Protective Effects of Zhi Ciweipi Water Extracts on Dextran Sulfate Sodium Salt-Induced Ulcerative Colitis in Mice

XIAHOU Zhi-Kai, HAN Jun

(1)Teaching and Research Office, Scientific Research Center, China Sport and Health Research Institute, Beijing Sport University, Beijing100084, China;2)National and Local United Engineering Research Center of Key Technology of Traditional Chinese Medicinal Granules,Research Institute of Traditional Chinese Medicine, Beijing Tcmages Pharmaceutical Co., Ltd, Beijing 101301, China)

Abstract Ulcerative colitis (UC) is a disease characterized by chronic inflammation of the intestinal mucosa. However, its exact pathogenesis is not fully understood. Zhi Ciweipi (ZCWP), as a traditional Chinese medicine (TCM), has demonstrated suitable anti-inflammatory effects in the treatment of patients with bloody stool and hemorrhoids. However, its therapeutic effects on ulcerative colitis have yet to be investigated in depth. The aim of this study was to investigate the protective effect of the aqueous extract of ZCWP against dextran sulfate sodium salt (DSS)-induced ulcerative colitis in mice and its possible mechanism of action. Successful simulation of ulcerative colitis was achieved by providing mice with drinking water containing 3% DSS. The HE staining results revealed that the aqueous extract of ZCWP could significantly reduce DSS-induced colonic injuries. Transcriptome sequencing analysis identified 10 key inflammation-related genes (IL-6, IL-1β, CSF2, TNF, IL10, IFN-γ, CXCL1, CXCL2, CXCL9, CXCL10), all of which were significantly downregulated (P<0.05) in response to treatment with the aqueous extract of ZCWP according to qRT-PCR and Western blotting analyses. The immunofluorescence results further indicated that the aqueous extract of ZCWP was able to reduce the DSS-induced increase in the proportion of M1-type macrophages in the colon. Using single-cell sequencing, we thoroughly investigated the signaling relationships between different cell types, which revealed particularly strong communication between M1-type macrophages and fibroblasts. Subsequent qRT-PCR and Western blot analyses verified that the aqueous extract of ZCWP significantly downregulated the expression of DSS-induced fibrosis-related genes in colonic tissues (P<0.05). In conclusion, the results of this study suggest that the aqueous extract of prepared ZCWP exerts a protective effect against DSS-induced ulcerative colitis by inhibiting M1-type macrophage polarization, downregulating the expression of inflammatory factors, and preventing strong communication between M1-type macrophages and fibroblasts. These findings not only reveal the therapeutic mechanism by which ZCWP aqueous extract treats colitis, but also provide a new theoretical basis for its application in clinical practice.

Key words Hedgehog skin fried with talcum powder (ZCWP); transcriptome sequencing; single-cell sequencing; colonic inflammatory factor(CIF); colonic M1 macrophages

Inflammatory bowel disease (IBD) can be divided into specific inflammatory lesions and nonspecific inflammatory lesions. Ulcerative colitis (UC) is an IBD that is characterized by chronic and spontaneous inflammatory lesions of the colonic mucosa. Its etiology and pathogenesis are unknown, and the clinical symptoms include diarrhea, mucopurulent bloody stools, and abdominal pain. Studies[1]have shown that the incidence and prevalence of UC in Asian countries have been increasing yearly for the last 20 years. The occurrence and progression of colitis involve factors such as intestinal barrier damage, increased cell permeability, disruption of cytokine secretion, and upregulation of inflammatory signaling pathway markers. Therefore, inflammation control is of great importance to prevent and treat UC and improve quality of life. At present, the drugs used to treat UC are 5-aminosalicylic acid and glucocorticoids, but these drugs may produce certain side effects[2]. Therefore, there is an urgent need to find more effective and safer bioactive substances to prevent colitis.

Hedgehog skins fried with talcum powder, also known as Zhi Ciweipi (ZCWP), can stop bleeding and promote blood clotting, decrease cold and pain symptoms, enhance reproductive health and reduce urination and is used to treat stomach pain, vomiting, abdominal pain, hernias, hematochezia, hemorrhoids, spermatorrhea, and frequent urination. ZCWP and its compound formulations are commonly used in the clinical treatment of bleeding, swelling, and hemorrhoid pain caused by fever, reducing blood flow or hypertension. In traditional Chinese medical theory, hemorrhoids are caused by infection, which is the main cause of inflammatory reactions. ZCWP is considered an anti-inflammatory compound because it is used to treat hemorrhoids and has the effect of reducing infection.

