Identification of Bioactive Compounds and Potential Mechanisms of Fuzi in the Treatment of Ulcerative Colitis by Integrating Network Pharmacology and Experimental Validation

Abstract

Ulcerative colitis (UC) is a chronic inflammatory bowel disease without efficient treatment. Fuzi has anti-inflammatory and immunomodulatory properties. However, the bioactive compounds and mechanisms of fuzi in the treatment of UC are not completely understood. The active components of fuzi were retrieved from Traditional Chinese Medicine Database System Pharmacology and Analysis Platform; PharmMapper was used to predict the targets of the active components of fuzi; UC-related disease targets were obtained from Online Mendelian Inheritance in Man and Genecards databases, and Venny 2.1 was used to obtain common targets; Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analyses were performed on the common targets using R 4.0.2. STRING and Cytoscape 3.9.0 was used to construct a protein–protein interaction (PPI) network for the intersection targets. We then determined the role of the candidate molecule from fuzi, Higenamine (Hig), in a mouse model of dextran sulfate sodium (DSS)-induced colitis. In total, 21 active components and 420 corresponding targets of fuzi were obtained, of which 224 common targets were identified by intersecting with UC-related targets. The GO, KEGG, and PPI results suggested that fuzi and Hig may target RAC-alpha serine/threonine-protein kinase (AKT) to regulate the phosphoinositide-3-kinase (PI3K)/AKT pathway in UC. Animal experiments have shown that Hig treatment greatly reduced DSS-induced colitis, as measured by the disease activity index score, colonic inflammation, and intestinal barrier integrity. Mechanistically, Hig downregulated the DSS-induced PI3K–AKT signaling pathway by inhibiting AKT phosphorylation. Altogether, Hig alleviated DSS-induced colitis in mice, possibly by inhibiting colon inflammation and improving the intestinal barrier by regulating the PI3K–AKT signaling pathway. The active component Hig from fuzi is likely to play a role in the treatment of UC.

INTRODUCTION

Ulcerative colitis (UC) is a chronic and highly recurrent inflammatory bowel disease characterized by persistent mucosal inflammation extending from the rectum to the more proximal colon, leading to mucopurulent and bloody stools, diarrhea, and abdominal pain.¹⁾ The global incidence and prevalence of UC has increased drastically in recent decades. Additionally, UC is a risk factor for cancer. As high as 13.91% of UC patients eventually develop colorectal cancer within 30 years of diagnosis.²⁾ Countries with the highest incidence of UC are Canada, Australia, and those in northern Europe, and there has been a recent increase in attention in Asia. Multiple factors including genetic susceptibility, epithelial barrier defects, dysregulated immune responses, and environmental factors contribute to the pathogenesis of UC.³⁾ In addition, we previously found that neutrophil infiltration is involved in UC.⁴⁾

Currently, there is no effective treatment for UC. The recommended drugs for UC are salicylic acid preparations, such as mesalazine (5-ASA), glucocorticoids, immunosuppressants, and biological agents. However, long-term use of these drugs has severe side effects including hypertension, diabetes, osteoporosis, and liver damage.⁵⁾ Therefore, the development of more effective therapies for UC is of great interest. Fuzi, a traditional Chinese medicine, is well known for its anti-inflammatory and immunomodulatory effects and is used to treat tumors and inflammatory diseases.⁶⁾ Fuzi is widely used to treat Spleen-Yang deficiency and heat-dampness syndrome in intermingled cold and heat syndromes of UC.^7,8) It has been demonstrated that fuzi reduces dextran sulfate sodium (DSS)-induced colitis by inhibiting the mitogen-activated protein kinase (MAPK)/nuclear factor-kappaB (NF-κB)/signal transducer and activator of transcription 3 (STAT3) signaling pathways.⁹⁾ However, it is not entirely clear which active component of fuzi plays a role in treating UC. Through network pharmacological analysis, we found that higenamine (Hig) in fuzi might be an important component in the treatment of UC. Hig belongs to the class of protoberberines extracted from Fuzi, Nelumbinis Plumula, and other plants.¹⁰⁾ Research indicated that Hig has antioxidant, anti-inflammatory, and anti-apoptotic properties.^11,12) Hig was also reported to reduce intestinal damage by inducing heme oxygenase-1 protein expression and inhibiting the expression of inflammatory factors in an intestinal ischemia-reperfusion injury model.¹³⁾

Therefore, we aimed to identify the effective components of fuzi and its therapeutic effect in UC. We used relevant databases to search for effective components and potential mechanisms of fuzi in the treatment of UC. We speculated that Hig may be an essential component of fuzi. We first performed protein–protein interaction (PPI) network construction and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of the possible targets of Hig. We speculate that Hig may affect UC by regulating the phosphoinositide-3-kinase (PI3K)-threonine-protein kinase (AKT) pathway. Next, we established a mouse model of DSS-induced colitis. We evaluated the therapeutic effect of Hig on UC by monitoring the disease activity index (DAI), histopathological changes, inflammation levels, and intestinal barrier integrity. Finally, we demonstrated that Hig can improve DSS-induced colitis by reducing the production of inflammatory cytokines and chemokines, neutrophil recruitment to the mucosa, and improving the intestinal barrier.

