Uncovering the Multitarget Therapeutic Mechanism of Tong-Xie-Yao-Fang on Irritable Bowel Syndrome

Background . Tis study investigated the pharmacological mechanisms of Tong-Xie-Yao-Fang (TXYF) against irritable bowel syndrome (IBS). Methods . Te chemical profle of TXYF was identifed through UHPLC-QTOF-MS. Next, the obtained chemical profle served as the basis for network pharmacological analysis. Finally, the predictive performance of network pharmacology was validated by conducting molecular docking and animal experiment. Results . Seven key compounds of TXYF, namely, quercetin, ellagic acid, nobiletin, formononetin, isorhamnetin, vestitol, and licochalcone, were confrmed as the key components acting on IBS. TXYF treatment on IBS was mainly realized through the regulation of some key pathways of immune system, such as infammatory bowel disease, cytokine-cytokine receptor interaction, HIF-1, and Tcell receptor signaling pathway. NOS2, ACHE, ESR1, PTGS2, and RELA were the target genes of TXYF to improve IBS. Stable bonds between the key components and the core target genes were further verifed by the results of molecular docking. In vivo experiments confrmed the efects of TXYF on IBS. Further Western blot analysis showed that NOS2, ACHE, and ER α were signifcantly upregulated in the model group in comparison with controls ( P < 0 . 001) but then signifcantly downregulated after treatment with TXYF for 14 days ( P < 0 . 001). Conclusion . Tis study applied an integrated method based on network pharmacology and experimental validation to examine the underlying “multicomponent, multitarget, and multipathway” mode of action of TXYF in treating IBS. Te current fndings provided indicative paradigms and new insights into exploring the multitarget therapeutic mechanism of Chinese herbal compound.


Introduction
Irritable bowel syndrome (IBS) refers to a functional gastrointestinal disorder that is characterized by recurrent episodes of abdominal pain with altered bowel habits [1].IBS is classifed into three types, specifcally, IBS-D (diarrhea-predominant), IBS-C (constipation-predominant), and mixed or alternating IBS (IBS-M).Rome IV criteria showed that the prevalence of IBS ranges from 5% to 10% worldwide, with a gradually increasing trend [2].IBS often causes gastrointestinal discomfort, dietary restrictions, mood disorders, and disturbances in patients' daily activities, leading to a heavy medical and fnancial burden [3].In treating IBS, physicians normally focus on symptomatic drugs, the efectiveness which is far from patient's expectations [4].Moreover, the multifactorial nature of the physiopathology of IBS could limit the development of more efective drugs [4,5].As a result, IBS patients begin to seek complementary and alternative therapies to relieve their discomfort [6].
Chinese herbal medicine (CHM) is gaining recognition around the world owing to increasingly reported health benefts [7].Tong-Xie-Yao-Fang (TXYF) is one of the most famous CHM formulas employed to treat functional gastrointestinal disorders and has demonstrated its efectiveness in IBS treatment [8].TXYF consists of 4 herbs of Baishao (Paeoniae Radix Alba), Baizhu (Atractylodes Macrocephala Koidz), Chenpi (Citrus Reticulata), and Fangfeng (Saposhnikoviae Radix), which have the advantages of multicomponents and multitargeting.However, the complexity of CHM compounds also poses great challenges to pharmacological research, as their material basis and mechanism of action are currently unclear.
Network pharmacology, which is a cutting-edge interdisciplinary discipline in the systematic study of drugs, could mechanistically link drugs and diseases as well as quantitatively represent the key aspects of overall regulatory mechanism of drug action [9].Terefore, network pharmacology has been widely used to elucidate the pharmacological mechanisms of CHM formula [10,11].In this study, network pharmacology in combination with experimental validation was utilized to investigate the pharmacological mechanism of TXYF against IBS.
Lyophilized powder (1.0 g) was extracted using 50 mL of methanol/water (1 :1, v/v) under ultrasonication for 0.5 h.Next, the extract was centrifuged at 13,000 rpm for 10 minutes (min) at 4 °C and the supernatant was fltered through a membrane flter (0.22 μm).Finally, 1.0 μL of the fltrate was subjected to UHPLC-QTOF-MS for further analysis.