The possible mechanism underlying the protective effects of ZCWP water extracts on DSS-induced UC model mice was investigated using transcriptome sequencing and single-cell sequencing data.

1 Experimental Materials and Methods

1.1 Experimental Materials

ZCWP (batch number: 210501, Anhui Huifeng National Pharmaceutical Co., Ltd.), TRIzol, reverse transcription kits, and SYBR green reagents were purchased from Hunan Accurate Biology Co., Ltd. iNOS and F4/80 antibodies were purchased from Abcam. qRT-PCR primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. Dextran sulfate sodium (DSS) was purchased from Yeasent Company. RIPA lysate and BCA Protein Concentration Assay Kit were purchased from Shanghai Beyotime Biotechnology Co., Ltd. The PVDF membrane and ECL hypersensitive chemiluminescence solution were purchased from Millipore in the United States. Rabbit monoclonal antibodies against Interleukin-1β (IL-1β), Interleukin-6 (IL-6), Tumor Necrosis Factor (TNF), and β-actin, rabbit polyclonal antibodies against Chemokine (C-X-C motif) ligand 1 (CXCL1), CXCL2, CXCL5, CXCL9, Transforming Growth Factor-1β (TGF-1β), Mothers against decapentaplegic homolog 2 (Smad2) and Smad3 and HRP-labeled goat anti-rabbit IgG secondary antibody were purchased from Abcam. The rabbit monoclonal antibodies against Interferon γ (IFN-γ), α-Smooth Muscle Actin (α-SMA) and Fibronectin 1 (FN1) were purchased from Cell Signaling Technology. The rabbit polyclonal antibody against Colony Stimulating Factor 2 (CSF2) was purchased from Solebro, and the rabbit polyclonal antibody against CXCL10 was purchased from Thermo Fisher.

1.2 Samples

Two hundred grams of ZCWP was placed in a casserole dish, soaked in 1 400 mL of water for 30 min, boiled at 150-220 ℃, incubated at 80-120 ℃ for 30 min to prepare the decoction, and then poured out. The dregs were added to 1 200 mL of water and boiled at the same temperature for another 20 min. Then, the liquid was poured out, the two solutions were mixed and filtered with a 150 mesh filter cloth. The liquid was concentrated at 50 ℃ with a rotary evaporator, and the concentrated liquid was put into a freeze dryer and lyophilized (20.5 g) to obtain ZCWP water extracts. The sample components were identified with reference to the literature[3].

1.3 Animal model and drug administration

Male C57BL/6 mice (25-28 g, 8 weeks old) were purchased from the Experimental Animal Center of Hangzhou Medical College (Zhejiang, China) and housed at 20-26 ℃. The temperature did not vary by more than 4 ℃. The humidity was controlled between 40% and 70%, and noise in the animal room was kept at or below 60 dB to avoid stimulation. All procedures were performed in accordance with the guidelines and approval of the Ethics Committee of Beijing Sport University. The animal experiments were approved by the Animal Experiment Ethics Review Committee of Beijing Sport University (approval number is 2023028A). All of the experiments complied with the ARRIVE guidelines. Animals were randomly divided into 3 groups for the following treatments. In the control group, mice were fed normal drinking water. In the model group, mice were fed drinking water containing 3% DSS for 7 days to induce UC. In the treatment group, mice were fed drinking water containing 3% DSS for 7 days to induce UC, and the drug dose was calculated according to the equivalent dose of the human body per unit surface area. The human body equivalent dose of water extracts of ZCWP to mice was 158 mg/kg. The drugs were intragastrically administered once every day. Drug administration started one week before modeling and lasted for 14 continuous days). After modeling, body weight changes and disease activity index (DAI) scores of mice in different groups were recorded every day.