MATERIALS AND METHODS

Fuzi Active Components Database Establishment

We retrieved and collected the composition of fuzi¹⁴⁾ from China’s Traditional Chinese Medicine Database System Pharmacology and Analysis Platform (TCMSP, https://tcmspw.com/index.php), we retrieve and collect the composition of fuzi.¹⁴⁾ Based on the aforementioned collection of components, 21 active components were obtained using oral bioavailability (OB)≥30% and drug-likeness (DL)≥0.18 as screening conditions, and downloaded as mol2 files for the preparation of molecular docking.

Target Prediction

Fuzi-related target genes were collected from PharmMapper (http://www.lilab-ecust.cn/pharmmapper/).¹⁵⁾ These fuzi-related target names were calibrated to standardized names using the UniProt database (https://www.uniprot.org/).¹⁶⁾ UC-related target genes were collected from 2 databases: GeneCards (https://www.genecards.org/) and Online Mendelian Inheritance in Man (OMIM, https://omim.org/search/advanced/geneMap).¹⁷⁾ Potential target genes of fuzi or Hig therapy for UC were acquired using the Venny 2.1 (https://bioinfogp.cnb.csic.es/tools/venny/) intersection.

Active Component-Target Network Construction

The potential active components and matching targets of fuzi were introduced into Cytoscape 3.9.0 to construct the fuzi component–target network. In the component-target network, each component or target is represented by a node, and the relationship between the component and target is represented by a connecting line.

PPI Network Construction

We constructed a PPI network map to predict the co-expression, fusion, neighbor, and colocalization of potential target genes with predicted gene interactions. The potential targets of the retrieved compounds and disease targets were intersected, and overlapping targets were selected and entered into the STRING database (https://string-db.org/). The specific settings of the system are as follows: select “Homo sapiens” in the column of organisms, select “evidence” in the column of meaning of the network edge, and set the confidence level to highest confidence (0.900), hide disconnected nodes in the network, and the collected data were imported into Cytoscape 3.9.0 and used for networking. The CytoNCA plugin in the Cytoscape software was utilized to calculate the betweenness centrality of nodes, which can provide an in-depth analysis of the attributes of nodes in the interactive network. Higher quantitative values of the parameters indicate greater significance of the nodes in the network.

Functional Enrichment Analysis of Fuzi or Hig Therapeutic Targets For UC

R software (R 4.0.2 for Windows) was used to perform GO and KEGG enrichment analyses of potential targets of fuzi or Hig in the intervention of UC. The GO analysis was divided into molecular function (MF), cellular component (CC), and biological process (BP). The data were saved and analyzed visually using R software.

Molecular Docking of Active Ingredients to Key Target Genes

CB-Dock online molecular docking (http://cao.labshare.cn/cb-dock//) was used to explore the active ingredients and RAC-alpha serine/AKT (AKT1) docking.¹⁸⁾ A lower Vina score indicates a more stable ligand binding to the receptor and is used to preliminarily evaluate the binding activity of the compound to the target.

Animal Experiments

This project passed the Experimental Animal Ethics Committee of Southern Medical University (Approval Number: LAEC-2021-201). Male C57BL/6 mice (20–25 g, 8 weeks old) were obtained from the Experimental Animal Center of Guangdong Province (Guangzhou, China). Animals were housed at 24 ± 1°C and humidity of 50–70% in a specific pathogen-free environment with a 12 h light/dark cycle and free access to a standard laboratory diet and water. The experimental protocols were approved by the Institutional Animal Care and Use Committee of Southern Medical University (Guangzhou). For the experiments, 25 mice were randomly divided into 5 groups (5 mice per group): normal, 3.5% DSS, 3.5% DSS + 5-ASA, 3.5% DSS + 5 mg/kg Hig, and 3.5% DSS + 10 mg/kg Hig. Mice in the normal and 3.5% DSS groups were administered dimethyl sulfoxide (DMSO) intraperitoneally for 7 d, and mice in the 3.5% DSS + 5-ASA group were administered 5-ASA (200 mg/kg/d, dissolved in phosphate-buffered saline (PBS)) by gavage for 7 d, while those in the Hig-treated groups were intraperitoneally injected with Hig (dissolved in DMSO) for 7 d. Mice in the normal group received 3.5% DSS in drinking water. On Day 7, the mice were anesthetized by an intraperitoneal injection of 1.5% pentobarbital sodium and sacrificed for subsequent experiments.

