Exploring the Mechanism of Berberine Intervention in Ulcerative Colitis from the Perspective of Inflammation and Immunity Based on Systemic Pharmacology

Hindawi Evidence-Based Complementary and Alternative Medicine Volume 2021, Article ID 9970240, 11 pages https://doi.org/10.1155/2021/9970240

Research Article Exploring the Mechanism of Berberine Intervention in Ulcerative Colitis from the Perspective of Inflammation and Immunity Based on Systemic Pharmacology

Yan Jiang,1,2 Li Zhao,2 Qing Chen,2 and Lihong Zhou 1

1School of Medicine, Hunan Normal University, Changsha, Hunan, China 2e First Aﬃliated Hospital, University of South China, Hengyang, Hunan, China

Correspondence should be addressed to Lihong Zhou; [email protected]

Received 11 March 2021; Revised 3 April 2021; Accepted 9 May 2021; Published 9 June 2021

Academic Editor: Guang Chen

Copyright © 2021 Yan Jiang et al. )is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background. Ulcerative colitis (UC) is a chronic nonspecific inflammatory disease of the colon and rectum. Recent studies found that berberine had effects on inflammatory diseases and immune diseases. Methods. )e PharmMapper database was used to predict the berberine potential target and GeneCards database and OMIM database were utilized to collect UC genes. )e Cytoscape software was used to construct and analyze the networks and DAVID was utilized to perform enrichment analysis. )en, animal experiments were performed to validate the prediction results. )e experimental rats were randomly divided into normal group (control group), model group, and berberine group. )e general condition, body weight, gross morphology of colon tissue, and colonic mucosal damage index (CMDI) score were observed. )e pathological changes of colon tissue were observed by H&E staining. )e levels of serum interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and IL-4 were detected by ELISA. )e expressions of IL-1β, TNF-α, and IL-4 protein in colon tissue were detected by immunohistochemistry. Results. A total of 211 Berberine’s potential targets and 210 UC genes were obtained. )e enrichment analysis showed that berberine may regulate inflammation, inflammatory cytokines, and their mediated inflammation signal pathways such as inflammatory bowel disease (IBD), rheumatoid arthritis, cytokine-cytokine receptor interaction, TNF, T cell receptor, Toll-like receptor, and JAK/STAT signaling pathway. Compared with the model group, the body mass of rats in the berberine group was significantly increased (P < 0.05); the general morphology and pathological changes of colon tissue were significantly improved; CMDI score, serum and colon tissue IL-1β, TNF-α content, and protein expression were decreased significantly (P < 0.05); and IL-4 content and protein expression increased significantly (P < 0.05). Conclusion. Berberine can interfere with UC through related biological processes and signal pathways related to inflammation and immunity. In-depth exploration of the mechanism of berberine in the treatment of UC will provide a basis for clinical application.