1.4 Evaluation of pathological damage to the colon by HE staining

A 0.5 cm-long colonic tissue sample was fixed in 4% paraformaldehyde at room temperature and then embedded in paraffin. Five micrometer-thick sections were stained with hematoxylin for 3 min, dehydrated in 70% and 90% alcohol for 10 min each, stained with eosin for 3 min, dehydrated in alcohol, and then cleared by xylene to observe the histopathological features under a fluorescence microscope.

1.5 Measurement of colonic inflammatory factor expression by qRT-PCR

Ten milligrams of colonic tissue was taken from each group, 1 mL of TRIzol was added, and the tissue was thoroughly ground and crushed at -30 ℃ in a freezing grinder. Total RNA was extracted from the colonic tissue, and the concentration and purity of RNA were determined by an enzyme marker. Reverse transcription to cDNA was used. qRT-PCR experiments were performed using the SYBR green reagent. Reaction conditions: predenaturation at 95 ℃ for 30 s, denaturation at 95 ℃ for 10 s, annealing at 55 ℃ for 30 s, and extension at 72 ℃ for 15 s, with 45 cycles. Relative mRNA expression levels were calculated according to the 2-△△Ctmethod, and the primers and sequences are shown in Table 1.

Table 1 qRT-PCR primers and sequences

1.6 Transcriptome sequencing and data processing

RNA quality was checked using the Qubit Fluorometer and Agilent Bioanalyzer 2100, and RNA samples with RIN values greater than 7.5 were used for subsequent library construction. The NEBNext® Ultra ? RNA Library Prep Kit for Illumina® was used to build the library, and the samples were double-end sequenced (2×150 bp). The raw data obtained from sequencing were analyzed for quality control using FastQC, and sequence comparison was performed using HISAT2 with the reference genome of hg19. The list of differentially expressed genes between different treatment groups was found using String Tie and Ballgown.

1.7 Screening of differentially expressed genes (DEGs)

We selected and analyzed the DEGs in two groups: controlvs. model and modelvs. treatment. In our study, genes with a correctedP<0.05 and |log fold change (FC)| >1 were considered DEGs.

1.8 Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis

GO annotations (including BP, CC, and MF) and KEGG pathway (www.kegg.jp/feedback/copyright.html) analysis of DEGs were performed using a G: profiler database (https://biit.cs.ut.ee/gprofiler/gost), and aPvalue of 0.01 was considered statistically significant.

1.9 Protein-protein interaction (PPI) network construction

We used the STRING database to build a PPI network (https://string-db.org/). High confidence (0.700) was chosen as the threshold. Then, the PPI network was imported into Cytoscape 3.9.1 software, and the hub genes were further screened through the cytoHubba plug-in.

1.10 Immune infiltration analysis

The CIBERSORT (https://cibersortx.stanford.edu/) algorithm was used to estimate the proportion of immune cell types in the transcriptome. This method quantifies the proportion of 22 infiltrating immune cell types in the tissue based on 547 marker genes. We quantified the relative proportion of 22 immune cell types in the sample by using standardized gene expression data. The perm (degree of a permutation group) was set to 1 000, and the samples withP<0.05 in the CIBERSORT analysis results were used for further analysis.

1.11 Analysis of single-cell transcriptome sequencing data

The single-cell sequencing data were obtained from the GEO database (GSE162335) (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi), and the data analysis process was completed in R 4.2.1 software. First, the ‘SCTransform’ function in the ‘Seurat’ package was used to standardize the expression matrix, and the ‘RunPCA’ function was used for principal component analysis. After the UMAP method was reduced to clustering, the ‘harmony’ function was used to remove the batch effect from the sample. The cell group was annotated according to the cell type marker provided in the data. Then, a difference analysis was performed based on the cell type. Cell communication analysis was completed by the ‘cellchat’ package.

1.12 Determination of colonic M1 macrophage proportions by IF staining

Paraffin-embedded colon samples were prepared into 5 μm thick sections, and the sections were attached to polylysine-treated slides. The sections were baked at 60 ℃ for 2 h, dewaxed in xylene I for 30 min, dewaxed in xylene II for 10 min, rehydrated in gradient alcohol, and subjected to antigen retrieval. Afterward, blocking serum was added dropwise for 30 min at 37 ℃. Primary antibodies (iNOS: 1∶200, F4/80: 1∶200) were added dropwise and incubated overnight at 4 ℃ in a wet box. On the next day, fluorescent secondary antibodies were added dropwise and incubated at room temperature for 1 h. DAPI was added dropwise and incubated for 2 min at room temperature. The slides were washed 3 times with PBS and observed under a fluorescence microscope.