Ethics Approval and Consent to Participate

This experiment was approved by the Experimental Animal Ethics Committee of Southern Medical University (Approval Number: LAEC-2021-201). In addition, mice were kept to the highest standards for humane use of animals. Mice were not subjected to any unnecessary, painful, or scary manipulation. Before the mice were sacrificed, the mice were anesthetized to avoid pain and suffering.

Drugs and Reagents

DSS was purchased from MP Biomedicals (Los Angeles, CA, U.S.A.). Hig was purchased from GLPBIO (Montclair, CA, U.S.A.). Mesalamine was purchased from APExBIO (Houston, TX, U.S.A.). Primary antibodies against AKT and phospho-AKT (Ser473) were purchased from Cell Signaling Technology (Danvers, MA, U.S.A.), and secondary antibodies were purchased from Fude Biological Technology Co., Ltd. (Hangzhou, China). Primary antibodies against zonula occludens-1 (ZO-1) and occludin were purchased from Signalway Antibody (Greenbelt, MD, U.S.A.). The antibody against lymphocyte antigen 6 complex locus G (Ly6g) was purchased from ServiceBio (Wuhan, China). Goat anti-rabbit immunoglobulin G and horseradish peroxidase (HRP) secondary antibodies were purchased from HUABIO (Hangzhou, China).

DAI

During the intervention period, the mice were weighed daily, and fecal traits and hematochezia were recorded. The DAIscore was calculated as the sum of the body weight loss, stool consistency, and hematochezia scores, as shown in Table 1. For body weight loss: 0, <1%; 1, 1–5%; 2, 5–10%; 3, 10–20%; and 4, >20% weight loss. For stool consistency: 0, normal feces; 2, mushy stool; and 4, watery stool. For hematochezia: 0, no bleeding; 2, positive occult blood; and 4, gross bleeding.

Table 1. Scoring of Disease Activity Index

Score	Weight loss	Stool consistency	Hematochezia
0	<1%	Normal	No bleeding
1	1–5%
2	5–10%	Loose	Positive occult blood
3	10–20%
4	>20%	Diarrhea	Gross bleeding

Histopathology and PAS Staining

The distal colon (approximately 0.5 cm) of mice was fixed in 10% formalin, embedded in paraffin, sectioned on a microtome, and stained with hematoxylin–eosin (H&E) or Alcian blue-periodic acid-Schiff (PAS). Histological scores were calculated according to epithelial damage, the degree of crypt injury, and inflammatory cell infiltration in the colonic mucosa. The scoring system was based on a previously described scoring system.¹⁹⁾ PAS staining score was rated as excellent to poor (0–10) based on the number of goblet cells secreting mucin in the intestinal epithelium.²⁰⁾

Immunohistochemistry and Immunofluorescence Staining

Paraffin-embedded colon tissue samples were deparaffinized, dehydrated, and treated with a citrate buffer for antigen retrieval. Endogenous peroxidase activity was blocked with 3% H₂O₂ for 15 min, followed by incubation with goat serum for 30 min. The sections were subsequently incubated with the primary antibody overnight at 4°C. After washing thrice with PBS, the slides were incubated with HRP-labeled goat anti-rabbit secondary antibody for 30 min at room temperature. Sections were developed using 3,3′-diaminobenzidine tetrahydrochloride and counterstained with hematoxylin. Images were acquired using a light microscope. For immunofluorescence staining, a Leica DM4000 (Germany) was used. ImageJ (Bethesda, MD, U.S.A.) was used for the quantitative analysis of fluorescence intensity.

Western Blot

Total protein was extracted from the colon of lysates containing phosphatase and protease inhibitors. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10–12%) was used to separate proteins of different molecular weights, and the proteins were transferred to polyvinylidene fluoride membranes. Then, the membranes were blocked with 5% skim milk for 120 min at room temperature, followed by incubation with the primary antibody overnight at 4°C and with the corresponding secondary antibody for 2 h at room temperature. Western blotting was performed using an ECL chemiluminescence kit (Beyotime, China).

Enzyme-Linked Immunosorbent Assay (ELISA)

Colonic chemokine (C-X-C motif) ligand 1 (CXCL1), CXCL2, and myeloperoxidase (MPO) levels were measured using commercially available ELISA kits (CUSABIO, Wuhan, China), and colonic interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) levels were measured using commercially available ELISA kits (Excell, Shanghai, China). The optical density of the colorimetric reaction was measured using a 450 nm laser with a flat microplate reader.

