Systematically Investigating the Pharmacological Mechanism of Momordica grosvenori in the Treatment of Spinal Cord Injury by Network Pharmacology and Experimental Verification

Objective This study aimed to explore the molecular mechanism of Momordica grosvenori (MG) in spinal cord injury (SCI) by network pharmacology analysis. Methods We searched for potential active MG compounds using the TCMSP database and the BATMAN-TCM platform. The Swiss target prediction database was used to find MG-related targets and the targets of SCI from the CTD, GeneCards, and DrugBank databases. Following that, a protein-protein interaction (PPI) study was carried out. Cytoscape software was used to calculate the hub gene, and R software was used to evaluate the Gene Ontology (GO) and KEGG enrichment pathways. Finally, molecular docking between the hub protein and important compounds was performed. We verified STAT3, MAPK1, HSP90AA1, PIK3R1, PIK3CA, and RXRA potential targets by quantitative PCR. Results We obtained 293 MG-anti-SCI targets with potential therapeutic utility by intersecting 346 MG-related targets and 7214 SCI-related targets. The top 10 identified genes, ranking in descending order of value, were SRC, STAT3, MAPK1, HSP90AA1, PIK3R1, PIK3CA, RXRA, AKT1, CREBBP, and JAK2. Through enrichment analysis and literature search, 10 signaling pathways were screened out. The molecular docking of important drugs and hub targets revealed that some had a higher binding affinity. The results of quantitative PCR indicated that MAPK1, RXRA, and STAT3 were expressed differently in in vitro experiments. Conclusion In conclusion, the current work indicated that MG might play an anti-SCI role via multicomponent, multitarget, and multichannel interaction, which presents a novel idea for further research into the precise mechanism of MG-anti-SCI interaction.


Introduction
Spinal cord injury (SCI) is defned as structural damage and loss of whole or partial functional impairment of the spinal cord produced by various factors, often resulting in quadriplegia, paraplegia, or defecation dysfunction. Te incidence of SCI on a global scale ranges from 10.4 to 83 cases per million people per year. SCI can afect patients' quality of life and consume plenty of healthcare resources [1,2]. Complications such as lung infection, urinary tract infection, and pulmonary embolism are common after traumatic SCI and might result in death if the situation is severe [3,4]. SCI has signifcantly harmed society and individuals, becoming a major worldwide health and medical issue [2,5].
Clinical medical research has traditionally focused on the best approaches to treat spinal cord injuries. Within eight hours of the injury, methylprednisolone (MPS) can be utilized in some contraindication-free SCI cases, and patients should be advised of potential consequences [3]. Studies, however, indicate that MPS may increase gastrointestinal bleeding and that it provides no appreciable long-term benefts for those with acute traumatic SCI [6]. Highdose MPS treatment may increase the risk of adverse events in acute SCI patients but may not improve neurological recovery. Terefore, we advise against consistently using high-dose MPS early in acute SCI [7]. Riluzole can substantially improve the motor score, locomotor function, and neuropathic pain in the preclinical model of SCI [8]; however, insufcient data suggest riluzole's therapeutic effcacy for SCI. Te efect of riluzole on traumatic and nontraumatic SCI is only verifed in preclinical models [8]. Single-cell treatment for SCI has few advantages [9]. Numerous promising studies are being conducted to alleviate the severe efects of SCI. Positive clinical results have not yet been validated, and most trials are still in adolescence [10]. We need further study, as was already stated, to investigate and verify potential treatments.
Traditional Chinese medicine (TCM) has attracted the attention of more and more researchers in recent years as a potential treatment for SCI [11]. Additionally, the current study suggests that TCM is a successful treatment for SCI [11]. Our objective has always been to create efcient, affordable, and secure drugs. Long utilized as a food and medicine equivalent by the populace, MG is a perennial herbaceous vine of the Cucurbitaceae family that is exclusive to China. It is also very safe and reasonably priced. It is mostly grown in the Chinese province of Guangxi, in the counties of Yongfu, Lingui, and Longsheng. In Guangxi's traditional medicine, MG is homologous in medicine and food. It acts as an antioxidant [12], a hypoglycemic [13], an antitussive [14], a sputum reducer [15], an antiinfammatory [16], an antimicrobial [17], etc. Kaempferol is one of the active ingredients of MG, which has neuroprotective [18], cardioprotective [19], anti-infammatory [20], antioxidant [21], and anticancer efects [22]. Kaempferol can promote the recovery of motor function in SCI rats [23], indicating that MG may have great potential to repair SCI.
Nevertheless, MG's primary components and pharmacological efects have not been thoroughly identifed, limiting future research and application. As a result, a thorough examination of MG's primary pharmacological components and implications is necessary. System analytic methodologies, such as network pharmacology, are promising for developing fresh approaches to elucidating the drug-genedisease relationship [24]. Te network pharmacology approach of TCM presents a novel study methodology [25]. Tis strategy will promote the use of evidence-based medicine in TCM, helping to prove MG's therapeutic value and improving the current drug discovery process [21].
Te study aimed to develop an analytical approach for validating the efects of MG on SCI using network pharmacology, molecular docking, and experiments. Figure 1 depicts the workfow used in this study. Te study plan intends to achieve the following objectives: (1) Discovering bioactive components in MG; (2) predicting the related targets of MG and SCI; (3) identifying SCI and MG-related biological processes, potential therapeutic targets, molecular mechanisms, and signal pathways using comprehensive network analysis; (4) and validating the above-analyzed data through molecular docking. Terefore, in the investigation of this study, we used monomer components of MG for experimental verifcation in consideration of better efcacy and evaluation methodologies.