1.13 Western blot detection of colonic inflammatory factor protein expression

Five milligrams of colon tissues from each group was ground thoroughly and 300 μL of precooled RIPA lysate solution was added to lyse the tissue fully. The tissue was placed on ice throughout this process. The tissue lysate was collected and centrifuged at 4 ℃ for 15 min at 12 000 g. The supernatant was extracted, and the protein concentration was quantified using the BCA Protein Concentration Assay Kit. Fifty micrograms of protein samples were added to loading buffers according to a volume ratio of 1∶1, heat denaturation was conducted at 100 ℃ for 10 min, and then the sample was loaded, followed by SDS-PAGE electrophoresis to separate the proteins. Then, the separated proteins were transferred through Western transfer buffers to PVDF membranes, which were blocked with 5% skimmed milk powder at room temperature for 1.5 h, and then primary antibodies against IL-1β, IL-6, CSF2, TNF, IFN-γ, CXCL1, CXCL2, CXCL5, CXCL9, CXCL10, TGF-1β, Smad2, Smad3, α-SMA and FN1, all at 1∶1 000 dilution, were added and incubated overnight at 4 ℃. The next day, the membranes were washed 3 times with TBST for 10 min each time, incubated with HRP-labeled secondary antibody (1∶5 000 dilution) for 2 h at room temperature and then washed 3 times with TBST for 10 min each time. After adding ECL ultrasensitive chemiluminescent solution, exposure photographs were taken on a gel imager, the grayscale values were analyzed by ImageJ software, and the relative expression of each protein was calculated using β-actin as the internal reference.

1.14 Statistical analysis

The data were statistically analyzed using GraphPad Prism 6.0 software and expressed as the mean ± standard deviation. All data were subjected to at least three independent replicate experiments. Moreover,Pvalues less than 0.05 were considered statistically significant.

2 Results

2.1 Composition of ZCWP water extracts

ZCWP water extracts contained 147 proteins, among which collagen alpha-1(I) chain, E3 ubiquitin-protein ligase RNF180, myosin-2 isoform X3, keratin-associated protein 8-1, serum albumin and actin, cytoplasmic 2 isoform X1 and nine other proteins were found to be in high relative abundance (Table 2).

Table 2 Search information of the top ten proteins in ZCWP water extracts

2.2 ZCWP water extracts relieve DSS-induced UC

According to daily monitoring results of body weights, the control group showed no obvious changes in body weights, but the model group presented decreasing body weights as the modeling time progressed. The body weight reduction in the treatment group was less severe than that in the model group (P<0.05, Fig.1A). According to the daily DAI monitoring results, the control group had no DAI score, and the DAI scores of the model group increased gradually with the progression of modeling (P<0.05). The DAI scores of the treatment group were decreased compared to those of the model group (P<0.05, Fig.1B).

Fig.1 Comparison of body weight changes and disease activity index scores of mice in each group Male C57BL/6 mice were randomly divided into the control group, model group and treatment group. The mice in the control group were given normal drinking water. Mice in the model group were given drinking water containing 3% DSS for 7 days to induce acute mouse colitis. Mice in the treatment group were given drinking water containing 3% DSS for 7 days to induce acute mouse colitis, and drug treatment (158 mg/kg) was administered once daily by gavage starting one week before modeling and continuing for 14 consecutive days. Body weight changes as well as disease activity index scores were recorded daily for each group of mice since the beginning of modeling. Data are expressed as the mean ± standard deviation (n=6).* indicates P<0.05,** indicates P<0.01, and*** indicates P<0.001

The HE pathology of the colon showed that the control group had intact colonic structures, and the villi and crypt structures were not disrupted. In the model group, the villi and crypt structure were completely destroyed. However, the crypt structure was partially recovered in the treatment group (Fig.2).

