Based on network pharmacology, molecular docking and experimental validation to reveal the potential molecular mechanism of quercetin for the treatment of diarrheal irritable bowel syndrome

FENG Min-chao, LUO Fang, XIE Sheng, CHEN Zu-min, TAN Jin-xuan , LI Kai , CHEN Guo-zhong, WANG Dao-gang?

1.Guangxi University of Traditional Chinese Medicine, Nanning 530001, China

2.The First Affiliated Hospital of Guangxi University of Traditional Chinese Medicine, Nanning 530001, China

Keywords:

ABSTRACT Objective: To explore the potential mechanism of action of quercetin in the treatment of diarrhea irritable bowel syndrome (IBS-D).Methods: The potential targets of quercetin were obtained from the TCMSP, SwissTar-getPrediction, and BATMAN-TCM databases.The targets of IBS-D were obtained by searching the GeneCards database with ＂diarrhea irritable bowel syndrome＂ as the keyword, and the targets of quercetin and IBS-D were intersected.The PPI network was constructed by Cytoscape 3.7.1 software.The intersected targets were imported into the DAVID database for GO functional analysis and KEGG pathway enrichment analysis.The binding ability of quercetin to the core targets was observed using molecular docking.Based on this, we established an IBS-D rat model, administered quercetin for intervention,and experimentally validated the network pharmacology prediction results by HE staining and ELISA assay.Results: Network pharmacology analysis showed that TP53, TNF-α,AKT1, VEGF-A, IL-6 factors and MAPK, PI3K-Akt signaling pathway as the core targets and pathways of quercetin for the treatment of IBS-D.The results of animal experiments revealed that quercetin could inhibit the secretion of TP53, TNF-α, AKT1, VEGF-A, IL-1β and IL-6,reduce the inflammatory response and improve IBS-D.Conclusion: Quercetin could protect colon tissue by regulating the expression of TP53, TNF-α, AKT1, VEGF-A, IL-1β and IL-6,thereby treating IBS-D.

1.Introduction

Irritable bowel syndrome (IBS) is a common functional gastrointestinal disorder characterized by diarrhea, constipation,and pain that affects 9-23% of the world’s population, of which Diarrhoeal irritable bowel syndrome (IBS-D) is the Diarrhoeal irritable bowel syndrome (IBS-D) is the most common subtype of IBS, accounting for approximately 40% of cases] At present,the pathogenesis of IBS-D has not been fully revealed.Quercetin,as a flavonoid compound, has anti-inflammatory, antioxidant,and inhibitory effects on cell proliferation.It has been previously confirmed that quercetin can alleviate the inflammatory response in IBS rats and can be used as an adjuvant drug for the treatment of irritable bowel syndrome] Network pharmacology is a method used to analyze and predict the potential mechanisms of drug intervention in diseases from a macro perspective.Therefore, this study explores the mechanism of quercetin intervention in IBS-D based on network pharmacology and conducts preliminary verification through animal experiments to provide evidence to support the treatment of IBS-D by traditional Chinese medicine containing quercetin components.

2.Materials and methods

2.1 Experimental animals

Fifty SPF-grade male SD rats, weighing 160-200 g, were purchased from Hunan Slaughter Scene Da Laboratory Animals (Changsha,China; Certificate No.SCXK (Xiang) 2019-0004).All rats were kept in the Experimental Center for Medical Molecular Biology of the First Affiliated Hospital of Guangxi University of Traditional Chinese Medicine [SYXK(Xiang)2019-0001], with light sources alternately cycling between light and dark every 12 h.They were fed with conventional standard feed, and were free to feed and drink.The study was conducted in accordance with the Principles of Laboratory Animal Care and was approved by the Experimental Animal Ethics Committee of Guangxi University of Traditional Chinese Medicine(Approval No 2021-066).

2.2 Experimental drugs and reagents

Quercetin and sodium pentobarbital were purchased from Sigma(batch number: Q4951-10G; AM00469); Hematoxylin and Eosin(H&E) staining kit (Solarbio, G1120); Rat IL-1β, IL-6, TNF-α,VEGFA ELISA kit (Proteintech, batch number: KE10003;KE10007; KE10002; KE20014); rat TP53 ELISA kit (CUSABIO,CSB-E08336r); Rat AKT1 ELISA kit (Shanghai Coabio, CB13377-Ra).