Key Active Ingredient and Screening of Target Genes.
Te names of all compounds identifed by UHPLC-QTOF-MS/MS method were entered into the TCMSP (https://www.tcmsp-e.com/)for screening key active ingredients and their corresponding target genes.Drug-like ≥0.18 and oral bioavailability ≥30% were the screening criteria [12].We used UniProt (https://www.uniprot.org/)[13] to normalize the obtained genes for gene symbols.

PPI Network Construction.
Common target genes between drug and disease were defned as potential targets of TXYF against IBS and used for subsequent analysis.Potential targets were introduced into the STRING (https:// string-db.org/)[17] for PPI analysis.Te species were limited to "Homo sapiens" and the minimum interaction score required was set at high confdence (0.400).PPI networks were visualized using Cytoscape 3.7.2software [18].

GO and KEGG Enrichment Analysis.
To explore the biological functions related to potential targets, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were performed in the DAVID platform (https://david.abcc.Ncifcrf.gov/)[19].GO enrichment analysis was used to interpret and annotate genes from three dimensions of cellular components, molecular functions, and biological processes.KEGG database was mainly applied for pathway analysis.P < 0.05 was regarded as statistically signifcant.

Drug-Compound-Target Network Construction and
Screening of Core Genes.A drug-compound-target network was developed to examine the relationship between the ingredients of TXYF and targets of IBS.CytoHubba, the plugin of Cytoscape [20], was used to perform the screening of core genes and key components.Te degree values were calculated, and the target nodes with the top 12-degree values were taken to generate a new core network.

Molecular Docking.
In the present study, to validate the compound-target correlation, molecular docking was carried out in Discovery Studio 2019.Te compounds structures of TXYF and macromolecular protein target receptors related to IBS were downloaded from PubChem (https:// pubchem.ncbi.nlm.nih.gov/) and RCSB PDB (https://www.rcsb.org/),respectively.

Animals and Experimental
Protocol.Ten litters of newborn SPF SD rats (5 male ofspring per dam) were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co.All the experimental animals were housed in a barrier environment at the Laboratory Animal Center of Xiyuan Hospital, Chinese Academy of Traditional Chinese Medicine, at the temperature of 22-25 °C with a relative humidity of 50%-60% under controlled 12-h light/dark cycle.Adequate food and water were provided for the experimental animals.
Two litters of rats were randomly selected as the control group, while the rest of the rats were used to develop IBS-D model of liver depression and spleen defciency syndrome.
Firstly, the suckling rats were separated from the dams for 3 h a day (9 am to 12 am) from postnatal day 2 to day 14.On day 22, the suckling rats were weaned and caged separately from dams.On day 30, the body weight of all the rats 2 Journal of Food Quality was measured and randomly divided into model group, high-dose TXYF group (HL), medium-dose TXYF group (TM), and low-dose TXYF group (LH).When the rats were >6 weeks old and weighed >220 g, the enema procedure was performed using 4% acetic acid solution (1 ml) once a day for 30 seconds (s) for 2 weeks.Te rats in the control group underwent the same procedure using 1 mL of normal saline.Te rats were stimulated using the chronic unpredictable psychological stress method [20] starting from day 56, and another diferent stimulation method was selected for daily operation for 21 days.Te rats were given a gavage intervention on day 77.Te administered doses of TXYF were converted to TL (0.47 g/ml), TM (0.94 g/ml), and TH (1.88 g/ ml).Te rats were intragastrical administered at a dose of 1 mL/100 g once a day for a total of 14 days.Te other groups were given equal amounts of saline using the same method.
At the end of the experiment, after fasting for 12 h, the mice were sacrifced to harvest large intestine tissues, which were stored at −80 °C for subsequent studies.