Statistical Analysis

IBM SPSS 23.0 software was used for statistical analysis. GraphPad Prism 7 software was used for the graphic design. One-way ANOVA was used for statistical analysis among multiple groups. All data are presented as mean ± standard deviation. p < 0.05 was considered statistically significant.

RESULTS

Fuzi Active Components Database Establishment and Active Component-Target Network Construction

Active components of fuzi were retrieved from TCMSP, and OB≥30% and DL≥0.18 were selected as screening conditions. Twenty-one active components were identified (Table 2). The possible targets of the active components of fuzi were screened using the PharmMapper database, and 420 targets were identified. An active component-target network was constructed, as shown in Fig. 1.

Table 2. Basic Information of Active Components in Fuzi

No.	Molecule ID	Molecule name	MW	OB (%)	DL
1	MOL002211	11,14-Eicosadienoic acid	308.56	39.99	0.2
2	MOL002388	Delphin_qt	303.26	57.76	0.28
3	MOL002392	Deltoin	328.39	46.69	0.37
4	MOL002393	Demethyldelavaine A	700.91	34.52	0.18
5	MOL002394	Demethyldelavaine B	700.91	34.52	0.18
6	MOL002395	Deoxyandrographolide	334.5	56.3	0.31
7	MOL002397	Karakoline	377.58	51.73	0.73
8	MOL002398	Karanjin	292.3	69.56	0.34
9	MOL002401	Neokadsuranic acid B	452.74	43.1	0.85
10	MOL002406	2,7-Dideacetyl-2,7-dibenzoyl-taxayunnanine F	776.9	39.43	0.38
11	MOL002410	Benzoylnapelline	463.67	34.06	0.53
12	MOL002415	6-Demethyldesoline	453.64	51.87	0.66
13	MOL002416	Deoxyaconitine	629.82	30.96	0.24
14	MOL002419	Higenamine	271.34	82.54	0.21
15	MOL002421	Ignavine	449.59	84.08	0.25
16	MOL002422	Isotalatizidine	407.61	50.82	0.73
17	MOL002423	Jesaconitine	675.85	33.41	0.19
18	MOL002433	(3R,8S,9R,10R,13R,14S,17R)-3-Hydroxy-4,4,9,13,14-pentamethyl-17-[(E,2R)-6-methyl-7-[(2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-[[(2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxymethyl]oxan-2-yl]oxyhept-5-en-2-yl]-1,2,3,7,8,10,12,15,16,17-decahydr	781.1	41.52	0.22
19	MOL002434	Carnosifloside I_qt	456.78	38.16	0.8
20	MOL000359	Sitosterol	414.79	36.91	0.75
21	MOL000538	Hypaconitine	615.79	31.39	0.26

Fig. 1. A Fuzi Active Component-Target Network

Potential Target Genes and the PPI Network Map of Fuzi Therapy for UC

The target genes associated with UC were screened using the GeneCards and OMIM databases, with a total of 5139 genes. Using Venny 2.1 software, 224 potential targets of fuzi were identified (Fig. 2A). Sequentially, 224 target genes were imported into the STRING database to obtain the PPI network map (Fig. 2B). The top 7 genes in the PPI network were heat shock protein HSP 90-alpha (HSP90AA1), AKT1, proto-oncogene tyrosine-protein kinase Src (SRC), retinoic acid receptor RXR-alpha (RXRA), thymidylate synthase (TYMS), estrogen receptor 1 (ESR1), and MAPK1.

Fig. 2. (A) Venn Diagram of Fuzi Active Component Targets and UC Disease Targets and (B) PPI Network of Key Targets of Fuzi in the Treatment of UC

GO and KEGG Pathway Enrichment Analyses of the Targets of Fuzi in the Treatment of UC

GO and KEGG pathway enrichment analyses were carried out on the 224 overlapping targets to evaluate the pharmacological mechanism of fuzi against UC. A total of 2550 biological functions were obtained from GO enrichment analysis (p < 0.05). The top 4 BP, CC, and MF enrichment results are shown in Fig. 3A. BP includes responses to xenobiotic stimuli and lipopolysaccharides (LPSs). CC included the vesicle lumen, membrane raft, cytoplasmic vesicle lumen, and secretory granule lumen. Furthermore, the enriched MF included protein serine/threonine/tyrosine kinase activity, protein tyrosine kinase activity, ligand-activated transcription factor activity, and nuclear receptor activity.

