Uncovering the Mechanism of Curcuma in the Treatment of Ulcerative Colitis Based on Network Pharmacology, Molecular Docking Technology, and Experiment Verification

Aim The incidence of ulcerative colitis (UC) is increasing steadily in developed countries, it is plaguing nearly 1 million people in the United States and European countries, while developing countries have had a rapidly increased incidence over the past decades. Curcuma is widely used in treating malaria, UC, Crohn's disease, and colon cancer, which lead to diarrhea and bloody stool. However, the systemic mechanism of curcuma in treating UC is still unclear. Our work was supposed to expound how does curcuma alleviate UC in a comprehensive and systematic way by network pharmacology, molecular docking, and experiment verification. Methods Traditional Chinese Medicine System Pharmacology Database (TCMSP), Shanghai Chemistry & Chemical Industry Data Platform (SGST), and papers published in Chinese Network Knowledge Infrastructure (CNKI) and PubMed were used to collect the chemical constituents of curcuma based on ADME (absorption, distribution, metabolism, and excretion). And effective targets were predicted by Swiss Target Prediction to establish the curcuma-related database. The disease targets of UC were screened by GeneCards and DrugBank databases, and Wayne (Venn) analysis was carried out with curcuma targets to determine the intersection targets. AutoDock software and TCMNPAS system were used to dock the core chemical components of curcuma with key UC targets. Protein interaction (PPI) network was constructed based on the STRING database and Cytoscape software. Gene function GO analysis and KEGG pathway enrichment analysis were carried out by using Metascape database. Finally, HE staining was performed to identify the inflammatory infiltration and expression difference in TNF-α and STAT3 before and after the treatment of curcuma which was verified by immunoblotting. Results Twelve active components containing 148 target genes were selected from curcuma. Potential therapeutic targets of curcuma in the treatment of UC were acquired from 54 overlapped targets from UC and curcuma. Molecular docking was used to filter the exact 24 core proteins interacting with compounds whose docking energy is lower than −5.5 and stronger than that of 5-aminosalicylic acid (5-ASA). GO and KEGG analyses showed that these targets were highly correlated with EGFR tyrosine kinase inhibitor resistance, PI3K-Akt signaling pathway, JAK-STAT signaling pathway, MAPK signaling pathway, and inflammatory bowel disease (IBD). Experiments verified curcuma relieved pathological manifestation and decreased the expression of TNF-α and STAT3. Conclusion Curcuma relieved the colon inflammation of ulcerative colitis via inactivating TNF pathway, inflammatory bowel disease pathway, and epithelial cell signaling in Helicobacter pylori infection pathway, probably by binding to STAT3 and TNF-α.


Background
UC is characterized by abdominal pain, diarrhea, and bloody stool [1]. Globally, the annual incidence of UC is about 9-20/ 100,000 and the prevalence is 156-291/100,000 [2]. However, the exact pathogenesis is still not fully clear for the etiology is prototypically diverse. It is known that many factors are involved in the development of UC, interacting environmental, genomic, microbial, and immunological elements [3]. With the deterioration of UC, it will eventually lead to colorectal cancer. erefore, to prevent before the occurrence of UC, to avoid the complications of UC, and to prevent the recurrence of UC after recovery, a new treatment is urgently needed.
TCM is gaining its popularity in the ameliorating subhealth state and treating disease. Much more attention has been focused on the treatment of UC by Chinese herb; curcuma is one of the popular ones. In ancient China, curcuma was widely used to invigorating the circulation of blood in clinical applications; nowadays, the function of the positive regulation of inflammatory cytokines in inflammatory diseases [4] and its safety [5] attracted many more clinical trials and experimental verifications.
It is well known that TCM is guided by the theory of TCM and characteristics of being multicomponents, multitargets, and multipathways in the treatment of diseases, which meets the requirements of systematically tackling complex diseases such as colorectal cancer. Guo et al. established the model of colorectal cancer and predicted the traditional Chinese medicine components of inhibiting inflammation-induced tumorigenesis by using network pharmacology method [6]. Gupta et al. gathered curcumin's pleiotropic activities from many research studies to conclude its ability to modulate numerous signaling molecules such as proinflammatory cytokines, apoptotic proteins, cyclooxygenases, and C-reactive protein in human participants [7]. To improve the "one target and one drug" mode to "network targeting multicomponent" mode and to discover traditional Chinese medicine from the perspective of system and molecular level [8], a systemic overall approach of curcuma in the treatment of UC is still needed to verify the previous results and broaden the mechanism of curcuma in the treatment of UC.