2.3 Instrumentation

Tissue slicer (Leica, Germany, RM2016); enzyme labeler (Rayto,RT-6100); optical microscope (Olympus, BX43).

2.4 Network Pharmacological Analysis

2.4.1 Screening of Quercetin Target and Construction of Traditional Chinese Medicine Quercetin Target Network Diagram

Using the search term “Quercetin” in TCMSP (https://tcmspw.com/tcmsp.php), SwissTarget getPrediction (http://www.swisstargetprediction.ch/) and BATMAN-TCM database (http://bionet.ncpsb.org/batman-tcm/) to obtain potential targets of quercetin and corresponding traditional Chinese medicine.The TCMIP v2.0 database (http://www.tcmip.cn/TCMIP/index.php),Chinese Pharmacopoeia version 2020, and related literature were used to obtain the the properties, flavors, and meridians of traditional Chinese medicines.UniProt database (http://www.uniprot.org/) was used to standardize the names of genes and proteins.Cytoscape 3.7.1 was used to construct the TCM-quercetin-target network diagram.

2.4.2 Collection of IBS-D related targets

Relevant targets of IBS-D were obtained from the GeneCards database by using the search term “diarrhea irritable bowel syndrome” (https://auth.lifemapsc.com/).

2.4.3 Venn diagram mapping of cross-targeted genes

The Venny tool (http://bioinformatics.psb.ugent.be/webtools/Venn/)was used to map the Venn diagrams of quercetin target genes and IBS-D target genes and compare them to obtain the intersection of target genes.

2.4.4 Construction of Protein Interaction Network (PPI)

Based on the cross-targeting results, a PPI network was constructed in the string database (http://string-db.org/) with the species restriction of “Homo sapiens” and the minimum required interaction score of 0.4.The PPI network data were then imported into Cytoscape 3.7.1 software.A core set of targets for the network was obtained using CytoHubba and the MCODE algorithm plug-in.

2.4.5 GO functional analysis and KEGG pathway enrichment analysis

To identify potential pathway pathways for quercetin regulation of IBS-D, GO enrichment analysis of cross-targets was performed using the DAVID database (https://david.ncifcrf.gov/).We also performed KEGG analysis, plotted signaling pathway histograms based on P 0.01, and selected the top 20 signaling pathways to plot KEGG bubble plots.The data were then visualized through the OmicShare website (https://www.omicshare.com/).

2.5 Molecular docking simulations

The top five core genes identified in the PPI network analysis were selected for molecular docking simulations with quercetin.The 2D structure of one of them, quercetin, was downloaded from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/) and the mechanical structure was optimized using ChemBio3D.The crystal structure of the target protein was obtained from the RCSB database(https://www.rcsb.org/).Ligand and receptor files were converted to pdbqt format using AutoDockTools 1.5.6 and their structures were improved by replacing water molecules with hydrogen atoms.Finally, molecular docking simulations were performed in Discovery Studio software.The LibDock score calculated by Discovery Studio indicates the degree of ligand-receptor binding.

2.6 Animal experiments

2.6.1 IBS-D model preparation and experimental grouping Rats were randomly divided into blank group, model group, low,medium, and high-dose quercetin groups using a random number table method, with 10 rats in each group.Reference method for rat IBS-D model establishment] Briefly, rats were subjected to 1 hour+1 mL of 4% acetic acid enema daily for 7 d.After successful modeling, the rats in the quercetin group were given quercetin 5, 10,and 20 mg/kg by gavage[3], the rats in the blank group and model group were given equal amounts of physiological saline by gavage once a day for 2 weeks.

2.6.2 Histopathological observation of colon tissue in IBS-D rats

Before preparing the sections (5 μm thick), colon tissues of rats were fixed with 4% paraformaldehyde and embedded in paraffin.Sections were cut, baked for 30 min, then dewaxed and hydrated with xylene and anhydrous ethanol for H&E staining.Pathological changes in the colonic tissues of IBS-D rats were assessed by in light microscopy.

2.6.3 ELISA for serum levels of IL-1β, IL-6, TNF-α, TP53,AKT1, VEGFA in rats

Under aseptic conditions, blood was taken from the abdominal aorta of rats, and the blood samples were allowed to stand for 2 hours at room temperature, and then centrifuged in a high-speed centrifuge at 3 000 r/min at 4 ℃ for 15 min.The upper layer of serum was removed and the OD values of IL-1β, IL-6, TNF-α, TP53, AKT1,and VEGFA were measured at 450 nm using an enzyme marker according to the manufacturer’s instructions, and the standard curve of the samples was plotted and the concentrations were converted.

