Exploring the Effects of Changping Decoction on Irritable Bowel Syndrome by Pathway and Pharmacology Network Bioinformatics Analysis

Background An increasing body of research has conrmed the effectiveness of Traditional Chinese Medicine (TCM) for the treatment of irritable bowel syndrome (IBS). Methods We explored the potential mechanism of Changping decoction (CPD) in the treatment of IBS through pathway analysis based on a network pharmacology approach. Public databases, including the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform, Gene Expression Omnibus, and STRING, were used to screen the active ingredients and targets of CPD. Enrichment analysis was performed using the R-3.6.0 software to expound the biological functions and related pathways of CPD targets. The Cytoscape software was used to construct a “disease-CPD-target” network and identify hub genes of CPD relevant for the treatment of IBS. Employing rat models, pathological observation and abdominal withdrawal reex tests were used to verify the effectiveness of CPD in the treatment of IBS. Immunohistochemistry was used to conrm the relationship between the CPD treatment and hub genes. Results Network pharmacological analysis of CPD for the treatment of IBS identied 159 active ingredients. A total of 118 key targets were identied, including MAPK8, VEGFA, PTGS2, and others. A series of signaling pathways, such as MAPK, Kaposi sarcoma-associated herpesvirus infection, and IL-17 signaling pathway were found to play an important role in the therapeutic mechanism of CPD in the treatment of IBS. Pathological observation and abdominal withdrawal reex tests conrmed that the symptoms of IBS in rats were relieved by CPD. Moreover, immunohistochemistry conrmed that CPD could inhibit the expression of inammation-associated factors, such as VEGFA, MAPK8, and PTGS2. Conclusions Based on network pharmacology analysis, the present study provides insights into the potential mechanism of CPD in the treatment of IBS after successfully screening for associated key target genes and signaling pathways. These ndings establish a theoretical basis for the development of CPD-derived therapeutics.

Despite considerable research efforts, the treatment of IBS remains a signi cant challenge mainly owing to its poorly de ned pathophysiology. Traditional Chinese medicine (TCM) has provided various therapeutics for treating gastrointestinal disorders over centuries [5,6] . An increasing body of evidence has revealed that natural medicinal active ingredients may have bene cial effects for the treatment of IBS, and these effects are likely to be multifactorial. Among host-related factors, intestinal in ammation, central alterations (i.e., aberrant stress responses and cognitive dysfunctions), peripheral alterations, intestinal mucosal barrier integrity, and intestinal ora, as well as neurotransmitters and hormones in the enteric nervous system (ENS), are believed to be involved [7,27,28,29] .
In this conceptual framework, herbs appear as an attractive option in terms of both e cacy and safety, while prebiotics, synbiotics, and antibiotics require further study [45] . A variety compounds found within herbs regulate complex targets, in turn modulating known and unknown mechanisms. Among herbal therapies, Changping decoction (CPD), which includes herbs widely used for treating gastrointestinal disorders, is a classic TCM prescription. The aim of this study was to provide evidence for the bene cial effects of CPD on IBS through pharmacology and pathway bioinformatics analyses, highlighting the relationship between immune-related pathways and IBS pathophysiology, which was further con rmed in vivo. Our study aimed to systematically explore the potential mechanism of CPD in the treatment of IBS and, thus, provides a theoretical basis for the development of CPD-derived therapeutics.

Prediction of IBS-related targets
The Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/) is an international public repository for high-throughput microarray and next-generation sequencing functional genomic datasets [11] . In this study, we selected a dataset (GSE36701 [12] , GPL570) from the GEO database using the following keywords: "IBS" and "Homo sapiens." The data of normal samples and IBS samples from GSE36701 were used for differential gene expression analysis by employing the Wilcoxon test from the "limma" package in the R-3.6.0 software. The false discovery rate was set at <0.05, and an absolute value of log2 | fold change |> 1 was set as the cut-off value.

Screening the intersection of CPD targets and IBS targets
We screened the intersection of CPD targets and IBS targets through the VENNY2.1 online tool (https://bioinfogp.cnb.csic.es/tools/venny/index.html).
Construction a protein-protein interaction (PPI) network and screening of hub genes through which CPD regulates IBS The drug-target interaction network was constructed using Cytoscape 3.7.2 software. The network had 129 nodes, including 9 drugs, 118 common target genes, 1 disease, and 1786 edges ( Figure 2B). The STRING database (http://string-db.org) provides a critical assessment and integration of PPIs, including physical as well as functional associations [13] . The "CytoHubba" package in Cytoscape 3.7.2 software is a tool for exploring the core genes of the PPI network based on 11 calculation methods. MCC, a newly proposed method, has better performance with regard to precision of hub gene prediction from the PPI network [14,15] . In this study, we input the intersection of CPD targets and IBS targets in the STRING database to obtain a PPI network (Organism set to Homo sapiens) and then screened hub genes using the "CytoHubba" package.
Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis The "clusterPro ler" package is an ontology-based R package that not only automates the process of biological term classi cation and the enrichment analysis of gene clusters but also provides a visualization module for displaying analysis results [16] . We used the "clusterPro ler" package in the R-3.6.0 software to perform enrichment analysis on the intersection of CPD targets and IBS targets to con rm the potential mechanism of CPD in the treatment of IBS. To determine the core mechanism of action of CPD in IBS, we analyzed the core 10 targets to determine the major signaling pathways by enrichment analysis.