Active Compound Screening. Traditional Chinese
Medicine System Pharmacology Database [9] (TCMSP, http://lsp.nwu.edu.cn/tcmsp.php), Shanghai Chemistry & Chemical Industry Data Platform (SGST, http://www. organchem.csdb.cn), and papers published in Chinese Network Knowledge Infrastructure (CNKI, https://www. cnki.net/) and PubMed were used to collect the chemical constituents of curcuma. We screened curcuma compounds based on absorption, distribution, metabolism, and excretion (ADME) [10], and pharmacokinetic information retrieval filters were used to retrieve bioactive compounds for further analysis under the conditions of OB ≥ 30% and DL ≥ 0.18 in TCMSP [9]. We further screen the active ingredients by their effects on the human body. However, the compounds were searched from CNKI, PubMed, and SGST without ADME parameters, so we obtained chemical formula of those components from PubChem (https:// pubchem.ncbi.nlm.nih.gov/) to finish Swiss ADME prediction [11], which was requested that OB degree was equal to HIGH and at least two terms of druglikeness were YES [12].

Screening of Possible Targets for Curcuma.
PubChem was used to search the chemical structures of the active compounds. Potential targets of curcuma were predicted by Swiss Target Prediction (STP, http://www. swisstargetprediction.) [11]. Probability was used to balance the connection between compounds and targets, which was closer to 1, and it was more connective. We screened targets by the median of probability to establish potential target database related to curcuma.

Predicting the Possible Targets of UC.
Data of UCassociated target genes were gathered from GeneCards (https://www.genecards.org/) [13] and DrugBank [14] (https://www.drugbank.ca/) with the keyword "ulcerative colitis." In addition, articles published in CNKI and PubMed about the known targets of its active compounds were counted [15]. Genes from GeneCards were provided with scores, and genes were selected as UC-related ones whose scores were above the median degree [16].

Gathering Compound-Disease Overlapped Targets.
e screened curcuma targets and UC targets were imported into Bioinformatics [17] (http://www.bioinformatics.com. cn/), and the overlapped targets of compound-disease were obtained as the potential targets for further analysis.

PPI Network of Compound-Disease Overlapped Targets.
Protein-protein interaction (PPI) network was derived based on the STRING database (https://string-db.org/), which covered almost all functional interactions between the expressed proteins [18]. Species were set as "Homo sapiens," and the target interaction information was obtained according to the results of analysis.

Gene Ontology (GO) and KEGG Pathway Enrichment
Analysis.
e biological process (BP), molecular function (MF), cell component (CC), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were carried out using Metascape system (https://metascape. org/) [19]. In this research, GO functional annotation and KEGG pathway enrichment analyses were performed using the P value less than 0.05. Target-Pathway  Network. A visual network was constructed through   2 Evidence-Based Complementary and Alternative Medicine Cytoscape software to reflect the complex relationship between active compounds, filtrated targets [17], and pathways based on KEGG pathway enrichment analysis to reflect the relationship between top pathways, included targets, and active compounds. Nodes represented the compounds, targets, and pathways, while edges indicated the interactions between pathways, targets, and components potentially included in the treatment of UC by curcuma.

Molecular
Docking. Using TCMNPAS system [20] and AutoDock [21] software, the docking energy between the overlapped proteins and chemical ingredients of curcuma was calculated. Between the component and the target by docking score value the binding activity should meet two standards: bind tighter than 5-ASA and binding energy was lower than −5.5 to further filtrate the targets related to the treatment of UC by curcuma.

Preparation of Curcuma.
e native herb was selected, and standard decoction pieces were prepared with reference to the Chinese Pharmacopoeia (2015 edition). Extract was prepared by boiling the samples in 8 times amount of water for 30 min. e procedure was repeated 3 times.
2.3.6. Groups and the Construction of the UC Model. SD rats were accepted to the laboratory for 7 days before the experiments. According to the random number table, the rats were divided into 3 groups of 4 rats each: control, model, and curcuma. Except for the control group, the UC model was prepared with 5% DSS, and the intervention was given according to the group after 7 days.

Drug Administration.
e curcuma group was administered continuously by gavage with 2 ml 0.1 g/ml curcuma suspension for 7 days. e other two groups were given saline 1.08 g/kg.

