Pharmacology Mechanism of Polygonum Bistorta in Treating Ulcerative Colitis Based on Network Pharmacology

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Background
Ulcerative colitis (UC) is a chronic infammatory disease of the colon [1] and is characterized by abdominal pain, bloody diarrhea, the passage of mucus, and tenesmus [2]. Te extraintestinal organs, including the eyes, mouth, skin, joints, and liver, can be involved in the progression of UC [3][4][5][6]. Patients with UC have impaired colorectal function, poor quality of life [7], an increased risk of colon cancer [8], and a higher economic burden [9]. UC is more popular in western countries than in other countries and areas of the world [10][11][12]. However, the incidence of UC has increased gradually in Asia, the Middle East, and South America in the last two decades [13][14][15][16]. 5-aminosalicylic acid preparations, corticosteroids, immunosuppressants, biological agents, and small-molecule medications [2,10] are the main medications used to treat UC. However, due to the side efects, primary failure, or secondary failure of the medications, a proportion of UC patients lack efective treatment [17,18]. Terefore, it is necessary to develop new medications for UC. Tere is increasing evidence to support that Chinese herbal medicine can be efective in UC treatment [19][20][21][22][23]. Terefore, Chinese herbal medicines may be a potential source for developing new medications for UC. Polygonum bistorta (PB) is a member of the genus Polygonaceae [24]. In ancient China, it was used to treat dysentery, diarrhea, and hematochezia, which were similar to the symptoms of UC. Te pharmacological studies have shown that PB can inhibit infammation by reducing the release of proinfammatory cytokines [24] such as interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), which are involved in the infammation of UC [25][26][27]. Quercetin (Que), one of the main active compositions of PB, can inhibit infammation by reducing the release of reactive oxygen species and myeloperoxidase from human neutrophils [28]. Terefore, it is speculated that PB may be efective on UC, and direct evidence from the experiments and the clinical trials is needed.
Andrew L. Hopkins, a pharmacologist at Dundee University, proposed the concept of network pharmacology in 2007. Network pharmacology elucidates the occurrence and the development of diseases from the perspectives of systems biology and biological network balance, understands the interactions between the medications and the body from the holistic perspective of improving or restoring biological network balance, and guides drug discovery. It can efectively screen the bioactive compositions and the potential targets and has an advantage in analyzing the mechanisms of Chinese herbal medicine with multicompositions and multitargets [29]. Terefore, network pharmacology is suitable for developing medications for multifactor and multigenemediated diseases. Although the pathogenesis of UC has not been clearly elucidated, UC is considered to be caused by the interaction of multiple factors, including genetic susceptibility, immune abnormalities, enteric fora disturbances, and environmental factors [30][31][32].
In our study, the potential targets and the signaling pathways of PB in treating UC were searched and identifed with network pharmacology, molecular docking, and in vitro experiments in order to provide the foundation for conducting animal experiments on treating UC with PB. Te workfow of this study is summarized in Figure 1. [33] or a composition from CNKI or PubMed with an OB degree of HIGH and at least two terms of DL being YES is defned as having pharmacokinetic activity. Te chemical structures of the bioactive compositions were obtained from TCMSP and PubChem (https://pubchem.ncbi.nlm.nih.gov/) [34], then imported into Swiss Target Prediction (https://www. swisstargetprediction.ch/) [35] to obtain the potential target proteins. Te gene names of the target proteins were obtained from the reviewed human genes in the UniProt Knowledgebase (UniprotKB, https://www.uniprot.org/ ) [36].

Identifying the Overlapping Targets of PB and UC.
Te overlapping targets of PB and UC were identifed by importing the targets of PB and UC into the Bioinformatics website (https://www.bioinformatics.com.cn/).

Constructing Protein-Protein Interaction (PPI)
Network. Te overlapping targets of PB and UC were analyzed by the Search Tool for the Retrieval of Interacting Genes (STRING, https://www.string-db.org/) [42] to construct a PPI network. Te species were set as "Homo sapiens." Cytoscape [43] software was used for visualization. Te target whose degree was more than 2 times the median was defned as the core target.

GO and KEGG Pathway Enrichment
Analysis. Te metascape system (https://metascape.org/) [44] was utilized for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Te P value was set to less than 0.01.

2.1.6.
Constructing PB-Composition-Target-Pathway Network. Te bioactive compositions of PB and the corresponding targets and signaling pathways were imported into the Bioinformatics website, and the PB-compositiontarget pathway network was formed automatically.

Molecular Docking.
Te chemical structures of bioactive compositions were processed with ChemOfce software for energy minimization. Te protein structures of the core targets were obtained from the RCSB protein database (RCSB PDB, https://www.pdb.org/) and optimized by PyMOL software for ligand extraction, water extraction, and hydrogenation. Te molecule docking of bioactive compositions and target proteins was performed by AutoDock Vina 1.1.2 software [45]. Te results were visualized by PyMOL software.