1. Introduction the lesions are limited to the large intestine mucosa and submucosa. Current studies have shown that the patho- Inflammatory bowel disease (IBD) mainly includes ulcera- genesis of UC is complex, involving the combined effects of tive colitis (UC) and Crohn’s disease (CD). )e number of various factors such as genetic susceptibility, epithelial IBD patients in Europe and the United States has accounted barrier defects, dysregulated immune responses, and envi- for 0.5% of the world’s population. At present, retrospective ronmental factors [4, 5]. )e main treatments for UC in- studies on the incidence and prevalence of IBD worldwide clude the following: (1) Sulfasalazine salicylic acid show that the incidence in Europe and the United States has preparations, such as Mesalazine; (2) corticosteroids, such as reached a plateau, while other countries are in the first stage prednisone or dexamethasone; and (3) immunosuppressants of the growth of new cases [1]. By 2025, IBD patients in [6, 7]. However, these drugs are limited due to their limited China will reach 1.5 million [2, 3]. UC is a chronic non- clinical efficacy and large side effects. Meanwhile, TNF in- specific inflammatory disease of the colon and rectum, and hibitors are only effective in about half of patients [8]. At 2 Evidence-Based Complementary and Alternative Medicine present, natural plant products, especially plant component interaction (PPI) data [29]. )e Cytoscape software was monomers, are getting more and more attention in the utilized to construct and analyze the networks [30]. )e treatment of UC [9, 10]. nodes in the network represent targets, genes, and so forth, Berberine is an isoquinoline alkaloid extracted from the and edges show the relationship among nodes. )e “Net- root and bark of the Coptis chinensis plant of the Ranun- work Analyzer” plug-in was used to analyze the network culaceae family [11]. Recent studies have shown that ber- topology. )e “MCODE” plug-in was utilized to detect the berine has high value in the clinical application of tumors, closely connected part of PPI network. diabetes, cardiovascular diseases, inflammatory diseases, and Targets and genes were imported into DAVID (https:// immune diseases. Its main mechanism is anti-inflammatory david.ncifcrf.gov/), and the species was defined as “human” immune response and antioxidant [12–14]. Studies have also for gene ontology (GO) enrichment analysis and Kyoto shown that berberine can prevent the production of proin- Encyclopedia of Genes and Genomes (KEGG) pathway flammatory cytokines in colitis [15–19]. )e mechanism may enrichment analysis [31]. be that berberine inhibited lipopolysaccharide- (LPS-) induced cytokine production and the activation of MAPK and 2.3. Experimental Materials NF-κB in macrophages [17]. Meanwhile, berberine regulates the balance of Treg/)17 to treat UC by regulating the in- 2.3.1. Experimental Animal. Sixty specific-pathogen-free- testinal flora in the colon [20]. Berberine also exerts a pro- (SPF-) grade SD rats, male and female, weighing 220 + 10 g, tective effect on the colon of UC by regulating the interaction were purchased from Changsha Tianqin Biotechnology Co., of enteric glial cells-intestinal epithelial cells-immune cells Ltd., animal license number SCXK (Xiang), 2018–0002. SD [21]. Berberine can also interfere with mucosal inflammation rats were raised in the animal room of the First Affiliated driven by oncostatin M to treat chronic UC [22]. However, Hospital of University of South China, with a temperature of the network biological mechanism of berberine in the 26 ± 10°C and a relative humidity of 50% ± 10%. )e rats treatment of UC is still unknown. were adaptively fed for one week. )e procedures for care Systemic pharmacology, as a product of multidisciplinary and use of animals were approved by the Ethics Committee intersection, includes multiple research methods such as of the First Affiliated Hospital of University of South China classical pharmacology, chemical biology, and bioinformatics (USCAF-047) and all applicable institutional and govern- and covers a large number of experimental disciplines in- mental regulations concerning the ethical use of animals cluding research techniques from cells and tissues to organs were followed. [23, 24]. Systemic pharmacology studies the occurrence and development of diseases from the perspective of biological networks, understands the interaction between drugs and the 2.3.2. Experimental Drugs, Reagents, and Instruments. body, and guides the discovery of new drugs [25]. )erefore, )e experimental drugs, reagents, and instruments are as this study hopes to explore the molecular mechanism of follows: berberine hydrochloride (Shanghai Xinyi Tianping berberine intervention in the UC disease network through Pharmaceutical Co., Ltd., National Medicine Standard systemic pharmacological strategies. H31021444, 0.1 g/tablet), dextran sulfate sodium salt (DSS) (Sigma Inc., batch number 31404-5G-F), H&E staining kit (Wuhan Boster Biological Technology Co., Ltd., batch 2. Materials and Methods number AR1180), interleukin-1β (IL-1β), tumor necrosis 2.1. Berberine’s Potential Target Prediction and UC Genes factor-α (TNF-α), IL-4 ELISA kit (Ambion company, catalog Collection. PubChem Compound database search was used numbers xyR013, xyR017, and xyR097), IL-8 ELISA kit to retrieve the PubChem CID of berberine. )en PubChem (Nanjing Senbega Biotechnology Co., Ltd., batch number: CID was imported into Open Babel software to obtain the SBJ-R0700), Rat IL-6 ELISA kit (Cat. No. EK0412), Rat IL-12 3D structure of the compound and saved as a mol2 format (P70) ELISA kit (Cat. No. EK1652), Rat IL-10 ELISA kit file. )en, the mol2 format file of berberine was uploaded to (Cat. No. EK0418) (Wuhan Boster Bioengineering Co., Ltd.), the PharmMapper online database (http://lilab-ecust.cn/ rabbit anti-mouse IL-1β, TNF-α, IL-4 antibodies (Beijing pharmmapper/) [26], and the reverse pharmacophore Boaosen Biotechnology Co., Ltd., batch numbers bs-10859R, method was used for target prediction. bs-10802R, and bs-1740R), horseradish enzyme-labeled goat “Ulcerative colitis” was used as a key word, and the anti-rabbit IgG secondary antibody (Beijing Zhongshan OMIM (http://omim.org/) database [27] and GeneCards Jinqiao Biotechnology Co., Ltd., batch number ZB1208), (http://www.genecards.org) [28] were searched to obtain rotary microtome (Leica RM2235 rotary microtome), optical UC-related genes. In GeneCards, the genes with relevance scanning microscope (Zeiss Optical Instruments Beijing score > 12.0 were selected for sequence research. )e Uni- Branch), and microplate reader (Rui Xuan Electronic Prot database is used to correct all target names into Official Technology Co., Ltd.). Symbol (Table S1 and Table S2). 2.4. Experimental Methods