Animals and treatment
Thirty-two SPF Wistar rats [17] weighing 220 (±20) g were provided by Pengyue Laboratory Animal Center, Jinan, Shandong, China (License: SCXK20190003). The rats were raised in the SPF animal room of the medical college, Qingdao University, Qingdao, Shandong, China (License: SYXK20150003). The rats were isolated and fed for 1 week while they adapted to the environment and were then randomly divided again into a blank group (n = 8), model group (n = 8), CPD treatment group (n=8), and control treatment group (n = 8). According to the random number table method, 4 rats were fed in one cage for a total of 4 weeks.
All rats had free access to drinking water and food. The room temperature was 24 (±2) °C, the relative humidity was 50 (±5)%, light and dark conditions were alternated every 12 h, and the rat cages were cleaned every 2 days. The model group, CPD treatment group, and control treatment group underwent water avoidance stress (WAS) for 1 h/day for 10 days [18,19] . Brie y, rats were weighed and placed on a platform (8 × 6 cm) a xed to the center of a plastic container (55 × 50 cm) which was lled with water to 1 cm below the platform. The conversion of the human dose to that used in rats was calculated based on body surface. The CPD treatment group was administered CPD for 14 consecutive days. CPD was prepared by mixing a solid aqueous extract in distilled water (1.125 mg/ml) and administered at a dose of 1.0 ml/220 g body weight. The control treatment group was given saline (1.0 ml/220 g) for 14 consecutive days.
Abdominal withdrawal re ex (AWR) The AWR is mainly assessed to detect visceral hypersensitivity by dilating the rectum and colon to con rm whether the IBS model was successfully established [20,21] . In this study, all rats were fasted for 24 h but did receive drinking water. A small balloon dipped in liquid para n was then slowly inserted up to 4 cm into the colon. Subsequently, the rats received 10-80 mmHg pressure stimulation successively, each time increasing by 10 mmHg. The Al-Chaer method was used to evaluate the AWR score. When the abdominal wall of the rat is lifted from the bottom and contracted, or the rat body arches with elevation of the pelvis and scrotum, it is considered that the rat has a visceral pain threshold. The same pressure stimulations were performed three times, and the average pressure value was taken as the nal score.

Histological observation and Immunohistochemistry
After the rats were sacri ced, colonic biopsies were obtained and xed in cold 4% paraformaldehyde, embedded in para n, sectioned, stained with H&E, followed by gradient alcohol (100% twice, 95%, 70%, and 50% for 5 min each) and xylene dehydration (three times, for 5 min each). The colonic biopsy samples were observed under a light microscope (Olympus, Tokyo, Japan).
Immunohistochemistry experiments were performed on para n-embedded, 4-mm-thick sections. The sections were rst incubated with the primary antibody at 37 °C for 60 min. The primary antibodies used were as follows: rabbit polyclonal anti-mouse VEGFA (1:100 dilution; A nity Biosciences, Cincinnati, OH, USA), rabbit polyclonal anti-mouse MAPK8 (1:100; A nity Biosciences), rabbit polyclonal anti-mouse PTGS2 (1:100; A nity Biosciences). Thereafter, sections were incubated with a universal secondary antibody (1:200; A nity) at 37 °C for 30 min and then visualized using diaminobenzidine (DAB). Finally, the nuclei were labeled by counterstaining the sections with hematoxylin. Three randomly selected elds from each section were scanned under an Olympus CX31 microscope (Olympus, Tokyo, Japan), and quantitative analysis of immunohistochemistry results was performed using Image-pro plus software.

Statistical analysis
Graph Prism version 5.0 software was used to analyze the results. The statistical data are expressed as the mean ± standard deviation and were analyzed using the Wilcoxon rank-sum test.

Results
Results of CPD target protein and IBS disease target protein screening A total of 159 active ingredients (Table 1) and 189 related target proteins were screened in the TCMSP database. Differential expression analysis identi ed a total of 13,634 differentially expressed genes, of which 6140 were downregulated, and 7494 were upregulated ( Figure 1). According to the VENN diagram, a total of 118 target proteins were screened at the intersection of IBS and CPD ( Figure 2A).