Fig.2 Comparison of colonic pathological damage in each group of mice Male C57BL/6 mice were randomly divided into a control group, model group and treatment group. The mice in the control group were given normal drinking water. The mice in the model group were given drinking water containing 3% DSS for 7 days to induce acute mouse colitis. Mice in the treatment group were given drinking water containing 3% DSS for 7 days to induce acute mouse colitis, and drug treatment (158 mg/kg ) was administered once daily by gavage starting one week before modeling and continuing for 14 consecutive days. Colonic tissue samples from mice were collected and subjected to HE staining (200×, scale bar = 500 μm) to assess the colonic pathological damage in each group of mice. The red arrow indicates that the mucous layer of the mouse colon of the model group was completely destroyed, with the villus and crypt structures completely disappearing. The black arrow indicates that the mucous layer of the mouse colon in the treatment group was restored, with evident villus and crypt structures

2.3 Transcriptome sequencing pathway enrichment analysis and screening of key genes

We found 633 shared DEGs between the upregulated DEGs in the model group (compared with the control group) and the downregulated DEGs in the treatment group (compared with the model group) and 444 shared DEGs between the downregulated DEGs in the model group (compared with the control group) and the upregulated DEGs in the treatment group (compared with the model group) (Fig.3A). These 1 077 DEGs may be the most relevant genes to the effects of drug therapy. Next, we conducted GO and KEGG enrichment analyses on these 1 077 DEGs (Fig.3B, 3C). Then, we imported the 1 077 DEGs into the STRING database for PPI analysis. We found the 10 most important hub genes (Fig.3D).

Fig.3 Transcriptome sequencing analysis of the mouse colon (A) Venn diagram showing the shared upregulated differentially expressed genes in the model group (compared with the control group) with the downregulated differentially expressed genes in the treatment group (compared with the model group) (total 633) and the shared downregulated differentially expressed genes in the model group (compared with the control group) with the upregulated differentially expressed genes in the treatment group (compared with the model group) (total 444). We used |log fold change (FC)| >1 and P<0.05 as the criteria for significance. (B) GO enrichment analysis plot of differentially expressed genes. Significant correlations between differentially expressed genes and biological functions were revealed. Different colors were used to distinguish different GO classifications. The screening criterion for significant enrichment was P<0.01. (C) KEGG enrichment analysis of differentially expressed genes. Enrichment pathways of the top ten differentially expressed genes were mapped. The screening criterion for significant enrichment was P< 0.01. (D) Top hub genes. Nodes represent DEGs, while lines represent interactions of DEGs. The size and color of the boxes are set according to the “degree” value of the nodes. The higher the degree, the darker the red color of the DEGs, indicating that these DEGs play a more central role. Hub genes were filtered by the cytoHubba plugin

2.4 The CIBERSORT algorithm was used to estimate the proportion of immune cell types in the transcriptome.

Through immune infiltration analysis (Fig.4A), we found that drug treatment can reduce the proportion of M1 macrophages and increase the proportion of M2 macrophages. M1 macrophages mediate inflammation, and M2 macrophages can promote tissue repair. These results indicate that drug therapy can reduce the inflammatory reaction of pathological sites and promote tissue repair.

2.5 Single-cell sequencing analysis

A total of 8 cell types were obtained from single-cell transcriptome data after dimensionality reduction clustering, which included B cells, fibroblasts, GC cells, macrophages, mast cells, monocytes, plasma cells and T cells (Fig.4B). To explore whether the signal communication between different cell types is abnormal under disease conditions, we carried out cell communication analysis at the single-cell level. The results of cell communication analysis showed that macrophages and fibroblasts had strong cell communication with other types of cells (Fig.4C). To further explore the communication relationship between macrophages and fibroblasts, we showed the communication relationship between macrophages and fibroblasts and other cell types (Fig.4D-E). Moreover, we further explored the receptors and ligands involved in the communication between macrophages and fibroblasts (Fig.4F).

2.6 ZCWP water extracts downregulate colonic inflammatory factor expression

The expression levels of colonic inflammatory factors in each group were measured by qRT-PCR, and the results showed that the expression levels ofIL-1β,IL-6,CSF2,TNF,IFN-γ,CXCL1,CXCL2,CXCL9, andCXCL10 increased in the colonic tissue of the model group compared with those in the colonic tissue of the control group (P<0.05). The expression levels ofIL-1β,IL-6,CSF2,TNF,IFN-γ,CXCL1,CXCL9, andCXCL10 decreased in the treatment group compared with the model group (P<0.05), as shown in Fig.5. The results showed that there was high expression of inflammatory factors in UC model mice, and treatment with ZCWP water extracts downregulated the expression of these inflammatory factors.