2.2. Network Analysis and Enrichment Analysis Methods. 2.4.1. Animal Modeling. According to [32], 40 rats were )e berberine targets and UC genes were input into String randomly selected and fed with a normal diet for 7 days. On (http://string-db.org/) to obtain the protein-protein the 8th day, they were given distilled water supplemented Evidence-Based Complementary and Alternative Medicine 3 with 2% DSS for 7 days and then given only distilled water binding. )en the corresponding biotinylated antibody was for 14 days. )e above process is carried out in 3 cycles, and added to form an immune complex. )en horseradish the last two cycles are 1.5% DSS/distilled water for 5 day- peroxidase-labeled streptavidin was combined with biotin, s + distilled water for 6 days. Rats with reduced foraging, and the enzyme substrate OPD was added. After the yellow decreased body weight, loose stools, mucus pus, and blood in color appeared, the stop solution was added. )e absorbance the stool represented the successful modeling. )e (A) was measured at 450 nm wavelength of the microplate remaining 20 rats were given an equal volume of normal reader, and the contents of IL-1β, TNF-α, IL-4, IL-6, IL-8, saline instead of DSS as normal group. )e changes in diet, IL-10, and IL-12/P70 were calculated according to the A body mass, and stool traits of experimental animals were value of the sample to be tested. observed and recorded.

2.4.7. Detection of IL-1β, TNF-α, and IL-4 Protein Expression 2.4.2. Animal Grouping and Intervention. )e successfully in Colon Tissue. 1 cm3 rat colon tissue was fixed in 4% modeled rats were randomly divided into 2 groups (model paraformaldehyde for 30–60 min, washed twice with PBS, group and berberine group) according to the random dehydrated at 50°C, embedded in paraffin, and sectioned number method, with 20 rats in each group, half males and (about 4 μm). All samples are deparaffinized, hydrated, half females. )e 20 rats that were not modeled represented inactivated, and processed for antigen retrieval. After the the normal group. )e berberine group was given berberine sections were stained by immunohistochemistry, they were 1.8 g/kg by gavage 7 days after the model was established. observed under a microscope. )e Motic Advanced 6.0 )e normal group and the model group were given an equal image analysis system was used for analysis. volume of normal saline. )e intervention was performed once a day for 17 consecutive days. 2.5. Statistical Analysis. SPSS19.0 statistical software was 2.4.3. Specimen Collection. After fasting for 24 hours after used for statistical analysis. )e measurement data is the last administration, the rats were anesthetized with 2% expressed as mean ± SD (x�± s). )e samples were first tested sodium pentobarbital 0.3 ml/100 g. After blood collection, for normality and homogeneity of variance. If the variance the rats were sacrificed by cervical dislocation; colon tissues was uniform, the LSD method was used for multiple were collected for index detection and stored at −20°C. comparisons between groups; if the variance was not uniform, the nonparametric rank-sum test was used for comparison, and the Kruskal-Wallis H test was used to compare 2.4.4. General Morphological Observation of Rat Colon. the total difference. )e colon was cut along the longitudinal axis and rinsed with normal saline. According to Luketal’s standard [33], the degree of colonic mucosal damage was scored. 0 points 3. Results indicate no inflammation and ulcers. 1 point indicates local 3.1. Berberine’s Potential Target Prediction and UC Genes. hyperemia but no ulcers. 2 points indicate hyperemia and A total of 211 berberine’s potential targets and 210 UC genes thickening of the intestinal wall but no ulcers. 3 points were obtained. )ere are some intersections between the indicate 1 ulcer and inflammation, about 0 to 1 cm in di- berberine target set and the UC gene set (Figure 1(a)). )e ameter. 4 points indicate 2 or more ulcers and inflamma- targets shared by the two target sets are MMP9, HRAS, tions, about 1.1 to 2 cm in diameter, but there is no adhesion STAT1, PRKCQ, MMP2, VDR, S100A9, IL2, PLA2G2A, between the intestine and peripheral organs. 5 points in- ELANE, CTSG, PPARG, MMP3, AURKA, SRC, ALB, and dicate that the ulcer extends more than 2 cm, the intestine is BRAF. thickened, and the adhesion to the surrounding organs is serious. 3.2. Berberine-UC PPI Network Analysis 2.4.5. Pathological Observation of Rat Colon. )e colon tissue was fixed with 4% paraformaldehyde, decalcified, 3.2.1. Berberine-UC PPI Network. )is network is composed dehydrated, permeabilized, and embedded in paraffin for of 191 berberine potential target nodes, 151 UC gene nodes, H&E staining. )e histological changes were observed under 17 berberine-UC target nodes, and 6742 edges. )e top 20 the microscope. targets (according to the degree) can be divided into three categories: (1) berberine target set: EGFR (140 edges), CASP3 (128 edges), and MAPK14 (113 edges); (2) UC gene set: IL-6 2.4.6. Serum IL-1β, TNF-α, IL-4, IL-6, IL-8, IL-10, and IL-12/ (185 edges), TNF (181 edges), AKT1 (173 edges), TP53 (163 P70 Contents Detection. )e IL-1β, TNF-α, IL-4, IL-6, IL-8, edges), VEGFA (157 edges), STAT3 (146 edges), IL-10 (139 IL-10, and IL-12/P70 contents were detected according to edges), TLR4 (133 edges), CXCL8 (128 edges), IL-1β (125 the instructions of the ELISA kit. Rat IL-1β, TNF-α, IL-4, IL- edges), PTGS2 (118 edges), and IL-4 (114 edges); and (3) 6, IL-8, IL-10, and IL-12/P70 monoclonal antibodies were berberine-UC target set: ALB (169 edges), MMP9 (126 coated on the ELISA plate, and the corresponding proteins edges), SRC (123 edges), IL-2 (120 edges), and HRAS (111 in the standards and samples are monoclonal antibody edges) (Figure 1(b)). 4 Evidence-Based Complementary and Alternative Medicine