Potential Active Compound Screening.
As TCM, it is very important to screen drugs with appropriate ADME characteristics [27]. Te percentage of oral medications that are absorbed by the digestive tract and reach the systemic circulation blood in an oral dose is referred to as oral bioavailability (OB) [28]. Te term "drug-like properties value" (DL) refers to the similarity between compounds and known drugs [29]. Te probability that a compound will become a drug increases with its DL. Te pharmacological activity must occur under certain parameters, including appropriate OB and DL values. According to the TCMSP database, we must utilize OB ≥ 30% and DL ≥ 0.18 as ADME guideline ranges. As a result, we used OB ≥ 30% and DL ≥ 0.18 as screening criteria to identify potential compounds for further investigation.

Target Identifcation.
Te predicted targets of active compounds screened from MG were obtained from the SwissTargetPrediction database (https://www. SwissTargetPrediction.ch/) [30]. Te fnal therapeutic target was then obtained by converting the target into UniProt ID and removing duplicate values.

Network Construction and Topological Analysis.
Using the STRING website, we calculated and constructed the PPI network interaction diagram [34]. A network of disease-compound-target is used to elucidate the pathophysiology of MG using Cytoscape software (version 3.7.2) [35]. A fundamental network topology attribute called degree is utilized to assess the properties of various interventions [36]. Degree in an undirected graph is the number of nearby nodes. In an interactive network, betweenness is the quantity of shortest pathways between two nodes. We identifed the essential nodes of the entire interaction network and comprehended the primary mechanism underlying its interaction efect by studying the degree and intermediate number [35]. Ten the Gene Ontology Biological Process (GOBP) analysis method was applied to further investigate the biological characteristics of target genes.
2.6. Molecular Docking. Te 3D structure of the ligand was imported from Protein Data Bank (PDB, https://www.rcsb. org/) [37], the ligands were then prepared, and the water was drained. Hydrogen and small molecule ligands were used to construct binding sites (active pockets), and molecular docking was conducted with active components.

In Vitro Experiments to Validate Compounds.
We performed in vitro experiments with rat astrocytes (AS). AS was acquired from the ATCC cell bank (American Type Culture Collection, ATCC). AS is cultured in DMEM medium (containing 5% penicillin-streptomycin and 5% serum; DEME: Gibco; serum: tetrasodium serum; penicillinstreptomycin: Solarbio). Cells are stored in a clean cell culture incubator containing 5% CO 2 at 37°C. AS were activated with 1 ug/ml of lipopolysaccharide (LPS) to form an infammatory model and treated with 10 uM kaempferol. Te efect of kaempferol on specifc target genes, including STAT3, MAPK1, HSP90AA1, PIK3R1, PIK3CA, and RXRA, was observed.

Quantitative PCR.
RNA from astrocytes in 6-well plates was extracted using the Axygen RNA kit (Amresco, China) according to the manufacturer's instructions. Te concentration and purity of the RNA, extracted cellular RNA, was measured using a spectrophotometer. Total RNA was reverse transcribed to cDNA using the TARAKA cDNA synthesis kit (Takara Bio) according to the manufacturer's instructions.
We have designed gene-specifc primer sequences based on the target genes (STAT3, MAPK1, HSP90AA1, PIK3R1, PIK3CA, and RXRA) on the NCBI website (https://www. ncbi.nlm.nih.gov/) and the primer sequences are arranged in Table 1. Te cells were extracted from intracellular RNA by adding cell lysate and other relevant operations after the drug concentration was applied. After the reverse transcription of RNA to cDNA was completed, specifc primers were added to the 96-well plates to co-act with the intracellular cDNA, which was analyzed and observed by quantitative PCR to see if the drug was regulated through these targets of SCI and drug coactivity.