Fig. 3. (A) GO Analysis of Key Targets of Fuzi in UC Treatment and (B) KEGG Pathway Enrichment Analysis of Key Targets of Fuzi in UC Treatment

To gain a profound understanding of the mechanism of fuzi in the treatment of UC, 165 signaling pathways were obtained from KEGG pathway enrichment analysis (p < 0.05). The top 15 KEGG enrichment results are shown in Fig. 3B. We found that the PI3K-AKT, MAPK, and Ras signaling pathways may play an essential role in the protection of fuzi in UC. Combined with the results of the PPI network and GO enrichment analysis, we speculated that fuzi most likely plays a vital role in the treatment of UC by regulating the PI3K/AKT pathway via acting on AKT1.

Potential Target Genes and the PPI Network Map of Hig Therapy for UC

We selected Hig, the active component of Fuzi, for subsequent analysis. Figure 4A shows the structure of Hig. A total of 162 potential targets of Hig for the treatment of UC were identified via the Venny 2.1 software (Fig. 4B). These 162 target genes were successively imported into the STRING database to obtain a PPI network diagram (Fig. 4C). The top 7 genes in the PPI network were SRC, HSP90AA1, RXRA, MAPK1, ESR1, TYMS, and AKT1, suggesting that Hig may improve UC by regulating these genes.

Fig. 4. (A) The Structure of Hig; (B) Venn Diagram of Hig Targets and UC Disease Targets; and (C) PPI Network of Key Targets of Hig in the Treatment of UC

GO and KEGG Pathway Enrichment Analysis of the Targets of Hig in the Treatment of UC

Potential BP, CC, and MF of 162 target genes of Hig in the treatment of UC were identified by GO enrichment analysis. The top 4 BP, CC, and MF enrichment results are shown in Fig. 5A. The response to molecules of bacterial origin and LPS in the BP, the membrane microdomain and membrane raft in the CC, and the protein serine/threonine/tyrosine kinase activity in the MF all serve critical biological functions.

Fig. 5. (A) GO Analysis of Key Targets of Hig in UC Treatment and (B) KEGG Pathway Enrichment Analysis of Key Targets of Hig in UC Treatment

Using KEGG enrichment analysis, 150 target gene enrichment pathways were identified (p < 0.05). The top 15 KEGG signaling pathways with the highest enrichment are shown in Fig. 5B. Combined with the results of the PPI network map and GO enrichment analysis, we hypothesized that Hig might treat UC through the PI3K-AKT pathway by acting on AKT1.

Validation of Hub Genes AKT1 Affinity to Hig through Molecular Docking

CB-Dock (http://cao.labshare.cn/cb-dock//) was used to verify the interactions between Hig and AKT1. The Vina score was –6.4, showing that Hig may easily access and stably bind the active pockets of AKT1 (Fig. 6).

Fig. 6. Stereoview of the Binding Mode for Hig with AKT1

Effects of Hig on the DSS-Induced Colitis Mice

DSS treatment results in weight loss, diarrhea, hematochezia in mice, as shown by the increase of DAI scores (Figs. 7A, 7B). The colon length of the 3.5% DSS group was shorter than that of the normal group (Figs. 7C, 7D). Hig (10 mg/kg) and 5-ASA significantly reduced the DAI score, as shown by improved weight loss, diarrhea, and bloody stools (Figs. 7A, 7B). In addition, Hig (10 mg/kg) and 5-ASA ameliorated DSS-induced colon shortening (Figs. 7C, 7D). In conclusion, Hig alleviated the pathological signs of the colon in DSS-induced colitis.

Fig. 7. Effects of Hig on the Pathological Signs of the DSS-Induced Colitis Mice

(A) Daily body weight curve of mice; (B) scoring curve of mouse disease activity index; (C) representative images of the colon; and (D) colon length. *p < 0.05 vs. 3.5% DSS; **p < 0.01 vs. 3.5% DSS; ^#p < 0.05 vs. normal group; ^##p < 0.01 vs. normal group.

Effect of Hig on Colonic Histopathology in DSS-Induced Colitis Mice

We performed H&E staining to determine the histopathological changes after DSS treatment (Fig. 8A). In the 3.5% DSS group, the integrity of the colonic mucosa and crypts was obviously absent, and the inflammatory cells were seriously infiltrated. Both Hig (10 mg/kg) and 5-ASA treatment improved DSS-induced colonic mucosal injury, as evidenced by normal gland arrangement and less infiltration of inflammatory cells. Furthermore, Hig (10 mg/kg) and 5-ASA significantly decreased the colonic histology scores in DSS-treated mice (p < 0.05) (Fig. 8B). These results indicated that Hig ameliorated colon injury in DSS-treated mice.

Fig. 8. Effect of Hig on Colonic Histopathology in DSS-Induced Colitis Mice

(A) Representative images of colon H&E staining (×50, ×200) and (B) colon histology score. *p < 0.05 vs. 3.5% DSS; **p < 0.01 vs. 3.5% DSS; ^#p < 0.05 vs. normal group; ^##p < 0.01 vs. normal group.