Berberine UC

194 17 193

(a)

GPR35

SEC14L2 DTYMK BCAT2 SDS FKBP1B DCPS

FECH PDE4B GMPR2 NT5MGMPRGGMM HNMT DCK UCK2 PNMT METAP2 PDE4D DHODH FKBP3FFKB SORD HINT1 BHMT NMNAT1 MT-CO1 OAT ADK ADH5 RXRB TCF4 FABP6 SULT1E1 OTC FABP7 SULT2A1 HSD11B1 NR1H2 MTHFD1 DHFRD NR1H3 ADAM33GREM1GGRE STS CFD RARG DUT SHBG SPRSP MAOB TPMT ENSG00000160200 PPIA TYMS ADH1C CRABP2 DAPK1 MTAP TGM3 SIRT5 FABP3 AKR1B1 PPP1CCPP1CCC KAT2B PAPSS1 NR1I2 AKR1C3 GSR BCHE GSTP1 PRKACA POLD1 RAP2A ESRRG ESR1 HSD17B1 ACADM GSTA1 F10 IGF1R GPI NR3C1 ERBB4 MUTYH THRA CYP19A1 NOS2 MSH2 SERPINA1 PAH MLH3 SOD2 GSK3B MUC5AC PMS2 F11 RARB CA2 APCMSH6 SULT2B1 HNF4G CD4 MST1 HMOX1PPARGIGF1 TEK BUB1B RBP4 ODC1 XIAP CRP CHEK1 KIF11 SLC22A5 ALB CASP3 PIM1 TP53SRCCCND1HRAS HADH STAT1VEGFA MUC12 REL ANXA5 RIPK2 TGFBR1 MAP2K1JAK2 TGFB2 ELANE CTSG POLEP PTGS2 AKT1 SOCS1MUC1 PGR BST1 TTR PIK3CATGFB1 CDH1 PROCRR NR1H4 RAC1AC1AAC INSR PLCG2 GALNT12 DLG5 F2 NFE2L2IL1B CTNNA1NANA1A1 PDK2 CSF2 TNF RELA LCK KRAS LTA4H CXCR2EGFR SOCS3 IL4 MMP1 NFATC1PRKCQ MET MMP12 ABL1 GC IL1R1 IFNGIL2RA JAK3 AMY2A SLC22A4 CASP1 CCL2 PTK2 MMP2 BRAF MUC3A PCK1 IL2 BACE1 PTPN11 IL10 DPP4 AKR1C1 CLDN18 S100A12 ICAM1TLR4 NOD2 TLR5 TNFSF15 MMP3 CCL11 IL6R CXCL5 IRGM CASP7 CCR3 KRT7 NOD1 IL17A MME HLA-DRA AXIN2AAXI HLA-B FOLH1BPI HCK FCGR3A HLA-DRB1IL10RB VIP IL1RN CTLA4 RORA IL18RAP IL21 ADAM17MADCAM1 IL23A TRAPPC3 TNFRSF1BRIPK1 ITK BRINP3 IL23R NLRP3 IL10RA NOX1 NQO2 DUOX2 IL26 NKX2-3 HLA-A HLA-DQB1 CARD9 IL22 PTGER4S100A9DEFB4A EPHX2 MAPKAPK2 CTSS DPEP1

MICA

LTF TAP1 CTSF ABO BTNL2 LACC1 MYO9B REG4

RFK

FUT2

ZFP90 (b)

Jak-STAT Insulin signaling Leukocyte signaling pathway Insulin transendothelial pathway resistance migration Infammatory MAPK mediator signaling regulation pathway of TRP IL21 TP53 IL23R IL22 ITGA4 JAK2 channels Infammatory IL18 CCND1 NF-kappa B IL17A IL1R1 bowel signaling CXCL5 IL6R PTGS2 IL15 disease pathway RIPK1 CTLA4 RORACTNNA1PDE3B1 ERBB4 (IBD) TGFB2 ABL1 TNFRSF1B CCL5 HSPA8 CCL2 CTSK ACADM IL2RA TLR2 TEK FABP7 Non-small HLA-B CASP7 FABP6 HLA-DQB1 HIF-1 CASP3 FABP4 cell lung HLA-A HLA-DRB1 signaling cancer MAP2K1 FABP3 pathway CLDN2 HLA-DRA MAPK14 APOA2 CLDN18 IL23A MAPK10 MAPKAPK2 NOD2 NFATC1 PI3K-Akt GSK3B HCK FoxO signaling IL18RAP ITK PRKACA IL13 signaling pathway pathway SMAD3 PDPK1 RARB IL12B

