The possible mechanism of Hippophae fructus oil applied in tympanic membrane repair identified based on network pharmacology and molecular docking

Abstract Objective This study aimed to explore the mechanisms of Hippophae fructus oil (HFO) in the treatment of tympanic membrane (TM) perforation through network pharmacology‐based identification. Methods The compounds and related targets of HFO were extracted from the TCMSP database, and disease information was obtained from the OMIM, GeneCards, PharmGkb, TTD, and DrugBank databases. A Venn diagram was generated to show the common targets of HFO and TM, and GO and KEGG analyses were performed to explore the potential biological processes and signaling pathways. The PPI network and core gene subnetwork were constructed using the STRING database and Cytoscape software. A molecular docking analysis was also conducted to simulate the combination of compounds and gene proteins. Results A total of 33 compounds and their related targets were obtained from the TCMSP database. After screening the 393 TM‐related targets, 21 compounds and 22 gene proteins were selected to establish the network diagram. GO and KEGG enrichment analyses revealed that HFO may promote TM healing by influencing cellular oxidative stress and related signaling pathways. A critical subnetwork was obtained by analyzing the PPI network with nine core genes: CASP3, MMP2, IL1B, TP53, EGFR, CXCL8, ESR1, PTGS2, and IL6. In addition, a molecular docking analysis revealed that quercetin strongly binds the core proteins. Conclusion According to the analysis, HFO can be utilized to repair perforations by influencing cellular oxidative stress. Quercetin is one of the active compounds that potentially plays an important role in TM regeneration by influencing 17 gene proteins.


| INTRODUC TI ON
Tympanic membrane (TM) perforation is a common finding in otology clinics that causes hearing loss and infections and consequently decreases the quality of human life. 1,2 Persistent TM perforation with infection, if left untreated, usually requires surgical interventions. 2,3 Patients who undergo tympanoplasty may suffer from pain and potential postoperative complications and might incur high medical costs. 3,4 For simple perforations without other middle ear disorders (e.g., cholesteatoma), an alternative to rupture closure that is less invasive and expensive is preferable to surgical intervention. [5][6][7] Hippophae fructus oil (HFO), a traditional Chinese herb extracted from Hippophae fructus, [8][9][10] is composed of vitamin C, amino acids, and essential trace elements (e.g., zinc), which can enhance the proliferation of TM epithelial stem cells, promote their migration to lesions and induce blood vessel dilatation. 9,10 During the past few decades, HFO has been applied for the clinical treatment of TM perforation with satisfactory outcomes. 8 However, the biological mechanism of HFO therapy for TM regeneration remains unclear, and the main compounds of HFO that promote TM healing require further exploration.
The development of biological informatics engineering has made it possible to explore the mechanisms of Chinese herbs used for disease treatment. Network pharmacology combines pharmacokinetic and pharmacodynamic properties with the field of systems biology to study drugs, protein targets, and their pharmacological activities. 11,12 Hence, we explored the underlying mechanism of HFO in TM repair using the technologies of network pharmacology and molecular docking.

| Identification of active compounds of HFO and corresponding target genes
Detailed information on the compounds, targets and other secondary data related to HFO was obtained from the Traditional Chinese Medicine System Pharmacology Database (TCMSP, http://tcmspw. com/tcmsp.php) with the keywords Hippophae Fructus. 13 Specifically, oral bioavailability (OB) and drug likeness (DL), which are the most important indicators for evaluating the characteristics of absorption, distribution, metabolism, and excretion (ADME), were used to filter the candidate active compounds based on the thresholds OB ≥ 30% and DL ≥ 0.18. 14 For each active compound, we searched related target genes in the TCMSP database. An HFO gene set was then mapped using the UniProt database by limiting the species to "Homo sapiens" (https://www.unipr ot.org/) to obtain gene symbol annotation. 15