Histological Analysis.
Te colonic tissues were fxed in 10% neutral-bufered formalin, and 4-μm-thick sections were stained with hematoxylin-eosin (HE) according to standard procedures.Morphological changes of colonic mucosa were observed under light microscope (Nikon, Japan).

Western Blot
Analysis.Tissue samples from three rats were randomly selected from each group, followed by homogenization of colonic tissue proteins with cold lysis bufer and PMSF protease inhibitor.Subsequently, the BCA protein concentration assay kit was performed to detect the protein concentration.Te proteins were fractionated using sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred to a nitrocellulose membrane.After blocking with 5% skimmed milk for 2 h at room temperature, the membranes were incubated with primary antibody (rabbit anti-NOS2 (1 : 1000 dilution, YT3169, 131KD, Immunoway), rabbit anti-ACHE (1 : 500 dilution, AF5274, 67KD, Afnity), rabbit anti-Erα (1 : 500 dilution, BS1113, 66KD, Bioworld), and mouse antiβ-action (1 : 5000 dilution, YM3028, 43KD, Immunoway)) at 4 °C overnight, followed by further incubation with secondary antibodies for 2 h at room temperature.Bound antibodies were visualized by an enhanced chemiluminescence (ECL) system.

Statistical Analysis.
Te data were presented as the mean ± standard deviation.Statistical analysis was carried out using GraphPad Prism 7 (GraphPad Software, San Diego, CA, USA) by one-way analysis of variance for comparison.A signifcant diference was considered when P < 0.05.

Active Ingredients and Target Genes.
In this study, a total of 231 compounds of TXYF were identifed using the UHPLC-QTOF-MS protocol.A total of 42 key active ingredients and 218 corresponding target targets were obtained after importing the identifed components into the TCMSP database for screening.Figure 1 presented the compound-target network of TXYF.

Target Genes Associated with IBS.
Using the available resources (GeneCards, CTD, and DisGeNET), we fltered 5273 target genes correlated with IBS.Further analysis identifed 70 common target genes between the six resources by a Venn diagram (Figure 2).

PPI Network and Core Gene Analysis.
Te Venn diagram revealed (Figure 3) 24 overlapping genes between the disease targets and the drug targets.Subsequently, the 24 targets were imported into the String database for PPI analysis, and we generated a network (Figure 4) containing 24 nodes and 312 edges.5 and 6 present the results of GO and KEGG enrichment analysis, respectively.In GO analysis, biological processes mainly enriched in positive regulation of gene expression, response to drug, and positive regulation of ERK1 and ERK2 cascade, negative regulation of apoptotic process.In KEGG analysis, infammatory bowel disease, cytokine-cytokine receptor interaction, HIF-1 signaling pathway, T cell receptor signaling pathway, and pathways in cancer were the signifcantly enriched pathways.

Construction of Drug-Compound-Target Network and
Identifcation of Core Targets.A drug-compound-target network (Figure 7) containing 34 compounds, 24 targets, 58 nodes, and 121 edges was developed.Using Cytoscape plugin cytoHubba, the core active compounds and core targets were identifed according to the top 12-degree values.Te core targets were NOS2, ACHE, ESR1, PTGS2, and RELA, and the top active compounds were quercetin, formononetin, ellagic acid, isorhamnetin, vestitol, licochalcone, and nobiletin (Figure 8).