Cells.
Caco-2 cells were provided by the Shanghai Institutes for Biological Sciences and cultured in DMEM containing 20% FBS and 1% P-S.

Defning the Safe Intervening Concentration of Que for
Caco-2 Cells. Caco-2 cells were divided into 7 groups and cultured in the medium free of Que and the medium containing 5, 10, 20, 40, 80, and 160 μmol/l Que for 24 h, respectively. Te cell proliferation rate of each group was measured by the CCK8 assay. Te safe intervening concentration of Que was defned by the results of the cell proliferation rates.

Defning the Optimal Intervening Concentration of Que
for Caco-2 Cells. Caco-2 cells were divided into the control group, the model group, and 5 Que intervention groups with diferent concentrations. Except for the control group, Caco-2 cells were stimulated with 10% DSS [46] for 24 h to promote the expression of Act1 in the interleukin-17 (IL-17) signaling pathway. Ten, the cells were treated with 5, 10, 20, 40, and 80 μmol/l Que for 24 h, respectively, and the expression of Act1 in each group was detected by immunofuorescence staining (IF) to defne the optimal intervening concentration.

Observing the Efect of Que on the Expressions of TRAF6, FOS, and JUN.
Caco-2 cells were divided into the control group, the model group, and the Que intervention group. Te model group and Que intervention group were stimulated with DSS for 24 h. Afterwards, the intervention group was treated with the optimal intervening concentration of Que for 24 h. Te expressions of TRAF6, FOS, and JUN downstream of Act1 were detected by IF.

Compositions and Target Proteins of PB.
Six bioactive compositions and 139 corresponding target proteins are obtained and listed in Table 1 and Table S1, respectively. Te other compositions without bioactivity are listed in Table S2.

UC Associated Target
Genes. 3,235 UC-associated target genes were collected by GeneCards. Te higher the relevance score, the more closely the target gene is associated with UC. We screened the target genes according to the criteria that the relevance score was no less than 2 times the median score, and 804 target genes were obtained. Te numbers of the target genes obtained from the other websites were 118 (OMIM), 72 (Drugbank), 50 (TTD), and 15 (PharmGkb), respectively. Finally, 934 targets were obtained after removing the repetition (Figure 2, Table S3).  Evidence-Based Complementary and Alternative Medicine

Overlapping Targets of PB and UC.
Tere were 93 overlapping targets between PB and UC ( Figure 3, Table S4) for further analysis.

PPI Network of the Overlapping Targets of PB and UC.
Te PPI network was constructed by importing the overlapping targets of PB and UC into the STRING website with species set as "Homo sapiens." Te visualization was performed by Cytoscape software. In the PPI network, the core and the extending targets were represented by the nodes, and the connection between the genes was represented by the edges. Tere were 93 nodes (Figures 3 and 4; Table S4) and 1550 edges (Figure 4) in this network. Tere were 42 targets whose degree was higher than the median (Figures 3 and 5). Among them, there were 18 core targets whose degree was more than 2 times the median ( Figures 5-7, Table 2). Tere were 153 edges in the network of the core targets ( Figure 4).

GO and KEGG Pathway Enrichment Analysis.
Te biological functions and pathways enriched through GO and KEGG analysis of the core targets could be the potential intervening targets of the newly developing medication. GO analysis includes a biological process (BP), a cell component (CC), and a molecular function (MF). 467 BPrelated functions suggested that these core targets were associated with the infammatory response, cytokine production, and the response of cells to cytokine stimulation (P < 0.01, Figure 8). 10 CC related functions revealed that the targets could respond to the transcription regular complex, transcription repressor complex, nuclear envelope, membrane microdomain, and so on (P < 0.01, Figure 8). 30 MFrelated functions showed that the targets might have the following functions: cytokine activity, cytokine receptor binding, transcription factor binding, and ubiquitin protein ligase binding (P < 0.01, Figure 8). 102 pathways were enriched through KEGG analysis, such as the IL-17 signaling pathway, the colorectal cancer pathway, the TNF signaling pathway, and the nuclear factor kappa-B (NF-κB) signaling pathway (P < 0.01, Figure 9). Tese pathways were provided with an enrichment score and P value. Te higher the enrichment score and the lower P value of the pathway are, the Evidence-Based Complementary and Alternative Medicine higher its importance is. Te enrichment score of the IL-17 signaling pathway was higher, but its P value was lower than those of many other pathways. It suggested that the IL-17 signaling pathway might play an important role in the pharmacology mechanism of PB. Ten, we searched the IL-17 signaling pathway diagram through KEGG (Figure 10), and the interaction between several proteins of the IL-17 signaling pathway was identifed by the STRING website ( Figure 11). Te following targets were the overlapping parts of the core targets and IL-17 pathways: FOS and JUN (the main members of activator protein 1(AP-1)) [47,48], IL-1β, MMP9, CXCL8 and CCL2.