Effects of Hig on Levels of Inflammatory Cytokines, Chemokines, and Neutrophil Infiltration in DSS-Treated Mice

ELISA was used to detect inflammatory cytokines and chemokines in colon extracts (Figs. 9A–9D). Our results showed that the levels of TNF-α, IL-6, CXCL1, and CXCL2 were upregulated in the DSS-treated colon (3.5% DSS group) compared to the normal group. Interestingly, the elevation in inflammatory cytokines and chemokines was blocked by Hig and 5-ASA.

Fig. 9. Effects of Hig on Levels of Inflammatory Cytokines, Chemokines, and Neutrophil Infiltration in Mice with DSS-Induced Colitis

(A) Expression of IL-6 in colon; (B) expression of TNF-α in colon; (C) CXCL1 expression in colon; (D) CXCL2 expression in colon; (E) MPO expression in colon; (F) colon Ly6G+ neutrophil immunofluorescence staining; and (G) representative images of Ly6G+ neutrophil immunofluorescence staining of colon (×400). *p < 0.05 vs. 3.5% DSS; **p < 0.01 vs. 3.5% DSS; ^#p < 0.05 vs. normal group; ^##p < 0.01 vs. normal group.

MPO expression was detected using ELISA to evaluate the infiltration of neutrophils in the colon (Fig. 9E). Compared with the 3.5% DSS group, Hig and 5-ASA reduced the expression of MPO. Immunofluorescence staining was performed to detect the expression and distribution of Ly6G+ neutrophils in the colon. The number of infiltrated neutrophils was the highest in the 3.5% DSS group, and neutrophils were found in the deep mucosa. In contrast, Hig and 5-ASA significantly abated neutrophil infiltration (Figs. 9F–9G). These results suggest that Hig ameliorates DSS-induced colitis by alleviating the secretion of inflammatory cytokines, chemokines, and neutrophil infiltration.

Effect of Hig on Tight Junction in DSS-Induced Colitis Mice

To evaluate whether Hig has any impact on intestinal tight junctions, we detected the expression levels of ZO-1 and occludin in the colon of mice by Western blotting and immunohistochemical staining. Western blotting results showed that the expression of occludin and ZO-1 was downregulated in DSS-treated mice (3.5% DSS group) than in the normal group. However, Hig (10 mg/kg) treatment significantly improved the expression of ZO-1 and occludin (Figs. 10A–10D). IHC staining also showed that the expression of ZO-1 and occludin restored to normal levels in the Hig-treated group (Fig. 10E). Therefore, Hig is capable of preserving the tight junctions of the colon in DSS-treated mice.

Fig. 10. Effect of Hig on Tight Junction in DSS-Induced Colitis Mice

(A) Representative image of the ZO-1 protein level in the colon tissues; (B) the ZO-1 protein level in the colon tissues; (C) representative image of the occludin protein level in the colon tissues; (D) the occludin protein level in the colon tissues; and (E) immunohistochemical staining of ZO-1 and occludin in colon of mice (×200). *p < 0.05 vs. 3.5% DSS; **p < 0.01 vs. 3.5% DSS; ^#p < 0.05 vs. normal group; ^##p < 0.01 vs. normal group.

Effect of Hig on Intestinal Barrier Integrity in DSS-Treated Mice

To evaluate colonic barrier integrity, PAS was used to determine the number and mucin secretion by the goblet cells (Figs. 11A, 11B). Compared to the normal group, the number of goblet cells in the 3.5% DSS group was significantly reduced (p < 0.01). However, the number of goblet cells and mucin secretion was significantly raised after treatment with 10 mg/kg Hig (p < 0.01). In conclusion, Hig blocked DSS-induced goblet cell depletion.

Fig. 11. Effect of Hig on Mucus Barrier in DSS-Induced Colitis Mice

(A) PAS staining of colon (×50, ×200) and (B) the PAS staining score. *p < 0.05 vs. 3.5% DSS; **p < 0.01 vs. 3.5% DSS; ^#p < 0.05 vs. normal group; ^##p < 0.01 vs. normal group.

Effects of Hig on PI3K–AKT Signaling Pathway in the DSS-Induced Colitis Mice

As shown in Figs. 12A, 12B, Western blot results showed that phosphorylation of AKT was significantly elevated in the 3.5% DSS group compared with that in the normal group, suggesting that activation of AKT was involved in DSS-induced colitis. Importantly, treatment with Hig (10 mg/kg) drastically inhibited AKT phosphorylation. Thus, our results demonstrated that Hig may improve DSS-induced colitis by targeting the PI3K-AKT signaling pathway.