Construction the PPI network and hub gene screening
We constructed the PPI network from the STRING database, and a total of 120 nodes and 1354 edges were present in this network. According to the MMC calculation method, MAPK8, VEGFA, MAPK1, CASP3, PTGS2, IL10, CXCL8, JUN, IL1B, and EGF were recognized as core genes, playing major roles in the mechanism of action of CPD in IBS. We believe that this PPI network can help us identify the potential mechanism of CPD in treating IBS (Figure 3).

Results of enrichment analysis
To explore the potential mechanism of CPD in the treatment of IBS, we performed GO enrichment analysis of the intersection of IBS and CPD using the R-3.6.0 software. For the biological process, terms such as response to nutrient levels (GO:0031667), response to extracellular stimulus (GO:0009991), and response to toxic substance (GO:0009636) were enriched ( Figure 4A)

CPD alleviates intestinal visceral hypersensitivity in the WAS-induced IBS rat model
To determine whether CPD treatment could reduce intestinal visceral hypersensitivity in IBS, CPD was continuously administered via the intragastric route to IBS group rats for 2 weeks. The degree of colon pain threshold pressure was assessed by the AWR test. The colon pain threshold for the blank group and CPD treatment group was signi cantly lower than those for the model group and control treatment group (P < 0.0001) (Figure 7).

Results of histological observation and immunohistochemistry
Through histological observation, we found that the colon tissue of model group and control treatment group rats exhibited different degrees of lymphocyte proliferation, and no lesions were observed in colon tissues of the blank group and the CPD treatment group (Figure 8).
Immunohistochemistry analysis revealed that the expression of MAPK8, VEGFA, and PTGS2 in the model group and control treatment group samples was higher than that in the blank group and CPD treatment group (Figure 9).