TLR5 ZAP70 FGFR1 CD40LG

MMP1 LCK MET MAP3K7 PPAR Fc epsilon signaling SELE TGFBR1 CDK6 SOCS3 RI signaling

pathway CD4 RAC1 PTK2 VEGFA pathway

CCL11 PIM1 HSP90AA1 ICAM1

CXCR2 JAK3 KDR CXCL8 Ras ErbB PTPN11 RXRA signaling CCR3 TGFB1 signaling pathway IL10RB SETD7 RXRB TNFRSF1A pathway

IL10RA EGFR XIAP FOS

IGF1R PPARA SOCS1 CSF2 PCK1 PPP1CC Cytokine-cytokine Rheumatoid HSPA1L TLR4 INSR BRAF receptor arthritis REL IFNG IGF1 PRKCQ interaction CTNNB1 IL10 SOD2 HRAS CDH1 CDK2 MMP9 IL4 T cell MADCAM1 NOS3 MMP3 STAT3 Chemokine receptor HMOX1 IL2 PTGER4 EIF4E STAT1 NFKBIA signaling PTPN1 MMP2 signaling NR1H2 PLA2G2A FOXP3 NR1H3 SRC PPARG IL1B pathway pathway IL26 PLCG2 TNFSF15 IL6 TLR9 TNF Cell TNF IRF5 KRAS SMAD4 NFKB1 NOS2 RELA adhesion signaling TIMP1 AKT1 PIK3CA pathway molecules Toll-like B cell (CAMs) receptor receptor signaling signaling pathway VEGF Autoimmune pathway signaling Adipocytokine thyroid pathway signaling disease pathway

Regulation of INF−y−mediated signaling pathway (d) Positive regulation of IL−17 production Positive regulation of T−helper 2 cell differentiation Positive regulation of IL−8 biosynthetic process Negative regulation of acute inflammatory response Regulation of cell proliferation Regulation of apoptotic process Negative regulation of IL−17 production Positive regulation of chemokine production Lipopolysaccharide−mediated signaling pathway Positive regulation of NF−kB import into nucleus Positive regulation of superoxide anion generation IL−6−mediated signaling pathway Positive regulation of transcription, DNA−templated Positive regulation of IL−10 production Positive regulation of cell proliferation Positive regulation of IL−8 production Positive regulation of NO biosynthetic process Negative regulation of cell proliferation Positive regulation of IL−12 production Positive regulation of IL−1 beta secretion Positive regulation of smooth muscle cell proliferation Protein kinase B signaling GO:0042517 GO:0043536 Apoptotic process Cellular response to organic cyclic compound JAK−STAT cascade Positive regulation of monocyte chemotaxis Positive regulation of activated T cell proliferation Positive regulation of JAK−STAT cascade Negative regulation of IL−6 production Positive regulation of IL−6 production Monocyte differentiation NegLog10_P value Dendritic cell chemotaxis Positive regulation of tumor necrosis factor production 30 Response to tumor necrosis factor Leukocyte cell−cell adhesion Cellular response to mechanical stimulus 20 Positive regulation of inflammatory response Cellular response to lipopolysaccharide Positive regulation of interferon−beta production Extrinsic apoptotic signaling pathway 10 Negative regulation of interferon−gamma production Positive regulation of vasoconstriction MyD88−dependent toll−like receptor signaling pathway Positive regulation of T cell proliferation Response to cytokine Cellular response to IL−1 Count Regulation of inflammatory response Positive regulation of interferon−gamma production Negative regulation of T cell proliferation 10 GO:2001240 Positive regulation of NF−kB transcription factor activity 20 Positive regulation of B cell proliferation Positive regulation of MAP kinase activity 30 Monocyte chemotaxis Movement of cell or subcellular component Positive regulation of JNK cascade Extracellular matrix disassembly neutrophil chemotaxis Negative regulation of inflammatory response I−kB kinase/NF−kB signaling Inflammatory response Positive regulation of mononuclear cell migration Response to lipopolysaccharide Cellular response to hypoxia GO:0006919 Defense response to bacterium Positive regulation of ERK1 and ERK2 cascade Cellular response to insulin stimulus Type 2 immune response Positive regulation of IL−23 production IL−1 beta production GO:0070431 GO:0034123 Activation of MAPK activity Immune response Cellular response to tumor necrosis factor Positive regulation of chemokine biosynthetic process Positive regulation of IL−12 biosynthetic process Macrophage derived foam cell differentiation Tumor necrosis factor−mediated signaling pathway Positive regulation of I−kB kinase/NF−kB signaling Positive regulation of NO synthase biosynthetic process Chemotaxis Negative regulation of apoptotic process Regulation of establishment of endothelial barrier Negative regulation of IL−8 production 0 50 100 150 (e) Figure 1: Continued. 6 Evidence-Based Complementary and Alternative Medicine