Fig. 12. Effects of Hig on PI3K/AKT Signaling Pathway in DSS-Induced Colitis Mice

(A) Representative image of the p-AKT protein level in the colon tissues and (B) the p-AKT protein level in the colon tissues. *p < 0.05 vs. 3.5% DSS; **p < 0.01 vs. 3.5% DSS; ^#p < 0.05 vs. normal group; ^##p < 0.01 vs. normal group.

DISCUSSION

UC is a refractory disease that requires long-lasting treatment, and novel drugs are urgently needed. Fuzi, a traditional Chinese medicine, plays an important role in UC treatment. It has been reported that the Yiyi Fuzi Baijiang decoction has an anti-inflammatory effect by regulating the balance of T-helper type 17/T-regulatory cells.²¹⁾ Furthermore, fuzi has a significant anti-inflammatory effect on DSS-induced colitis via inhibition of the MAPK/NF-κB/STAT3 signaling pathway.⁹⁾ To investigate the possible mechanism of fuzi in the treatment of UC, we first performed network pharmacological analysis. Through enrichment analysis, we identified the PI3K-AKT, MAPK, and Ras signaling pathways as the main pathways by which the active fuzi components exert their effects in the treatment of UC. The findings of the GO enrichment study suggested that the active components of fuzi may exhibit effects in the treatment of UC in response to xenobiotic stimulus and LPS. Fuzi may regulate HSP90AA1, AKT1, SRC, and other molecules to relieve colitis. Therefore, we speculated that the active components of fuzi that exert its therapeutic effect on UC are most likely by acting on the AKT1 molecule and regulating the PI3K/AKT pathway under the application of xenobiotic stimulus and LPS.

Traditional Chinese medicine and its extracts have great potential in the treatment of UC.^22–25) Hig is a benzylisoquinoline alkaloid extracted from fuzi that has various pharmacological effects, including anti-inflammatory, antioxidant, anti-apoptotic, lipid-lowering, anti-fibrotic, and antiplatelet activities.²⁶⁾ Hig exhibits a strong anti-inflammatory effect. Wei et al. found that Hig inhibited the secretion of IL-6 and IL-8 as well as the phosphorylation of NF-κB in allergic rhinitis mice.²⁷⁾ Yang et al. found that Hig significantly inhibited the production of TNF-α, IL-6, reactive oxygen species, nitric oxide, and prostaglandin E2 in LPS-activated mouse microglial cells, possibly by suppressing the NF-κB signaling pathway.²⁸⁾ To determine whether Hig can treat UC, we first performed network pharmacological analysis. Similar to the results of the network pharmacology analysis of fuzi, we found that Hig exerts its therapeutic effect on UC by acting on AKT1 and regulating the PI3K/AKT pathway under the influence of LPS and molecules of bacterial origin. For further verification, we used Hig intervention in mice with colitis. By analyzing daily recorded body weight and stool characteristics, colon length, and H&E-stained slices of the terminal colon, we found that Hig ameliorated weight loss, hematochezia, and other symptoms in DSS-treated mice. Hig also alleviates colonic shortening in mice with colitis. H&E staining showed that the colonic mucosa of mice was less damaged, and inflammatory cell infiltration was reduced after Hig treatment. Interestingly, Shao et al. also reported that Hig improves DSS-induced UC in mice through the galectin-3/TLR4/NF-κB pathway recently.²⁹⁾ Based on the above findings, we conclude that Hig can improve DSS-induced colitis in mice.

The intestinal barrier is mainly composed of a mucus layer secreted by goblet cells, antimicrobial peptides, secretory immunoglobulin A, epithelial cells, microbiota, and the mucosal immune system.³⁰⁾ Numerous studies have shown that damage to the intestinal barrier plays an indispensable role in UC occurrence and development. UC is a polymicrobial infectious disease characterized by sustained breakdown of the mucus barrier.^31,32) Bacterial products in the gut, such as LPS, inflammatory factors, and bacterial flagellin A, can activate NF-κB, thereby promoting goblet cells to oversecrete MUC2. Long-term oversecretion eventually leads to goblet cell function depletion and mucosal barrier destruction.³³⁾ In our study, PAS staining was used to stain goblet cells to observe the mucous barrier in the colons of mice. We found that the number of goblet cells was significantly reduced in DSS-treated mice. The number of colonic goblet cells was normal in DSS-induced colitis mice treated with Hig, indicating a significant improvement in the mucosal barrier. There are many tight junction proteins in intestinal epithelial cells, including transmembrane proteins, peripheral membrane proteins, and cytoskeletal proteins.³⁴⁾ Occludin is a transmembrane protein that regulates the permeability of tight junctions in epithelial cells and maintains their polarity. As a peripheral membrane protein, ZO-1 is mainly responsible for connecting claudins, occludins, and the cytoskeleton to maintain the integrity of tight junctions. Under inflammatory conditions, the expression of ZO-1 and occludin decreases, thereby increasing intestinal epithelial permeability.³⁵⁾ The decrease in tight junction proteins in UC patients leads to damage of the intestinal barrier, which increases the permeability of the intestinal epithelium. Damage to the intestinal barrier is an important pathological mechanism of UC.³⁶⁾ In our experiment, Western blot and immunohistochemical techniques were used to detect the expression levels and locations of the tight junction proteins ZO-1 and occludin. Compared to normal mice, the expression of ZO-1 and occludin in DSS-treated mice decreased drastically. Treatment with Hig, particularly at higher doses, reversed the expression of ZO-1 and occludin in DSS-induced colitis. In conclusion, Hig showed a significant protective effect on the intestinal barrier damaged by DSS, as indicated by the improvement of the mucus barrier as well as the tight junctions of epithelial cells.

