in human and mammal cells, which is closely related to inhibition of repair after optic nerve damage [24] .
The Rho/ROCK/LIMK pathway is central for the mediation of repulsive environmental signals in the central nervous system. Inhibition of neurite outgrowth is counteracted by downregulation of ROCK2 and LIMK1. Three major biological effects can be speci cally attributed to the knockdown of ROCK2: inhibition of apoptosis, attenuation of axonal degeneration and promotion of neurite outgrowth [25] .

CCKR signaling map
Gastrin and cholecystokinin receptor (CCKR) signaling events are indicated [26] , including proliferation, migration, differentiation, anti-apoptosis and in ammation. While central modules such as PKC, AKT1, Rho GTPase, MAP3K11, MAPK1/3 and AP1 are reportedly involved in all three cellular outcomes, other signaling mechanisms are more speci c in their effect, e.g. the BCL-module signaling is only observed to be involved in apoptosis.

Angiogenesis
Vascular endothelial growth factor (VEGF) [27] represents one of the main factors involved not only in angiogenesis and vasculogenesis but also in neuritogenesis. Modulating the local immune system to recruit cells to promote endogenous angiogenesis could have advantages, such as a greater degree of endothelial cell polarization. Furthermore, the immune system has a critical role in resolving in ammation following injury, where macrophages also have additional roles in this aspect of tissue regeneration.
Gonadotropin-releasing hormone receptor pathway Gonadotropin-releasing hormone (GnRH) is a decapeptide that acts not only as the principal hormone controlling puberty, but also as neuromodulatory hormone [28] . The current study addressed to the dorsal root ganglion(DRG)extended molecular pro ing towards gene expression analysis of genes known to be involved in the gonadotropin releasing hormone receptor and neuro infammatory (oxytocin/cytokines/chemokines) signaling pathways [29,30] . Serum levels of pro-in ammatory mediators such as IL-1β, TNF-α, and IL-6 were signi cantly increased compared to healthy individuals within the entire study period,IL-1β plays a critical role in neuropathic pain, metabolic disorders, heart failure, and depression [31] .
Growing evidence suggests that many representative growth factors, such as VEGF, insulin like growth factor (IGF), nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), and broblast growth factor-2 (FGF2) have dual neurotrophic and angiogenic effects and were even named angioneurins [31] .

Conclusions
The potential targets and molecular mechanisms underlying the effects of strychnos nux-uomical against peripheral neuropathy mediated by chemokine and cytokine signaling pathway, p53 pathway feedback loops 2, CCKR signaling map, ras pathway, angiogenesis, interleukin signaling pathway, gonadotropin-releasing hormone receptor pathway, VEGF signaling pathway, apoptosis signaling pathway, T cell activation in the treatment of peripheral neuropathy were systematically investigated by network pharmacology. This research provides a scienti c basis for further elucidating the mechanism underlying the effects of Strychnos nux-uomical in the treatment of Peripheral neuropathy and suggests that the network pharmacology approach can be an effective tool to reveal the mechanisms of TCM. However, to improve the reliability of the results, further experimental experiments are needed to validate these results.
Declarations Figure 1 Targets network of peripheral neuropathy The components-target network in strychnos nux-uomical. Blue and green nodes denote the compounds and targets. Biological processes GO annotation analysis results Page 18/18 Figure 5 KEGG pathway enrichment analysis results