Discussion
Identi cation of mechanisms that in uence the e cacy of Chinese herbal formulae (CHF) for IBS might enable clinicians to predict the patient response and devise new more rational and tailored treatment strategies. In this study, to our knowledge for the rst time, we report the anti-in ammatory effects of CPD mediated through the combined regulation of MAPK8, VEGFA, and PTGS2. The occurrence and development of IBS is a multilevel, multistep, complex process in uenced by various factors, such as the gut microbiome, intestinal permeability, gut immune function, motility, diet, brain-gut interactions, and psychosocial status [2,3,4,30] . This multitude of factors complicate the treatment of IBS. A recent study suggested some risk factors such as food allergy, abnormal microbiota, bile acid malabsorption, and increased intestinal permeability as contributors to low-grade intestinal in ammation, the major pathological manifestation in IBS.
Identi cation of important target proteins related to disease occurrence is essential for the exploration of IBS therapy-associated molecular mechanisms. Avoiding the restrictions of labor-intensive and timeconsuming clinical trials with human subjects, in silico candidate target screening was employed. The target network consisted of 120 nodes and 1354 edges, and several topological parameters were calculated to obtain 10 important targets including hub or bottleneck proteins based on their high degree centrality and betweenness centrality. Pathway enrichment analysis yielded similar results. Overlapping targets, including PTGS2, VEGFA, MAPK8, CASP3, MAPK1, IL1B, EGF, JUN, IL10, and CXCL8, connected multiple subgroups, which may play pivotal roles in the pathway network [31,32,33,34] . Importantly, several major targets were both hub bottleneck nodes in the structural network and key points in the protein and pathway networks. Based on gene function classi cation, our analysis results revealed that the e cacy of CPD therapy in IBS was based on the modulation of target proteins involved in the in ammatory response and oxidative stress regulation (MAPK8, PTGS2, VEGFA).
Histological observation revealed that the colons of model group and control treatment group rats exhibited low-grade in ammation characterized by lymphocyte proliferation, which was not observed in blank group and CPD treatment group rat colons. Increased numbers of lymphocytes have been reported in the colon and small intestine of patients with IBS. Our ndings con rmed these observations in rats, and we further investigated the anti-in ammatory potential of CPD. A history of acute diarrheal illness preceding the onset of irritable bowel symptoms in some patients has suggested that the development of IBS occurs following infectious enteritis. The increased risk of post-infectious IBS is associated with bacterial, protozoan, helminth, and viral infections [41,42,43,44,45] . Some meta-analyses have shown an increased risk of IBS in patients who had experienced an episode of acute gastroenteritis [46,47] . A review of 18 studies showed a pooled 10% incidence of IBS and that the odds of developing IBS increased sixfold following acute gastrointestinal infection. One study in full-thickness jejunal biopsies supported our results, showing lymphocyte in ltration in the myenteric plexus as well as neuron degeneration, which stimulated the ENS, leading to abnormal motor and visceral responses within the intestine. Chronic infections have been suggested to trigger IBS symptoms in tandem with predisposing factors. Therefore, targeting the in ammatory pathways in IBS holds promise as an effective therapeutic approach.
The MAPK signaling pathway, to which MAPK8 belongs, is considered one of the classical in ammatory pathways [44] . Stool examinations from patients with diarrhea-predominant IBS have revealed a high level of serine-protease activity. The role of intestinal serine-proteases in the pathophysiology of IBS remains under investigation. In our study, we found that MAPK8 could regulate the expression of PTGS2 and VEGFA in Kaposi sarcoma-associated herpesvirus infection (hsa05167), thus, participating in the process of in ammation and cell proliferation (Fig. 6B) in IBS. MAPK8 could also regulate the expression of PTGS2 in the IL-17 signaling pathway (hsa04657) and, thus, participate in the process of autoimmune pathology, neutrophil recruitment, and immunity to extracellular pathogens (Fig. 6C). Further, immunohistochemistry demonstrated the potential of CPD to downregulate the expression of MAPK8 in an IBS rat model. This notion is supported by the observation that CPD exerts its anti-in ammatory effects in colon tissue by inducing MAPK8 and PTGS2 in this experimental setup. PTGS2 was reported to play a critical role in regulating in ammatory responses by generating in ammatory mediator prostaglandin E-2 (PGE2) and contributed to the development of chronic in ammatory diseases [42,43] . VEGFA is heavily involved in several chronic in ammatory disorders, where it not only promotes angiogenesis but also directly fosters in ammation [38,39] . Angiogenesis is inherent to chronic in ammation and is associated with structural changes, including activation and proliferation of endothelial cells [40] , as well as capillary and venule remodeling, all of which result in the expansion of the tissue microvascular bed. Studies have shown that the relationship between PTGS2 and VEGF is closely associated with in ammation and angiogenesis. It is now well established that, in diseases such as rheumatoid arthritis, psoriasis, atherosclerosis, and chronic lung in ammation, VEGFA is intimately involved in the pathogenesis, and targeting VEGFA is a promising new therapeutic strategy to dampen in ammation. In this study, we demonstrated activation of the VEGF pathway in actively in amed IBD mucosa. The expression of both VEGFA and its receptor VEGFR-2 is enhanced in tissue biopsy specimens from in amed bowel segments [41] . We next investigated whether CPD reduced the expression of VEGFA to exert its anti-in ammatory effects on intestinal endothelium in vivo. Our results identi ed VEGFA as intimately involved in IBD pathogenesis and at the crossroads between in ammation-driven angiogenesis and mucosal in ammation. This suggests that the CPD-mediated downregulation of VEGFA signaling may represent a new strategy to dampen intestinal in ammation.
We detected the expression of VEGFA, PTGS2, and MAPK8 in each group via immunohistochemistry, and found that CPD could effectively inhibit their expression. Further, low-grade in ammation in the colon tissue of rats and visceral pain were alleviated with CPD. Moreover, we showed that the increased PTGS activity and PTGS2 protein levels in HUVECs treated with VEGF were mediated through protein tyrosine kinases, but not protein kinase C (Figs. 4 and 5). These ndings were consistent with those of previous studies showing that VEGF receptors on endothelial cells acted via protein tyrosine kinases, and the mitogen-driven induction of PTGS2 in endothelial cells was mediated through protein tyrosine kinases.
Therefore, the downstream regulation of PG production in HUVECs activated with VEGF was affected at several points such as PTGS2, tyrosine phosphorylation, and phospholipase. In summary, our study is the rst to show that PTGS2 can be induced through protein tyrosine kinase in HUVECs treated with VEGF.
VEGF is known to be involved in pathological processes such as in ammation, atherosclerosis development, and carcinogenesis. Thus, the use of speci c PTGS2 inhibitors in related conditions might have therapeutic potential.

Conclusion
Nowadays, a myriad of therapeutic options are available for IBS. However, treatment outcome remains unsatisfactory. Our research shows that CPD treatment can signi cantly alleviate intestinal visceral hypersensitivity and low-grade intestinal in ammation while also decreasing the expression of in ammation-related factors such as VEGFA and PTGS2 in IBS rats. These ndings provide evidence that CPD can effectively relieve symptoms of IBS in patients. CPD is a traditional CHF including a number of medicinal plants with various therapeutic properties, traditionally used for different diseases including the treatment and prevention of intestinal disease. CHF contains different active agents and may act on multiple targets, signaling pathways, and biological processes with potential synergistic effects and chemical reactions. A total of 118 potential targets and 159 pharmacologically active components were identi ed for CPD, supporting the clinical potential of this TCM. As an increasing number of people tend to use TCM as alternative treatment, more extensive and well-designed preclinical and clinical trials assessing the potential synergistic and adverse side effects of herb-drug interactions, as well as mechanisms of action, will highlight future directions in IBS therapy research.