HE Staining and Western Blot.
e steps to stain the samples are as follows: xylene I and xylene II, 10 min for each; 100% alcohol I, 100% alcohol II, 95% alcohol, and 85% alcohol, 5 min for each; water washing for 20 s, hematoxylin for 7 min, water washing for 1 min, 1% hydrochloric acid alcohol for 10 s, 50°C water washing for 5 min, eosin stain for 2 min, and water washing for 10 s; 85% alcohol, 95% alcohol, 100% alcohol II, and 100% alcohol I, each for 2 min; and xylene II and xylene I, 3 min for each. Neutral balsam was added after xylene was passerillaged.
For protein extraction, tissues were placed in homogenized tubes; 5 porcelain beads, 500 μL of RIPA, and 5 μL PMSF were added to each tube. After five times' homogenization, the colon tissue was basically broken. e supernatant was extracted after centrifugation to test the protein concentration and to collocate protein solution. Equivalent amounts of protein (200 μl) were denatured at 98°C for 10 min in sample loading buffer, then separated by electrophoresis in 15% gel, and electrotransferred onto 0.45 μm polyvinylidene difluoride membranes for 60 min at 350 mA. Subsequently, the membranes were blocked in blocking buffer (0.01 M phosphate-buffered saline, 0.05% Tween-20 with 5% skim milk) at 25 ± 5°C, followed by incubation with primary antibodies against STAT3 (1 : 5000) and TNF-α (1 : 5000) at 4°C overnight. After being washed with Trisbuffered saline containing Tween-20 (TBST) for 5 min three times, the membranes were incubated with a horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. After the membranes were washed three times in TBST for 20 min each time, the bands were visualized on X-ray film using an enhanced chemiluminescence western blotting (WB) detection system. e Image Lab ™ software was used for quantitative analysis. e workflow of this study is summarized in Figure 1. We built the ingredient-target collection of curcuma and UC-related genes, respectively. e overlapping targets of curcuma and UC were subsequently identified using molecular docking. And the PPI network was constructed according to the targets. Further, the gene ontology (GO) and the Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analyses were performed and also compound-target-pathway network was executed. Next, we performed animal experiments to verify the remission of UC by admitting curcuma through HE staining and immunoblotting. Last, key pathways were analyzed to elucidate the mechanism of curcuma in the treatment of UC.

Active Compounds and Targets of Curcuma. TCMSP database, SGST, and articles published in CNKI and
PubMed were used to gather the active components and targets, and 13 potential ingredients were discovered from curcuma (Table 1). We excluded 1,7-bis(4-hydroxy-3methoxyphenyl)-1,4,6-heptadiene-4-one and determined that 12 active components out of 13 can produce platelet aggregation and other functions in the human body, through literature review [24,25]. Furthermore, 148 targets were affirmed through PubChem and Swiss Target Prediction.

Searching the Potential Disease Targets.
We obtained the curcuma-related targets from GeneCards (whose relevance score is above the median point) and the ones from DrugBank, CNKI, and PubMed (Table S3). en the searched results were overlapped with UC targets to obtain the UC-related disease protein targets ( Figure 2). Fifty-four potential targets were obtained based on the degree of correlation between curcuma and UC, and their detailed information is shown in Table 2.

Molecular Docking.
We input 54 potential genes into the PDB database (http://www.rcsb.org/) to query their PDB ID [26]. en, we downloaded the molecular structure of the 12 effective compounds of curcuma in the treatment of UC in ZINC database [27] (http://zinc.docking.org/). Molecular docking was progressed in TCMNPAS to calculate the docking score. e interaction strength between compounds and gene corresponding proteins can be expressed by docking fraction, and the lower the score is, the higher the interaction intensity is. Further, we docked the 54 potential targets with 5-ASA. As shown in Figure 3, the 24 proteins whose docking energy with curcuma compounds matched two standards, the docking energy was above that of 5-ASA and lower than −5.5, were picked for further network analysis.

Establishment of PPI Network.
e molecular docking filtered 24 core targets for curcuma in the treatment of UC.  Figure 1: e workflow of the study. Chemical constituents of curcuma were collected from TCMSP, SGST, CNKI, and PubMed based on druglikeness and oral bioavailability. e active compounds were collected further and imported into Swiss Target Prediction to get protein targets of curcuma. en, we obtained UC-related genes from GeneCards and DrugBank, and coincident genes from curcuma and UC were collected for molecular docking to filter the proteins binding with compounds stronger than 5-ASA. PPI network was carried out by using the STRING database. GO and KEGG pathway analyses were performed by Metascape, and compound-target-pathway network was executed by Cytoscape. Further experimental verification and pathway analysis were carried out to interpret the mechanism. Evidence-Based Complementary and Alternative Medicine en, the 24 genes were uploaded into the STRING database for analysis. We selected protein targets with a medium confidence score of 0.400 and the selected protein targets were plotted as an interaction network. e network of protein-protein interactions (PPI) was established through the STRING database. As shown in Figure 4, 47 nodes and 274 edges were contained in the network; in detail, the average node degree is 12.5. Nodes represented the core targets and the extended targets, edges represented the connection between the genes, and the degree value represented the association intensity.