Rheumatoid arthritis TNF signaling pathway T cell receptor signaling pathway Non−small cell lung cancer VEGF signaling pathway Adipocytokine signaling pathway Toll−like receptor signaling pathway HIF−1 signaling pathway negLog10_P value NF−kappa B signaling pathway 25 Insulin resistance 20 PPAR signaling pathway 15 Fc epsilon RI signaling pathway 10 FoxO signaling pathway Jak−STAT signaling pathway 5 Autoimmune thyroid disease B cell receptor signaling pathway Count ErbB signaling pathway Cytokine−cytokine receptor interaction 10 Chemokine signaling pathway 20

Insulin signaling pathway 30 Leukocyte transendothelial migration Inﬂammatory mediator regulation of TRP channels PI3K−Akt signaling pathway MAPK signaling pathway Ras signaling pathway Cell adhesion molecules (CAMs) Inﬂammatory bowel disease (IBD)

2.5 5.0 7.5 10.0 12.5 (f)

Figure 1: Network analysis results. (a) Venn diagram of berberine’s potential targets and UC genes. (b) Berberine-UC PPI network. (c) Clusters of berberine-UC PPI network. (d) Signaling pathway of berberine-UC PPI network. (e) Bubble chart of biological processes in cluster 1. (f) Bubble chart of signaling pathway. Red diamonds stand for signaling pathways; pink circles stand for berberine potential targets; blue circles stand for UC genes; purple circles stand for berberine-UC target. x-axis stands for fold enrichment.

3.2.2. Biological Processes of Berberine-UC PPI Network. related biological processes. Cluster 13 failed to return any )e berberine-UC PPI network was analyzed by MCODE human biological processes (Table S3). )e biological pro- and returns 14 clusters (Figure 1(c)). )e berberine targets cesses in cluster 1 were shown in Figure 1(e) as an example. and UC genes in those clusters were input into DAVID to perform GO enrichment analysis. Cluster 1 is mainly related to inflammation (inflam- 3.2.3. Signaling Pathways of Berberine-UC PPI Network. matory cells, e.g., macrophages, neutrophil proliferation, )e targets and genes in berberine-UC PPI network were etc.), inflammatory cytokines (IL-10, IL-6, IL-8, IL-1β, IL-12, input into DAVID to perform pathway enrichment analysis and IL-17), and their mediated inflammation signal path- and return sixteen signaling pathways (Figure 1(d)). )e top ways (NF-κB, JAK/STAT, and Toll signaling pathway); 10 signaling pathways are as follows: inflammatory bowel immune response (immune cells, e.g., TH1 and TH17 cells disease (IBD), rheumatoid arthritis, TNF signaling pathway, activate and proliferate, B cells activate and proliferate) and T cell receptor signaling pathway, cytokine-cytokine re- immune cytokines (various chemokines and INF-α, INF-β, ceptor interaction, Toll-like receptor signaling pathway, INF-c and their mediated immune response); and apoptosis JAK/STAT signaling pathway, FoxO signaling pathway, and proliferation. Cluster 2 is mainly related to immune HIF-1 signaling pathway, and insulin resistance response (such as positive regulation of interleukin-17, (Figure 1(f)). )e details of the signaling pathway were T-helper type 17 cellular immune response activation, T cell shown in Table S4. proliferation, T-helper type 1 immune response activation, memory T cell differentiation, and chemokine-mediated signaling pathway). Cluster 4 is mainly related to purine 3.3. General Condition of Rats. One day after the estab- nucleotide metabolism. Cluster 5 is mainly related to mitosis lishment of the model, the rats were depressed, with dull and and tetrahydrofolate metabolism. Cluster 6 is mainly related curly hair and reduced activity, and accompanied by mucus to the metabolism of steroid hormones and their signal stools, bloody stools, and decreased body weight. Compared transduction. Cluster 8 is mainly related to steroid-mediated with the normal control group, the body weight of the model signaling pathways. Cluster 9 is mainly related to oxidative group was significantly reduced, and the difference was stress. Cluster 10 is mainly related to T cell proliferation and statistically significant (P < 0.01). Compared with the model signaling pathways mediated by immune cytokines. Cluster group, the body mass of the berberine group increased 11 is mainly related to the transforming growth factor significantly, and the difference was statistically significant pathway. Clusters 3, 7, 12, and 14 did not return any UC- (P < 0.01) (Figure 2(a)). Evidence-Based Complementary and Alternative Medicine 7

250 5 # 200 ∗ ∗ 4

150 3 ∗

100 2

50 1 Body weight comparison (g) 0 0 Colonic mucosal damage index (CMDI) Control Model Berberine Model Model Model Control Control Control Berberine Berberine Berberine

Before modeling 7d aer modeling 7d aer modeling

(a) (b)

(c)

Figure 2: (a) )e body weight. (b) Eﬀect of berberine on CMDI. (c) Pathological changes of colon (compared with normal control group, ∗P < 0.05 and ∗∗P < 0.01; compared with model group, #P < 0.0 and ##P < 0.01; H&E staining, x100; A, normal group; B, model group; and C, berberine group).