| Compound-target pharmacology network and functional analysis
After preparing two sets of target lists for the HFO-related genes and disease targets, a screening for drug-disease crossover was performed. The common targets were selected, and a Venn diagram was generated using the Venn Diagram package of R software to visualize the intersection sets. The compound-target-disease network diagram was prepared using Cytoscape version 3.8.0 software to show the relationships among the TM, HFO, and gene targets. 21 A functional enrichment analysis, including gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, was performed to elucidate the underlying mechanism through assessments of biological processes (BPs), cellular components (CCs), molecular functions (MFs), and key signaling pathways. The intersecting proteins were then evaluated by bioinformatics annotation using the "clusterProfiler" and "bioconductor" packages of R software. The significantly enriched GO and KEGG terms were identified, and p-values < 0.05 and q-values < 0.05 indicated a strong association with related BPs. 22,23

| Protein-protein interaction network and critical subnetwork
For identification of the potential targets of HFO and the interactions between them, the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) (http://strin g-db.org/) database 24 was used to analyze the intersecting protein-protein interactions (PPIs) through the intersection set between HFO targets and TM-related genes. After importing the PPI network into Cytoscape software, a critical subnetwork was investigated using the CytoNCA plugin. 25 The values of betweenness, closeness, degree, eigenvector, and local average connectivity-based method, as well as the network scores, were independently calculated, and the eligible genes with scores higher than the median value were selected to establish a critical subnetwork. 25

| Molecule docking technology
The compound with the largest number of critical genes related to TM was selected for further analysis based on molecular docking.
The 2D structure of the molecular ligands was downloaded from the PubChem database (https://pubch em.ncbi.nlm.nih.gov/), 26 and the 3D structure with minimum energy was calculated and exported using ChemBio3D software. 27 Furthermore, the 3D structure of the receptor protein encoded by the selected gene was obtained from the UniProt database and downloaded from the RCSB PDB database (https://www.rcsb.org/). 28 After preparing the files of the 3D structure, the receptor proteins were dehydrated, and the ligands were removed using PyMOL software. 29 AutoDock Tools was used to modify the receptor protein by calculating the hydrogenation and charges of proteins. Moreover, the parameters of the receptor protein docking site were set to include the active pocket sites where small molecule ligands bind, and the molecular docking between the compound and receptor proteins was displayed using AutoDock

| Active compounds and potential targets
Using the criteria DL ≥ 0.18 and OB ≥ 30%, 33 main and effective compounds were retrieved and selected (Table 1). A total of 182 compound-related targets were annotated into a gene symbol set using the UniProt database. In addition, 393 TM-related targets were extracted from the OMIM, GeneCards, PharmGkb, TTD, and DrugBank databases after removing duplicates. Moreover, an intersection of the compound-target and disease-related genes, which contained 22 target proteins, was obtained and is shown in Figure 1. As shown in Figure 2, PTGS2 and PGR were identified as two genes that were most frequently targeted by the HFO ingredients, and both of these genes were targeted by 13 active compounds.

| Network analysis of targets
Furthermore, quercetin may be one of the primary therapeutic compounds affecting 17 target proteins on the TM and may play a critical role in the healing process.

| GO and KEGG enrichment analysis
The GO enrichment analysis revealed the underlying BPs, CCs, and Using the filters p-value < 0.05 and q-value < 0.05, a KEGG enrichment analysis was performed to elucidate the related pathways.
The results revealed that 103 potential signaling pathways were enriched, and the top 30 of these pathways are shown in Figure 3. The bubble plot demonstrates that these gene targets affected pathways related to infection and wound healing, such as the IL-17 signaling pathway.

| PPI diagram and core subnetwork
Twenty-two overlapping genes associated with TM and HFO were inputted into the STRING database, and a PPI network diagram was established after selecting "Homo sapiens." As shown in Figure 4,

| Molecular docking
Referring to the results of the core gene network, we identified quercetin as a ligand of CASP3, MMP2, IL1B, TP53, EGFR, CXCL8, PTGS2, and IL6 protein receptors. A further calculation was then conducted to simulate the molecular docking of quercetin with these eight gene proteins, and the docking affinity values are listed in Table 3. A greater absolute value for the docking affinity indicates stronger binding ability between the active site of the protein receptor and the compound. The docking results indicate that quercetin can easily enter and bind the active pocket of the eight core target proteins, can form hydrogen bonds with the amino acid residues, and exhibits high binding affinity ( Figure 6).