PB-Composition-Target-Pathway Network. Te compositions and targets obtained by TCMSP and Swiss
Target Prediction and the targets and pathways enriched through KEGG were integrated (Table S5). All the information was imported into the Bioinformatics website to build the PB-component-target-pathway network ( Figure 12). Among the bioactive compositions of PB, quercetin (Que) played the most important role in intervening at the overlapping targets of PB and UC. It could intervene in the IL-17 signaling pathway through core targets such as FOS, JUN, IL-1β, MMP9, CXCL8, and CCL2.   (Table 3). Molecular docking sites, hydrogen bond lengths, and amino acid residues of PB compositions and proteins are shown in Figure 14. Figures 11 and 12, IL-17A, B, C, E, and F can bind to Act1/TRAF3IP2 and TRAF6 through diferent IL-17 receptors (IL-17RA, B, C, and E) and further regulate AP-1(Fos/Jun) to promote the release of proinfammatory cytokines and chemokines. DSS can induce colitis in mice [51][52][53] and rats [54][55][56]. Te expressions of Act1/TRAF6/FOS/JUN of the IL-17 signaling pathway in Caco-2 cells were increased after being stimulated with DSS in our experiment. Que is the main active composition of PB according to the results of network pharmacology and molecular docking. It may act on the proteins in the IL-17 signaling pathway. Terefore, we further observed the efect of Que on the IL-17 signaling pathway.

Experiment Verifcation. As shown in
Te CCK8 assay showed that the maximum noncytotoxic concentration of Que was 80 μmol/l ( Figure 15). Act1 expression was used as the efect indicator to determine the optimal intervention concentration of Que in the IL-17 signaling pathway. 10 μmol/l Que could maximally reduce Act1 expression in Caco-2 cells stimulated by DSS (Figures 16(a) and 16(e)). Terefore, 10 μmol/l was used as the optimal concentration of Que to intervene in the expressions of the downstream proteins (TRAF6, FOS, and JUN) in the IL-17 signaling pathway. 10 μmol/l Que could also efectively reduce the expressions of TRAF6 (Figures 16(b) and 16(f )), FOS (Figures 16(c) and 16(g)), and JUN (Figures 16(d) and 16(h)) in Caco-2 cells stimulated by DSS.
Many studies have confrmed that the IL-17 signaling pathway is involved in the pathogenesis of UC [82][83][84]. Nanki et al. found that the IL-17 signaling pathway was   [83]. Te results showed that the serum levels of IL-17 in UC patients were signifcantly higher than those in healthy controls. Besides, the levels of IL-17 in active UC patients were higher than those of patients in remission and were correlated with CRP levels. Te mRNA expression of IL-17R in the colonic mucosa of patients with active UC was also higher than that in healthy controls. Zhou et al. confrmed that IL-17A, IL-17RA, Act1, p-ERK, p-JNK, p-p38, and other proteins in the IL-17 signaling pathway were involved in the pathogenesis of UC in HT29 cells and DSS-induced colitis mice [84]. Te expressions of these proteins in the model group were higher than those in the control group. After TCM intervention, the infammation of the colon mucosa was alleviated, and the levels of the above signaling proteins were also decreased. Tese studies suggested that the IL-17 signaling pathway might be involved in the pathogenesis of UC and be related to disease activity. Terefore, it may be a potential target for treating UC.
In our study, GO and KEGG enrichment analysis showed that intervention of the cytokines and regulation of the infammatory response might be the mechanism of PB in the treatment of UC. Among the infammation-related pathways enriched by KEGG, the importance of the IL-17 signaling pathway was high. Te core targets FOS, JUN, IL-1β, MMP9, CXCL8, and CCL2 were all involved in the IL-17       [49,50,85]. Te hydrophobic interaction and hydrogen bond interaction between compounds and amino acids of target proteins can promote stable binding [49,50,85]. In addition, the active sites assist the compounds to form sufcient contacting points and stably bind to target proteins by maintaining optimal catalytic microenvironments [85]. It was verifed in vitro experiments that the expressions of Act1, TRAF6, and AP-1(Fos/Jun) in the IL-17 signaling pathway increased in the cells stimulated by DSS and decreased in Que-treated cells. Terefore, the IL-17 signaling pathway could enhance the infammatory response and aggravate UC. PB and its main active composition Que could treat UC by inhibiting the IL-17 signaling pathway. Te possible mechanism of Que treating UC is shown in Figure 17.

Conclusion
PB has been used to treat diarrhea and hematochezia effectively for hundreds of years in ancient China. It might be a potential source for developing new medications for UC.

Data Availability
Te datasets generated or analyzed during the current study are available from the corresponding author upon reasonable request.

Conflicts of Interest
Tese authors declare that there are no conficts of interest. Evidence-Based Complementary and Alternative Medicine 15