2.7 Statistical analysis

All data analysis was conducted using SPSS 25.0 software and GraphPad Prism 9.0 software.Data were expressed as (±s).Significance of differences between groups was analyzed using oneway ANOVA.P ＜ 0.05 was considered a statistically significant difference.

3.Results

3.1 Screening of quercetin potential targets

The TCMSP, SwissTargetPrediction and BATMAN-TCM databases were searched for potential targets of quercetin and corresponding TCMs, and a total of 311 potential targets and 188 TCMs were obtained.Subsequently, we constructed an herbal medicine-quercetin-target network, as shown in Figure 1, which included 500 nodes and 499 edges, including, for example, targets such as IL2, IL6, IL10, CXCL2, MYC, and herbs such as Eriocaulon buergerianum, Moutan cortex, Cyperi rhizoma, Caryophylli flos,Notoginseng radix et rhizoma.

3.2 Analysis of the Nature, Taste, and Meridian of Traditional Chinese Medicine Containing Quercetin

Search for traditional Chinese medicine containing quercetin in the TCMSP database, and use databases such as TCMIP to summarize the properties, flavors, and meridians of traditional Chinese medicine.The “four qi” of traditional Chinese medicine containing quercetin are mostly classified as cold; The five flavors are mostly bitter and sweet; The meridian of return often enters the liver and lung meridians, as shown in Figure 2.

3.3 Construction of PPI network

We searched the TCMSP, BATMAN-TCM, and Swiss target databases to predict potential targets of quercetin in the treatment of IBS-D, and identified 311 potential targets.By searching the GeneCards database (correlation score 16.26128387), 650 IBS-D related genes were obtained.After importing the target genes of quercetin and IBS-D into the OmicShare cloud platform, 77 overlapping target genes were identified, as shown in Figure 3a.Further explore the core goal of quercetin in treating IBS-D, and import these overlapping target genes into the STRING website to construct a PPI network, which includes 77 nodes and 1135 edges,including targets such as TP53, VEGFA, SRC, EGFR, and IL1B, as shown in Figure 3b and Table 1.

Fig 1 Chinese herbal medicine-quercetin-target network diagram; Left:Herbal medicines containing quercetin, Middle:Quercetin, Right:Quercetin-containing targets.

Fig 2 Analysis chart of Chinese herbal medicine’s sexual flavor and meridian

3.4 Search for quercetin core targets

Using MCODE to partition and cluster the PPI network, a total of 4 modules were obtained.The module with the highest score(score=33.632) included 39 nodes and 639 edges, as shown in Figure 4a and Table 2; Module 2 (score=3.750) includes 9 nodes and 15 edges, as shown in Figure 4b; Module 3 and 4 (score=3.000) include 3 nodes and 3 edges, as shown in Figure 4c-d; In addition, using the CytoHubba plugin to extract the top 10 nodes ranked by degree in the PPI network, including TP53, TNF-α, AKT1, VEGFA, and IL6 are shown in Figure 4e and Table 3.Combining the results of two algorithms, the above targets have been identified as the core targets for quercetin treatment of IBS-D.

3.5 GO functional enrichment and KEGG pathway enrichment analysis

77 overlapping target genes were submitted to the DAVID database and Om-icShare cloud platform for GO and KEGG pathway enrichment analysis.A total of 2793 related GO enrichment entries and 132 KEGG pathways were identified (P 0.01).The GO enriched entries related to IBS-D therapy include 2494 biological process entries (BP), 178 molecular functional entries (MF), and 121 cellular component entries (CC).The top 20 entries of BP, MF, and CC in GO enrichment analysis are shown in Figure 5.These analyses indicate that biological processes such as regulation of biological quality, regulation of programmed cell death, positive regulation of molecular function, and cell proliferation play important roles in the anti IBS-D effect of quercetin.Molecular functions include growth factor receptor binding, protein phosphatase binding,receptor binding, cytokine activity and kinase activity.The cellular components are mainly involved in plasma membrane protein complex, perinuclear region of the cytoplasm, plasma membrane and cell periphery.