Gene Ontology Enrichment Analysis and KEGG Pathway
Enrichment. We imported the selected potential 25 target genes into the Metascape system for GO and KEGG pathway enrichment analyses. e results revealed that the functions of these potential targets were related to many biological processes, molecular functions, cellular components, and pathways, which were of crucial importance in the development and treatment of UC. A total of 886 biological processes were enriched based on P < 0.05, such as cellular response to lipid, response to oxygen levels, and regulation of inflammatory response ( Figure 5(a)). A total of 58 molecular function GO terms were enriched ( Figure 5(b)). ese targets of molecular function mainly involved nuclear receptor activity, transcription factor activity, steroid hormone receptor activity, and many genes related to the molecular functions described above. In all, 18 cell component GO terms were enriched ( Figure 5(c)). e targets were closely related to RNA polymerase II transcription factor complex, transcription factor complex, nuclear transcription factor complex, and membrane raft, and many targets were ranked highly as potential related genes. e biological processes, molecular functions, cellular components, and pathways revealed the vital role of curcuma in the treatment of UC.
To further reveal the potential mechanism of curcuma on the effect of UC, we conducted KEGG pathway enrichment analysis on 24 targets and screened out 95 pathways based on the threshold of P < 0.05( Figure 5(d)). Numerous pathways for potential target genes were identified, such as epithelial cell signaling in Helicobacter pylori infection related to misregulation of intestinal flora. TNF signaling pathways and inflammatory bowel disease (IBD) are closely related to the inflammatory reaction process. p53 signaling pathway is included in the pathways in cancer. Moreover, MAPK signaling pathway and TGF-beta signaling pathway play a crucial role in immunological stress. In addition, we found some other pathways such as endocrine resistance, hepatitis B, serotonergic synapse, and longevity regulating pathway, which revealed that curcuma has a potential application in other related diseases.
e KEGG pathways verified that curcuma cured UC by regulating gut microbiota, inflammatory process, immunization, and inflammatory reaction.

Component-Target-Pathway Network.
To view the relationship between the components, common targets, and their corresponding pathways, a target-pathway network was constructed ( Figure 6). Twenty pathways, 47 core common targets, and 12 active compounds were connected. e network contained 88 nodes and 628 edges, in which the green colored circles represented the core common targets, the compounds colored in blue were pathways that contain the targets, and yellow nodes were effective components in curcuma. e pathways with more targets were pathways in cancer, EGFR tyrosine kinase inhibitor resistance, endocrine resistance, and HIF-1 signaling pathway, which correspond to 12,8,8, and 8 targets, respectively. e result suggested that these four pathways probably played significant therapeutic roles.

HE Staining and the Effect of Curcuma on the Expression of Key Protein in the Colon of UC Rats.
According to the HE staining (Figure 7), DSS could change the crypt structure and inflammatory infiltration in the model rats, which were characterized by the distortion and branch of crypt structures, loss of goblet cells, appearance of a large number of crypt abscesses, lymphocytes, and plasmacyte. e above results indicated that curcuma could ameliorate DSS-induced UC in terms of colon histopathological changes.
Compared with the control group, the protein expression of STAT3 and TNF-α in the colon samples of the model group was significantly increased (P < 0.01). Compared with the model group, the curcuma group can reduce the expression of STAT3 and TNF-α (P < 0.05) (Figure 8).