Potential Active Compounds Identifcation.
We extract 11 MG of active compound candidates from the TCMSP database (OB ≥ 30%, DL ≥ 0.18) [30]. However, no eligible components can be found in the BATMAN-TCM database [26]. Te details of database searches and data extraction are shown in Table 2. We explored the research on the role of each major component. Among them, M01 beta-sitosterol has many biological functions, such as antianxiety and sedation, analgesia, immunomodulation, antibacterial, anticancer, anti-infammatory, lipid-lowering, liver protection, protection against NAFLD, wound healing, antioxidant, and antidiabetic activities [38]. M02 kaempferol (3,5,7-trihydroxy-2 -(4-hydroxyphenyl) -4H-chromen4-one) is a polyphenol that is abundant in fruits and vegetables. Kaempferol has been reported to exert anti-infammatory efects diferently [39]. It can reduce oxidative stress and infammatory response and promotes recovery of motor function in rats with SCI [23]. M03 mandenol is an unsaturated fatty acid with antibacterial and anti-infammatory properties, which is used in many cosmetics [4]. M04

MG-Related Targets and SCI-Related Targets.
To directly retrieve the name of the active ingredient's target gene, we entered the SMILE number of the active components (UniProt ID) into the SwissTargetPrediction platform [30] and deleted the duplicated value. For the active ingredients whose SMILE number cannot be obtained directly from the website, we use Open Babel software to calculate [43]. Finally, analyzing the above data, we received 11 MG-related active compounds and 346 MG-related targets. Te GeneCards database (https://www.genecards.org/) was performed to predict the potential targets of compounds, and 5716 potential targets were obtained [31]. Ten, we used the same strategy to examine the Comparative Toxicogenomics Database (CTD, https://ctdbase.org/) for 3234 possible targets [32]. In addition, we obtained 15 potential targets of SCI from the DrugBank database (https://go.drugbank.com/) [33]. After converting the potential targets from the three databases into UniProt numbers and merging them, 7214 potential targets remained after the duplicate values were removed.

Terapeutic Targets for MG-Anti-SCI.
We discovered the therapeutic targets at the confuence of MG and SCI, which had potential therapeutic benefts for treating SCI with MG. Venny software was used to intersect the MG and SCI targets, as illustrated in Figure 2, and 293 prospective targets were found [44].
Te interaction network is then constructed using Cytoscape software to refect the connection between active compounds from MG and SCI targets (Figure 3). In Figure 3, the interaction relationship is shown by the line connecting the nodes.

Hub Genes of MG-Anti-SCI and Construction of PPI
Network. We retrieved 293 therapeutic targets of MG for SCI from the STRING database and constructed a PPI network (Figure 4) consisting of 270 and 584 nodes, and the average node degree is 5.04. Te TSV format fle acquired from the STRING website is then imported into the Cytoscape program for additional analysis and visualization.
Te key genes for MG anti-SCI are determined from the preceding PPI network using the CytoNCA App plugin of the Cytoscape software. Te sorting result of the top 10 hub genes (SRC, STAT3, MAPK1, HSP90AA1, PIK3R1, PIK3CA, RXRA, AKT1, CREBBP, and JAK2) is completely according to the algorithm of degree (Table 3).

GO and KEGG Pathway Enrichment
Analysis. GO and KEGG pathway enrichment analysis plays a signifcant role in network pharmacology research. Trough the DOSE, enrichplot, path view, KEGGgraph, Rgraphviz, Cluster-Profler, org.Hs.eg.db, and ggplot2 packages in the R Studio software, we operated GO enrichment analysis on the 293 obtained targets of MG-anti-SCI. As a result, we received a total of 2781 GO items, including 2426 BP, 98 CC, and 255 MF. Te top ten enrichment results of BP, CC, and MF are displayed using a bar chart or a bubble chart ( Figure 5).
Te results show that kaempferol has good docking results with AKT1, RXRA, HSP90AA1, CREBBP, and JAK2. For instance, the scoring function LibDockScore of LibDock molecular docking is based on the ligand and receptor's afnity, relative energy, and docking mode. It is commonly assumed that a LibDockScore greater than 100 implies a stronger binding, and the higher the score, the more stable the shape of the complex formed via docking. Te greater the afnity, the higher the ligand's binding strength to the protein (Table 4, Figure 7).

Evidence-Based Complementary and Alternative Medicine 5
Based on the analysis of kaempferol and hub target data through molecular docking software, the diagram in Figure 7 is obtained.