During colitis, natural killer T cells strongly express TNF-α, which stimulates the epithelial cells to express CXCL1, CXCL2, and CXCL3. These chemokines induce migration and infiltration of neutrophils into the intestine.³⁷⁾ Patients with UC without mucosal neutrophils had a 60% to 70% relative reduction in the risk of recurrence. Complete elimination of neutrophil inflammation should be defined as a therapeutic target for disease remission in patients with UC.³⁸⁾ Therefore, we examined the expression levels of inflammatory cytokines and chemokines, and the degree of neutrophil infiltration to detect the severity of colitis and to evaluate the effect of drug therapy. In addition, the reduction of neutrophil infiltration should be considered as a therapeutic goal. In this experiment, the expression levels of IL-6, TNF-α, CXCL1, and CXCL2 were detected using ELISA, and the level of colon neutrophils was evaluated using immunofluorescence staining. Our results showed that Hig significantly alleviated colon inflammation and reduced infiltration of colon neutrophils in mice with colitis.

The PI3K/AKT pathway is involved in the regulation and release of pro-inflammatory cytokines such as TNF-α and plays an important role in the progression of UC. Inhibition of the PI3K/AKT pathway can improve UC.³⁹⁾ Based on the results of network pharmacological analysis, we speculate that Hig may reduce intestinal damage by inhibiting the PI3K/AKT pathway. Furthermore, we found that Hig ameliorated AKT activation in mice with DSS-induced colitis. However, our study is contrary to Wu’s finding that Hig activates the PI3K/AKT signaling pathway in cardiomyocytes,⁴⁰⁾ which may be explained by the following reasons. First, animal models are different. Second, Hig may act on other signaling pathways, such as NF-κB at the same time, resulting in decreased cytokine secretion and thus decreased activation of the PI3K/AKT signaling pathway.²⁸⁾ Based on our results, we concluded that Hig alleviated DSS-induced colitis, possibly by regulating the PI3K/AKT pathway.

CONCLUSION

In this study, the active components of fuzi were analyzed using TCMSP, and the targets of the active components were predicted using PharmMapper. By intersecting with UC disease-related targets, we identified the possible target genes of fuzi for UC treatment. GO and KEGG pathway analyses were performed for these targets. We further studied whether Hig, the active component of Fuzi, could improve UC. First, we obtained a possible target for Hig therapy for UC by intersecting the target of Hig with the disease target of UC. Second, we predicted that Hig might intervene in the pathogenesis of UC by regulating the PI3K/AKT pathway via GO and KEGG pathway enrichment analyses of these related targets. Finally, we conducted animal experiments for further verification. We found that Hig not only improved the weight loss and hematochezia symptoms of DSS-induced colitis mice but also reduced damage to the colon mucosa and inflammatory cell infiltration. In addition, Hig also reversed decreased intestinal barrier protein expression and mucous barrier destruction. We also found that Hig reduced AKT phosphorylation and inhibited the PI3K-AKT signaling pathway. In summary, we found that Hig is an important active component of Fuzi in the treatment of DSS-induced colitis by inhibiting inflammation, reducing intestinal neutrophilic infiltration, and improving the intestinal barrier through regulating the PI3K/AKT pathway.

Funding

This study was supported by a Grant from the Natural Science Foundation of Guangdong Province, China (No. 2018A030313817).

Author Contributions

Miaomiao Ma and Leshi Liang designed and performed the experiments, wrote the original draft, and analyzed the data. Meihong Lin performed the data analyses, conceptualization, and review. Canhua Luo and Xingfeng Deng contributed to conceptualization and review. Changhui Yu contributed to study conceptualization, writing—review and editing, and acquisition of study funding.

Conflict of Interest

The authors declare no conflict of interest.

Data Availability

All the necessary data used to support the results of this study are included in the manuscript.

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