The 132 KEGG pathways involve human diseases (such as cancer, infectious diseases, cardiovascular diseases, and immune diseases), cellular processes (cell growth and death, transportationand catabolism), biological systems (such as the immune system,digestive system, and circulatory system), environmental information processing (such as signal transduction and signaling molecules and interactions), and metabolism (such as lipid metabolism).In addition, the Pathways in cancer, MAPK, PI3K-Akt, and TNF signaling pathways have been identified as the four main pathways playing a major role in the development of IBS-D, as shown in Figure 6.

Tab 2 Topological analysis of network core targets based on Mocde

3.6 Molecular docking analysis

Using five core targets (TP53, TNF- α, AKT1, VEGFA, IL6)were molecularly docked with quercetin, as shown in Figure 7.The calculation results based on Discovery Studio software show that TP53-quercetin , TNF-α-Quercetin, AKT1-quercetin,VEGFA-quercetin, and IL6-quercetin have good binding ability,with LibDock scores of 85.6984, 111.196, 72.4317, 79.8263, and 107.199, respectively.

3.7 Experimental verification results

3.7.1 General situation of rats

The blank group of rats showed better mental health, normal feces,regular diet, and ease of movement; The model group rats had poor mental state, loose feces, reduced diet and activity, and decreased body mass; After treatment, the rats in each dose group of quercetin showed good mental state, gradually improved fecal quality,increased diet and activity, and increased body weight.

3.7.2 Effects of Quercetin on Colon Tissue

Fig 3 Network pharmacological target analysis

Fig 4 PPI network core target analysis

Fig 5 GO enrichment analysis of network pharmacological targets of quercetin for IBS-D treatment

In the blank group, the epithelial cells of the colon tissue were closely connected, the intestinal mucosa was structurally intact, the glandular structure was neatly arranged, and no obvious organic lesions were seen.In the model group, the rats were seen to have cup-shaped cell plasma rich in fluid particles, a small number of neutrophils and inflammatory cells were present in scattered quantities, and the intestinal mucosal structure was relatively intact.Compared with the model group, no obvious organic lesions were seen in the colonic tissues of rats in all dose groups of quercetin, and the inflammatory reaction was reduced, as shown in Figure 8.

Fig 6 KEGG enrichment analysis of network pharmacological targets for quercetin treatment of IBS-D

Fig 7 Molecular docking of quercetin and core proteins

Tab 3 Topological analysis of network core targets based on Cytohubba

Fig 8 Effect of quercetin on colonic histopathology in IBS-D rats (HE, ×400)

3.7.3 Effect of quercetin on TP53, TNF-α, AKT1, VEGF-A,IL-1β, IL-6 in IBS-D rats

ELISA results showed that TP53, TNF-α, AKT1, VEGF-A, IL-1β, and IL-6 were elevated in rats of the model group compared with the blank group (P＜0.05); Compared with the model group,quercetin rats in each treatment group reversed the elevation of TP53, TNF-α, AKT1, VEGF-A, IL-1β, and IL-6 ( P＜0.05), with the quercetin high-dose group having the most significant inhibitory effect, as shown in Table 4.

Tab 4 Effect of quercetin on TP53, TNF-α, AKT1, VEGF-A, IL-1β and IL-6 in IBS-D rats (n=10, ±s)

Tab 4 Effect of quercetin on TP53, TNF-α, AKT1, VEGF-A, IL-1β and IL-6 in IBS-D rats (n=10, ±s)

Note: Compared with the blank group, *P＜0.05; Compared with the model group, #P＜0.05.

Group TP53 TNF-α AKT1 VEGF-A IL-1β IL-6 Blank 21.4±20 32.82±11.82 4.48±1.39 270.81±35.77 14.49±4.51 34.3±6.13 Model 114.41±15.38* 317.62±28.43* 65.13±8.54* 452.65±27.27* 120.45±15.36* 141.91±15.75*Low dose 69.48±4.91# 220.41±17.79# 42.08±3.98# 400.12±13.83# 74.13±7.65# 104.76±9.77#Medium dose 51.41±4.57# 157.31±10.39# 25.04±3.9# 358.76±7.43# 47.31±7.1# 76.58±5.27#High dose 34.57±3.64# 106.17±11.02# 13.53±2.29# 319.41±5.44# 31.2±3.37# 55.99±5.41#F 131.6 237.6 156.9 64.57 137.2 121.3 P＜0.05 ＜0.05 ＜0.05 ＜0.05 ＜0.05 ＜0.05