Discussion
UC is one of the autoimmune diseases affecting northern Europe, Canada, and Australia [28]. e occurrence and development of UC are related to commensal microflora, antigen recognition, dysregulation of immunological responses, leucocyte recruitment, and genetic factors [29]. A large number of murine experiments and clinical tests have been performed to identify the bright prospects for the treatment of UC by curcuma, and TNF-α and STAT3 are two research hotspots. It was reported that curcumin and semibionic extraction of compound turmeric can inhibit the proinflammatory signaling by STAT3 and TNF-α in  Evidence-Based Complementary and Alternative Medicine experimental colitis [30,31]. Similarly, clinical research studies verified that curcumin inhibits NF-κB expression by regulating tumor necrosis factor-α (TNF-α) in humans [5]. Further, meta-analyses identified that curcumin have the potential to induce and maintain remission in UC patients with no serious side effects [28]. e 12 found core compounds not only have anti-immune and anti-inflammation effects but also have the effect    Evidence-Based Complementary and Alternative Medicine of regulating intestinal flora. Scholars verified that the extension of UC is important for the positivity rate of H. pylori later [32]. Moreover, it was reported that curcumin downregulated the expression of tumor necrosis factor-α (TNF-α) through inhibiting NF-κB expression [32].
Meanwhile, curcumin enhanced the suppressive function of Treg cells and promoted the recovery of damaged colonic mucosa in UC [33]. Previous studies also suggested that CLRs revealed in the research can be included in many immune responses [34].    Molecular docking verified that the binding energy of 24 key targets was better than that of 5-ASA and lower than −5.5, meaning the interaction between the compounds and targets of UC can bind tighter than that of 5-ASA, indicating better therapeutic effects.  Figure 6: Component-target-pathway network of potential pathways in the treatment of UC by curcuma. e green colored circles correspond to the core common targets and the compounds calculated in blue were pathways that contain the targets, and the nodes in yellow are components in curcuma.  In order to predict the mechanism of curcuma in the treatment of UC, we analyzed the key candidate targets by performing GO enrichment results, such as biological processes, molecular functions, and cellular components. e GO terms (P < 0.05) indicated that the major hubs were significantly involved in multiple biological processes, including cellular response to lipid, cellular response to organic cyclic compound, and response to steroid hormone. Furthermore, molecular function enrichment analysis showed nuclear receptor activity, transcription factor activity, and steroid hormone receptor activity were contained in the healing process. e active targets involved were AKT1, EGFR, TNF, STAT3, and PTGS2, which mainly concentrate on the molecular processes of immunization, inflammatory reaction, gut microbiota, etc. Some of the genes and mechanisms have been verified in curcumin, while PTGS2 was proposed connecting with the treatment of UC by curcuma firstly as a gene with high possibility. Meanwhile, cellular components consist of RNA polymerase II transcription factor complex, transcription factor complex, nuclear transcription factor complex, and membrane raft, and EGFR, DPP4, ADAM17, BCL2, and CDK2 were contained in the cellular components. e verified impact of EGFR on UC by regulating epithelial barrier function could identify the reliability of this GO analysis [35].

12
Evidence-Based Complementary and Alternative Medicine e results of pathway analysis and KEGG pathway database [36] analysis showed that the potential mechanisms of curcuma in treating UC were mainly immune regulation, inflammatory reaction, intestinal flora regulation, and the signal pathways related to immunoregulation. Among them, the IBD pathway (Figure 9) was representative and verified in our research.
In the IBD pathway, antigen-presenting cells (APC) endocytosis the invading bacteria and secrete TGF-β and IL-6. en, TGF-β and IL-6 stimulate 17 activating STAT3 with RORγt and RORα for further inflammatory cytokines. e activated expression of STAT3 participates in the gene transcription and protein expression of a variety of inflammatory factors such as TNF-α and IL-1β, thus promoting the formation and persistent aggravation of inflammation [37][38][39]. Besides, STAT3, another gene symbol, participates in inflammation. TLR4, an important link in the development of the pathogenesis of UC, can nonspecifically bind to pathogen phase molecules, initiate signal transduction, and eventually lead to the release of nuclear factors [40]. However, lipopolysaccharides activate NK-κB pathway through TLR4 in intestinal epithelial cells and then induce inflammation by secreting TNF-α in Tolllike receptor signaling pathway. Curcuma may inhibit the inflammation through TLR4, TNF, STAT3, and RORct.

Conclusion
In summary, consistent with clinical experience in the long history, experimental verification, and our HE staining, as well as our WB results, curcuma has significant advantages in the treatment of UC. At the same time, the uncovered targets and pathways were excavated for a better research of how curcuma relieves UC. It has a significant value to provide theoretical basis for clinical treatment of UC and a potent evidence for further study of the mechanism of curcuma in the treatment of UC.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare that they have no conflicts of interest.

Authors' Contributions
SX L conceived and designed the study. SX L and QD L wrote the manuscript. FZ L accomplished statistical data. SX L, QD L, and FZ L gathered active components and target information. FZ L, QD L, and LS L reviewed and edited the manuscript. H C, J L, WC D, and JY S revised the article. All authors read and approved the manuscript.