3.4. Effect of Berberine on Colonic Mucosa Damage Index fibrosis in the intestinal wall; new glands can be seen around (CMDI). Compared with the normal group, the CMDI some colon ulcers, and new goblet cells and intestinal score of the model group was significantly increased mucosa can be seen in the intestinal wall (Figure 2(c)). (P < 0.05). Compared with the model group, the CMDI score of rats in the berberine group was significantly decreased (P < 0.05) (Figure 2(b)). 3.6.EffectofBerberineonSerumIL-1β,TNF-α,andIL-4Levels. Compared with the normal group, the serum levels of IL-1β and TNF-α in the model group increased significantly, and 3.5. Pathological Changes of Colon. )ere was no or rare the levels of IL-4 decreased significantly (P < 0.05). Com- inflammatory cell infiltration in the colon tissue of rats in the pared with the model group, the levels of serum IL-1β and normal group. In the model group, the mucosa of the colon TNF-α in the berberine group were significantly reduced, tissue of rats was obviously hyperemia, edema, extensive and the levels of IL-4 were significantly increased (P < 0.05) inflammatory cell infiltration, and decreased goblet cells; the (Figure 3(a)). glands around the ulcer are defective and disorderly distributed, and the intestinal wall is extensively fibrotic; the lesion reaches deep into the submucosa, muscle layer, and 3.7. Effect of Berberine on Serum IL-6, IL-8, IL-10, and IL-12/ even serosal layer; and inflammatory granuloma can be seen. P70 Levels. Compared with the normal group, the cytokines Compared with the model group, the pathological changes IL-6, IL-8, and IL-12 were significantly increased after in the colon of the berberine group were significantly im- modeling (P < 0.05), while IL-10 was significantly decreased proved; its manifestations as mild hyperemia, edema, re- (P < 0.05). )e expression and secretion of these cytokines duced inflammatory cell infiltration, and shallower are positively correlated with the degree of inflammation of inflammatory cell infiltration, mainly concentrated in the the disease, and they also show that the modeling is suc- mucosal layer and submucosa, with a small amount of cessful. After intervention, compared with the model group, 8 Evidence-Based Complementary and Alternative Medicine

400 500 ∗ ∗ 400 300

# 300 ∗ 200 # ∗ 200 # # ∗ # 100 ∗ # 100 # ∗ Level of infammatory in the serum 0 Level of infammatory in the serum 0 Model Model Model Model Model Model Model Control Control Control Control Control Control Control Berberine Berberine Berberine Berberine Berberine Berberine Berberine

IL-1β (pg/ml) IL-6 (pg/L) IL-10 (pg/L) IL-4 (pg/ml) IL-8 (pg/L) IL-12/P70 (pg/L) TNF-α (pg/ml) (a) (b) 150

IL-1β

100 ∗ # ∗ # ∗ # IL-4 50 in the colon (IM) Level of infammatory factors

0 TNF-α Model Model Model Control Control Control Berberine Berberine Berberine

IL-1β IL-4 TNF-α (c) (d)

Figure 3: (a) Eﬀect of berberine on serum IL-1, TNF-α, and IL-4 levels. (b) Eﬀect of berberine on serum IL-6, IL-8, IL-10, and IL-12/P70 levels. (c) IL-1, TNF-α, and IL-4 in colon tissue (immunohistochemistry, x200). (d) Expressions of IL-1, TNF-α, and IL-4 in colon tissue (∗compared with normal control group, P < 0.05; #compared with model group, P < 0.05; A: normal group; B: model group; and C: berberine group).