| DISCUSS ION
Tympanic membrane perforation can lead to severe morbidity and disability. has not yet been identified. 2,7 In traditional Chinese medicine, doctors use Chinese herbs to repair TM perforations, and these herbs have been reported to accelerate the wound healing process. [31][32][33] Previous studies have suggested that HFO may be an effective, safe and convenient herb for promoting the healing of TM perforations. 8 However, the underlying mechanism of HFO in TM repair remains unknown. Thus, in this study, we performed a network pharmacology-based analysis to explore the mechanism of HFO in TM repair.
The results from the GO enrichment analysis revealed that the mechanism through which HFO promotes TM regeneration may be associated with cellular oxidative stress. During the process of tissue wound healing, the biological regulation of cellular oxidative stress generally plays a critical role in cell proliferation and migration. 34,35 The signaling pathways identified from the KEGG enrichment analysis also suggested that these gene proteins may contribute to this biological process. Although the healing process of the F I G U R E 5 Core proteins subnetwork diagram  [36][37][38][39] This evidence may support our finding that HFO may promote TM healing by regulating biological processes related to cellular oxidative stress.
The PPI network diagram reflected the protein-protein relation- In addition to the EGFR protein, other gene proteins also play important roles in the TM healing process. Among these core proteins, IL1B and IL6 participate in the process of histological damage and inflammation, as demonstrated with acute animal models. 43,44 The activity of MMP2 is also hypothesized to be involved in destruction of the TM fibrous layer and the prognosis of otitis media. 45,46 According to our analysis, 21  HFO displays a variety of advantages in addition to healing benefits and is less invasive. [8][9][10]31,32 As an extract from berries and seeds of traditional medicinal plants, HFO is extensively available from many sources. In addition, HFO is easily prepared and does not require cold storage, which may significantly reduce the associated costs. In addition, this treatment has the ability to prevent ototoxicity and infection, which is crucial for TM healing and auditory reconstruction. 55 Although HFO shows great healing potential for TM perforations, the number of studies on the clinical application of HFO is not as high as that of studies that investigated other bioactive materials, which may be due to its complex composition and unknown mechanism related to the TM. 9,10 Therefore, further studies, if possible, should not only concentrate on exploring the underlying mechanism but also focus on a more detailed assessment of its clinical effectiveness. In addition to the healing and hearing results of HFO applied to acute or chronic perforations, the most effective dosage, duration and frequency of the application, and the patching materials have not yet been fully investigated. 1,4 In addition to exploring the mechanism and efficacy of HFO in further studies, investigations should assess whether the ingredients of this mixture can also be applied for TM regeneration based on our analysis. Quercetin, for instance, has been utilized as a medicine-loaded vehicle for topical delivery in wound healing according to previous studies. [47][48][49][50] The ability to quench reactive oxygen species and mitigate the inflammatory process is essential in wound healing after topical application. 49,50 Considering these advantages, the utilization of quercetin has led to the novel notion of using herb monomers combined with other bioactive materials and has resulted in the identification of promising topical prodrugs.
In this study, we employed network pharmacology to elucidate the multitarget effects of HFO on TM perforations. Additionally, we discussed several potential herb monomers for TM perforation treatment, which could contribute to the development of new therapeutic strategies. However, the possible effects of HFO revealed from the network analysis require further verification by in vitro biological experiments. This analysis provides theoretical evidence regarding the mechanism of HFO in TM regeneration that can be further explored in future studies.

| CON CLUS ION
According to the analysis, HFO can be utilized to effectively repair TM perforations by influencing the regulation of cellular oxidative stress. Quercetin is one of the active compounds of HFP that potentially plays an important role in TM regeneration by influencing 17 gene proteins. This pharmacology-based approach helps elucidate the underlying mechanisms of HFO in repairing TM perforations, but further experimental exploration is needed for verification of these mechanisms.

ACK N OWLED G M ENTS
This study was supported by the National Natural Science Foundation

CO N FLI C T O F I NTE R E S T
None.

AUTH O R CO NTR I B UTI O N S
All persons designated as the authors have participated sufficiently in the work to take public responsibility for the content of the manuscript. All the authors ensured that they all gave substantial contributions.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are openly available from TCMSP, GeneCardS, OMIM, TTD, PharmGkb, and DrugBank belong to public databases.