Molecular Docking Analysis.
According to the results of molecular docking, all the LibDock scores were over "50" (Figure 9), suggesting that all the key active ingredients were well docked to the corresponding target genes.3D and 2D molecular docking model of the key active ingredients with core target genes are presented in Supplementary Figures 1  and 2.  10.As shown in Figure 11, the body weight, daily food intake, abdominal withdrawal refex, and fecal water content were determined to evaluate the efects of TXYF on IBS-D.Before medication, the body weight, daily food intake, and abdominal withdrawal refex were lower in the model group compared with the control group (P < 0.05).Interestingly, the three indexes were signifcantly increased in the rats treated with diferent doses of TXYF for 14 days compared to the model group (P < 0.05).Similarly, fecal water content was higher in the model group than in the control group (P < 0.05), but it was signifcantly decreased in rats treated with diferent doses of TXYF for 14 days (P < 0.05).
According to the results of HE (Figure 12), no loss of epithelial structures and normal glands was observed in the colonic tissues of the control rats, while a small amount of infammatory cell infltration, submucosal edema, and gap enlargement were seen in the mucosa of the model group.It is noteworthy that the degree and extent of damage to the colonic tissues of the rats were alleviated by the intervention of TXYF, especially in the rats that received a high dose of TXYF.Journal of Food Quality 3.7.2.Western Blot Analysis.Tree potential targets (NOS2, ACHE, and ESR1) predicted by network pharmacology were further validated.As shown in Figure 13, NOS2, ACHE, and ERα were signifcantly higher-expressed in the model group compared with the control group (P < 0.05).Interestingly, NOS2, ACHE, and ERα were signifcantly low-expressed in rats treated with diferent doses of TXYF for 14 days than the model group (P < 0.05).Tese results all indicated that TXYF could be benefcial to IBS-D through NOS2 and ACHE as well as ERα among other potential targets.