IL-6, IL-8, and IL-12 in the berberine group were signifi- 4. Discussion cantly reduced (P < 0.05), and IL-10 was significantly increased (P < 0.05). )is shows that the cytokine changes after TNF-α, IL-6, IL-1β, and COX2 are the most important treatment and the treatment is effective (Figure 3(b)). targets for UC. Berberine may indirectly act on those disease targets by acting on multiple targets such as SRC, CASP3, MAPK8, and JAK2. )e levels of TNF-α, IL-6, and IL-1β 3.8. Effect of Berberine on Colon Tissue IL-1β, TNF-α, and IL-4 reflect the disease process. Clinically, the international Levels. Compared with the normal group, the expressions of guidelines for the medical management of UC recommend IL-1β and TNF-α protein in the colon tissue of the model the use of anti-TNF monoclonal antibodies and vedolizu- group increased significantly, and the expression of IL-4 mab for the treatment of UC [34]. protein decreased significantly (P < 0.05). Compared with GO enrichment analysis showed that berberine is mainly the model group, the expressions of IL-1β and TNF-α involved in biological processes and molecular functions protein in the colon tissue of the berberine group were such as cytokine regulation, cell survival, migration, pro- significantly reduced, and the expression of IL-4 protein was liferation, and apoptosis. KEGG enrichment analysis also significantly increased (P < 0.05) (Figures 3(c) and 3(d)). showed that these targets are mainly enriched in pathways Evidence-Based Complementary and Alternative Medicine 9 related to inflammation, immunity, and tumors, such as weight of the rats in the model group were significantly JAK/STAT3, NF-κB, and TNF signaling pathways, which are reduced, the CMDI score was significantly increased, and the related to the development of ulcerative colitis. NF-κB and colon tissue showed different degrees of pathological STAT3 mainly exist in the cytoplasm. When they are damage, which was consistent with the changes in UC. subjected to appropriate external stimuli such as TNF-α, IL- Berberine can improve the general condition and body 6, and LPS, NF-κB and STAT3 are activated. )e activated weight of UC rats to varying degrees, reduce the CMDI NF-κB and STAT3 enter the nucleus and bind with DNA to score, and improve the gross morphology and pathological mediate the expression of downstream inflammatory factors damage of the colon, suggesting that berberine can signif- (TNF-α, IL-1β, and COX2) [35]. Current research shows icantly improve the general morphological changes and that UC is mainly related to the immune and intestinal flora pathological damage of UC rats. caused by multiple signaling pathways such as PI3K/AKT/ Meanwhile, after modeling, the serum and colon tissue mTOR, NF-κB, TLR4/My D88/NF-κB, MAPK, JAK/STAT, IL-1β and TNF-α levels increased, and the IL-4 level de- Wnt, and Notch signaling pathway [36, 37]. )e current creased. After the intervention of berberine, the levels of IL-1 phase I clinical study shows that berberine can significantly and TNF-α and protein expression in rat serum and colon reduce the Geboes grade of colon tissue [38]. )e mecha- tissue were significantly reduced, and the IL-4 levels were nism may be related to the inhibition of )17 and the significantly increased. )is suggests that the improvement regulation of the interaction of enteric glial cells-intestinal of berberine’s immune function may be related to the epithelial cells-immune cells [21, 39]. Berberine mainly regulation of the protein expression of cytokines such as IL- relieves colitis by improving the intestinal barrier function Iβ, TNF-α, and IL-4. and promoting anti-inflammatory and antioxidative stress responses. Its anti-inflammatory mechanism may be related 5. Conclusion to blocking the IL-6/STAT3/NF-κB signaling pathway [40–42]. In summary, berberine can interfere with UC through re- In recent years, the incidence of UC in our country has lated biological processes and signaling pathways related to been increasing year by year, and it has become a common inflammation and immunity. Berberine can significantly clinical refractory disease. )e pathogenesis of UC is mainly improve the general morphology and pathological changes related to heredity, immunity, microorganisms, and so forth; of the colon tissue of UC rats induced by DSS, and it can immune factors occupy an important position among them regulate the immune function of the body by affecting the [43]. )e mechanism is that abnormal intestinal mucosal expression levels of inflammatory cytokines and intestinal immune system leads to infiltration of a large number of mucosal immune proteins. In the future, we will deeply inflammatory cells in the intestine. )e massive release of explore the mechanism of berberine in the treatment of UC inflammatory factors causes the necrosis and shedding of so as to provide a basis for clinical application. intestinal epithelial cells and reduces the intestinal mucosal barrier function [43, 44]. SIgA is an important immuno- Data Availability modulatory protein in the intestinal tract. It acts as the first )e data used to support the findings of this study are in- line of defense to maintain the homeostasis of the intestinal cluded in the article and the supplementary information mucosa by protecting the integrity of the intestinal biological files. barrier and regulating the immune response [45]. In the course of immune response, the activation and proliferation of colonic lamina propria (LP) T lymphocytes are extremely Conflicts of Interest important for maintaining intestinal immune homeostasis )e authors declare that there are no conflicts of interest. [45, 46]. In addition, the imbalance of proinflammatory cytokines/anti-inflammatory cytokines is an important Supplementary Materials reason for the disorder of intestinal mucosal immune function [47]. )e role of proinflammatory factor IL-Iβ and Table S1: berberine potential targets. Table S2: UC genes. anti-inflammatory factor IL-4 has become increasingly Table S3: biological processes of clusters. Table S4: signaling prominent. In particular, IL-1β causes the production of pathways. 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