4.Discussion

It is currently believed that IBS-D is associated with a variety of mechanisms including gut microbiota, visceral hypersensitivity,intestinal permeability, gut-brain interactions, and low-grade inflammation] Among other things, the inflammatory response is the basic response induced by the immune system to protect the body from pathogens, tissue damage, and stress.The network pharmacology results of this study showed that the core key targets of quercetin for the treatment of IBS-D were TP53, TNF-α,AKT1, VEGF-A, and IL-6, which are associated with inflammatory response, programmed cell death, and oxidative stress.Studies have shown that TP53, as a transcription factor, can increase the permeability of the outer mitochondrial membrane and induce apoptosis, as well as regulate the cell cycle, inflammatory response,and apoptosis through PI3K/Akt, MAPK, and other signaling pathways] AKT1 is an intracellular kinase expressed in almost all tissues that regulates cellular metabolism, homeostasis, survival and proliferation] Interestingly, it has been identified that AKT1 phosphorylation induces NLRP3 inflammatory vesicle activation to mediate the inflammatory response in colitis-associated cancers]Oxidative stress activates growth, chemotaxis, inflammatory cells,anti-inflammatory and other transcription factors to maintain homeostasis in the body’s internal environment] It was reported that VEGF-A is a highly conserved secreted signaling protein that is regulated by effectors such as hypoxia or growth factors, while VEGF is an important node in the oxidative stress process, which increases the expression of intracellular ROS, induces endothelial cell migration and proliferation, and regulates the balance between hypoxia/reoxygenation systems] In addition, VEGF deficiency increases apoptosis and affects endothelial cell survival and metabolism] As a promoter of the inflammatory response, TNF-α increases the production of more chemokines and cytokines, leading to amplification of the inflammatory cascade and exacerbating inflammatory damage in organs] IL-6 is a key cytokine that induces and maintains inflammation and can be released by macrophages in response to tissue injury, and high IL-6 expression promotes a systemic inflammatory response] In this study, quercetin significantly decreased the expression levels of TP53, TNF-α, AKT1, VEGF-A,IL-1β, and IL-6, suggesting that quercetin may intervene in IBS-D by inhibiting apoptosis, cellular autophagy, oxidative stress, and inflammatory responses.

KEGG pathway enrichment analysis showed that the MAPK, PI3K Akt, and TNF signaling pathways may be the main pathways in the development of quercetin induced IBS-D.The MAPK pathway has been shown to be an important pathway in the development of IBS-D and can play a role in regulating the secretion of immune-related molecules and analgesia in the IBS rat model] In addition, inhibitors of MAPK were effective in relieving inflammation and neuropathic pain in animal models] Studies have shown that PI3K/Akt signaling has an important role in apoptosis and autophagy in cells] According to reports, the PI3K Akt pathway activates autophagy to disrupt the intestinal mucosal barrier, leading to the occurrence of IBS syndrome.Inhibiting the PI3K Akt pathway can protect intestinal epithelial cells and improve the intestinal mucosal barrier] Previous studies have also shown that inhibition of the PI3K/AKT signaling pathway can reduce visceral sensitization and ameliorate IBS-D syndrome in IBS-D mice] The TNF signaling pathway controls the development of the immune system, cell survival signaling pathways, proliferation and regulates metabolic processes] Hou et al.found that inhibiting the TNF pathway induced inflammatory signaling can protect intestinal epithelial tight junctions and repair the damage caused by IBS-D to the intestinal epithelial barrier]Unfortunately, this study did not experimentally validate the MAPK,PI3K-Akt, and TNF signaling pathways, and we hope that further research can be conducted.

In summary, this study used a network pharmacology approach to reveal the key targets and pathways of action of quercetin in the treatment of IBS-D.Experiments verified that quercetin acted on the key targets of TP53, TNF-α, AKT1, VEGF-A, IL-6 and other key targets to regulate the inflammatory response, oxidative stress,cellular autophagy, and other pathways, and exerted a therapeutic effect in the treatment of IBS-D.

Authors’ contributions: Minchao Feng and Sheng Xie conceived and designed the study and experiments; Fang Luo and Jinxuan Tan were responsible for data collection and processing, and writing the article; Daogang Wang, Zumin Chen, Kai Li, and Guozhong Chen were responsible for the experimental conception and revision of the final article.

Conflict of interest statement: All authors declare no conflicts of interest.