Discussion
TXYF has been widely used in the treatment of IBS and has shown great efcacy.However, complex characteristics of CHM compound pose great challenges to the study of its mechanism of action.In this study, to investigate the pharmacological mechanisms of TXYF against IBS, network pharmacology in combination with experimental validation was performed.Network pharmacology screened a total of 34 active ingredients of TXYF acting on 24 IBS-related targets.Specifcally, the key active compounds were quercetin, formononetin, ellagic acid, isorhamnetin, vestitol, licochalcone, and nobiletin, and the core targets were ESR1, NOS2, ACHE, PTGS2, and RELA.Animal experiments validated the key results of network pharmacology.
In the present study, a total of 231 compounds of TXYF were identifed using the UHPLC-QTOF-MS protocol and served as the basis for further network pharmacological analysis.Finally, key active compounds, namely, quercetin, ellagic acid, nobiletin, formononetin, isorhamnetin, vestitol, and licochalcone, were screened based on the predictions from network pharmacology.Oxidative stress is closely related to the pathological mechanism of IBS, and the mechanism lies in the excessive reactive oxygen species (ROS) production produced by oxidative stress, which in turn damages gastrointestinal epithelial cells [21].Quercetin is a subclass of favonoids with a variety of pharmacological activities such as anti-infammation, antioxidation,  6 Journal of Food Quality anticarcinogenesis, and antivirus [22].Te addition of quercetin to diets has been reported to be able to improve antioxidant capacity and alleviate oxidative damage to the intestinal mucosa [23].In vitro studies have also observed that quercetin can inhibit ROS production in gastrointestinal epithelial cells, thereby preventing damage resulted from oxidative stress [24].Additionally, quercetin could contribute to the antioxidant capacity of the body through promoting the protein abundance of Nrf2 and regulating GSH-related redox homeostasis in enterocytes   Journal of Food Quality [23].Study also found that quercetin can signifcantly downregulate the colonic expression of ngn3, TPH, and pdx1, increase pain threshold pressure, and reduce visceromotor responses in IBS animals [25].Ellagic acid is a strong antioxidant with various properties such as antioxidation, reduction of infammation, and apoptosis [26].Receiving ellagic acid improves sleep quality and gastrointestinal symptoms in IBS patients, which may be realized through antioxidation and moist properties of ellagic acid [27].Other key components have also been discovered to have antioxidant efects by earlier studies [28][29][30][31][32], but studies on their efects on IBS are highly limited.Te present network pharmacology revealed their importance and can provide direction for their further research in IBS.
PPI network was developed, and NOS2, ACHE, ESR1, PTGS2, and RELA were identifed as the core targets of TXYF against IBS.NOS2 is a reactive free radical functioning as a biological mediator in several biological processes, including neurotransmission and antibacterial and antitumor activities.Recent studies have detected a close relationship between NOS2 and infammation and visceral hypersensitivity [33].NOS2 overactivation can result in 8 Journal of Food Quality a variety of infammatory diseases, and hypersensitivity can be controlled by modulating NOS2-associated infammation [33].In this study, animal experiments revealed that the level of NOS2 in the colonic tissue of rats in the IBS model group was signifcantly elevated compared to the control group but was then signifcantly decreased after TXYF treatment for 14 days.ACHE hydrolyzes the neurotransmitter acetylcholine at the neuromuscular junction and brain cholinergic synapses, thereby terminating signal transmission.ACHE is present in a variety of molecular forms with similar catalytic properties, but difers in the way how its oligomers assemble and cells attach to the cell surface.Conditions associated with ACHE include colonic pseudo-obstruction.With deepening understanding of the brain-gut axis in the pathogenesis of IBS [34], more attention should be paid to the role of ACHE in IBS [35].Te present study detected a signifcant increase in ACHE level in the IBS model in comparison to the control group, but interestingly, it was decreased after implementing 14 days of TXYF treatment.A growing body of data suggested that sex hormones may play a crucial role in the pathogenesis of IBS [36].ESR1 is a protein-coding gene that encodes an estrogen receptor and ligand-activated transcription factor.Concurrent alterations in immunomodulatory cytokines and microRNAs have been observed in IBS patients, indicating that dysregulation of local immune responses may be realized in an estrogendependent manner [37].Te results of in vivo experiments confrmed that TXYF had certain efects on IBS, and Western blot analysis revealed that the ERα level was signifcantly higher in the colon tissue of the IBS model group than that in the control group, but it was decreased signifcantly after 14 days of TXYF treatment.PTGS2 is a PTGS that infuences the development of chronic infammatory diseases through producing infammatory mediator prostaglandin E-2 (PGE ( 2)) and is a potential therapeutic target for IBS [38,39].RELA, also known as NF-κB, is a ubiquitous transcription factor involved in several biological processes.Previous studies have confrmed that TXYF can efectively improve intestinal permeability and enhance intestinal mucosal barrier function in IBS via the NF-κB pathway [40].Furthermore, molecular docking analysis demonstrated that TXYF can efectively bind to specifc proteins in IBS target genes, further validating the predictive results of network pharmacology.However, the limitations of the current study should be equally acknowledged.IBS is classifed into IBS-D, IBS-C, and IBS-M, but we only used IBS-D rats to verify the results of network pharmacology.Hence, whether the experimental results from IBS-D were applicable to IBS-C and IBS-M should to be further verifed.

Conclusion
Te present study applied an integrated method based on network pharmacology and experimental validation to uncover the underlying "multicomponent, multitarget, and multipathway" mode of action of TXYF for the treatment of IBS.Tis study provided indicative paradigms and new insights into the exploration of the multitarget therapeutic mechanism of Chinese herbal compound.

Figure 1 :Figure 2 :Figure 3 :
Figure 1: Te compound-target network of TXYF.Violet represents ingredients of TXYF, and green represents targets of IBS.

Figure 4 :Figure 5 :
Figure 4: PPI network from STRING.Te diferent colored circles indicate diferent protein molecules, and the connecting lines represent interactions between diferent protein molecules.

Figure 6 :
Figure 6: Results of KEGG pathway analysis.Te larger the shape, the greater the number of genes enriched.

Figure 7 :
Figure 7: Drug-ingredient-target genes network of TXYF.Violet represents ingredients of TXYF and green represents targets of IBS.
il e ti n i s o r h a m n e t i n qu erc eti n f o r m o n o n e t i n e ll a g ic a c c h a lc o n e

Figure 9 :
Figure 9: Results of libdock score of molecular docking.