Discussion
According to the report, the random aggregated yearly incidence of traumatic SCI in the Middle East and North Africa (MENA) Region was 23.24 cases per million individuals [45].
Individuals, families, and society bear huge costs due to these SCI patients. At the moment, signifcant progress has been made in the mechanism and clinical care plan of post-SCI, which includes decompression surgery, drugs, and rehabilitation. Te prognosis for SCI patients has improved as   [46]. Researchers are looking into pharmaceuticals, surgeries, medical bioengineering, and rehabilitation procedures to treat patients efectively. As is well known, TCM has been used in Asian countries to treat various diseases since ancient times [47]. MG is a traditional medicine used by Chinese people to treat respiratory tract infections. According to research, MG has anti-infammatory properties and can lower blood lipids and scavenge oxygen free radicals [48,49]. Kaempferol is one of the important active components of MG, which has been proven to be able to treat SCI in animal experiments [23]. However, considering its potential therapeutic value for SCI and its wide range of applications in daily life, the precise efect and specifc mechanism of MG in treating SCI in humans are unclear. Terefore, we used the technology of network pharmacology to deeply study the mechanism of MG working on SCI and lay the groundwork for further research. Initially, compounds and targets derived from MG were obtained by investigating TCMSP and BATMAN-TCM. OB ≥ 30 and DL ≥ 0.18 were the ADME principles. Ten, relevant target proteins were acquired from the Swis-sTargetPrediction databases (probability > 0), and their UniProt IDs were validated on the UniProt platform.
(2) Screening out related compounds according to ADME standards (TCMSP: 11, BATMAN-TCM: 0). (3) Te Swis-sTargetPrediction database is used to retrieve the target of the active component. Te likelihood that is higher than 0 is the selected standard. (4) Te fnal target is obtained by deleting duplicates (total number of targets: 346). (5) Checking for and recording the UniProt ID number of the obtained compound's target protein. Finally, 346 MG-related targets and 11 MG-related active molecules were identifed.
In the second step, 7214 SCI-related targets were acquired by exploring 3 disease databases, including CTD (number: 3234), GeneCards (number: 5716), and DrugBank (number: 15). Te website's online tool then constructs a Venn diagram of MG and SCI-related targets and extracts 293 targets from MG that may be benefcial in treating SCI.
Finally, the top ten hub genes (SRC, STAT3, MAPK1, HSP90AA1, PIK3R1, PIK3CA, RXRA, AKT1, CREBBP, and JAK2) were determined to have a higher therapeutic value for MG against SCI and will be examined subsequently.
AKT1 phosphorylates and activates the downstream pathway during SCI. Te PI3K/Akt signaling pathway can help patients recover from acute SCI [50]. Our fndings imply that AKT1 plays an important role in SCI healing. Tese fndings are consistent with the results of earlier investigations [51].
To lower oxidative stress and infammatory response, kaempferol can downregulate ROS-dependent MAPKs, NF-κB, and the pyroptosis signaling pathway, which shows that the active compound candidate kaempferol ofers potential for the treatment of SCI [23]. Te molecular docking analysis results revealed.
Molecular docking methods are generally considered useful techniques for screening drug candidates [52]. Molecular docking techniques can predict afnity and binding feasibility between candidate compounds and target proteins. Te score of molecular docking is determined by the interplay of Van der Waals forces, Coulomb interactions,    Te top 10 KEGG pathway enrichment analysis results are provided together with 293 MG targets against SCI. Te enriched pathways were categorized, and 10 highly associated pathways were selected as follows: lipid and atherosclerosis (hsa05417), EGFR tyrosine kinase inhibitor resistance (hsa01521), neuroactive ligand-receptor interaction(hsa04080), prolactin signaling pathway (hsa04917), apoptosis (hsa04210), AGE-RAGE signaling pathway in diabetic complications (hsa04933), insulin resistance (hsa04931), prostate cancer (hsa05215), chemical carcinogenesis-receptor activation (hsa05207), and endocrine resistance (hsa01522).
Based on our preliminary fndings from the above network pharmacology study, we believe that the primary active components of MG may play a role in the treatment of SCI via the pathways mentioned above and hub genes. Taken together, we anticipate that an increasing number of researchers will commit themselves to clarify the mechanism of TCM's action through network pharmacology, thereby increasing global awareness of this Chinese treasure. In the future, based on the above research results, we will explore the efects of MAPK1, RXRA, and STAT3 on SCI through more in vivo and in vitro experiments.

Data Availability
Te data used to support the fndings of this study are included within the article.

Conflicts of Interest
Te authors declare no conficts of interest.