Fig.5 Comparison of the gene expression of colonic inflammatory factors in each group of mice Total colonic RNA was extracted using TRIzol from each group of mice, and the expression levels of inflammatory factors were measured by qRT-PCR. Data are expressed as the mean ± standard deviation (n=6).** indicates P<0.01, and*** indicates P<0.001

The protein expression levels of colonic inflammatory factors were detected by Western blotting. The results showed that the expression levels of IL-1β, IL-6, CSF2, TNF, IFN-γ, CXCL1, CXCL2, CXCL9, and CXCL10 were increased in the colon tissue of the modeling group compared with the control group (P<0.05). The protein expression levels of the above inflammatory factors decreased in the treatment group compared with the modeling group (P<0.05), as shown in Fig.6. The results indicate that the water extracts of ZCWP can downregulate the expression of inflammatory factors in ulcerative colitis model mice.

2.7 ZCWP water extracts inhibit the infiltration of M1 macrophages

The IF results showed that the expression of F4/80 and iNOS was almost undetectable in the colon of the control and administration groups, while F4/80 and iNOS expression was found in the model group, as shown in Fig.7. This finding reflects that the proportion of M1-type macrophages in the colon is extremely low under normal conditions. DSS modeling can strongly increase the infiltration of M1-type macrophages in the colon, and treatment with ZCWP water extracts can decrease the infiltration of M1-type macrophages.

Fig.7 Comparison of the M1 macrophage infiltration in the colon of mice in each group The infiltration of M1-type macrophages in each group of mouse colons was determined using IF. Colon sections were taken and incubated overnight at 4℃ in a wet box after baking, dewaxing, antigen repair and dropwise addition of primary antibody (iNOS: 1∶200, F4/80: 1∶200). All samples were observed using fluorescence microscopy (400×, scale bar = 100 μm). The red arrows indicate the colocalization of iNOS and F4/80, representing M1-type macrophages

2.8 ZCWP water extracts inhibit colon fibrosis

The results of RNA-seq indicate that the expression levels ofTGF-1β,Smad2 andSmad3 in the colon tissue of the model group were increased compared with those in the colon tissue of the control group, while the expression levels ofTGF-1β,Smad2 andSmad3 decreased after treatment with ZCWP water extracts, suggesting that ZCWP can be used to reduce colon fibrosis caused by DSS. To clarify this phenomenon, we carried out verification through qRT-PCR and obtained similar results to the RNA-seq data. The expression ofTGF-1β,Smad2,Smad3,α-SMAandFN1 in the colon tissue of the model group was upregulated compared to that in the colon tissue of the control group (P<0.05). The expression levels ofTGF-1β,Smad2,Smad3,α-SMAandFN1 in the colon tissue of the treatment group decreased compared with those in the colon tissue of the model group (P<0.05), as demonstrated in Fig.8.

Fig.8 Comparison of colonic fibrosis-related gene expression in each group of mice Total colonic RNA was extracted from the colon of each group of mice using TRIzol, and the expression levels of fibrosis-related genes were measured by qRT-PCR. Data are expressed as the mean ± standard deviation (n=6).*** indicates P<0.001

These findings were subsequently verified by Western blot, which showed that TGF-1β, Smad2, Smad3, α-SMA, and FN1 expression levels were elevated in the colonic tissue of the modeling group compared to the control group (P<0.05), and that TGF-1β, Smad2, Smad3, α-SMA, and FN1 expression levels were decreased in the treatment group compared to the modeling group (P<0.05), as shown in Fig.9. The results indicate that ZCWP water extracts can reduce colonic fibrosis in ulcerative colitis model mice.

Fig.9 Protein expression of fibrosis-related factors in the colon of three groups of mice by Western blotting The total protein in the colon tissue of each group of mice was extracted, and the protein expression levels of fibrosis-related factors were detected using Western blotting. β-Actin was used as an internal reference. Data are expressed as the mean ±SD (n=3).*** indicates P<0.001

3 Discussion

The immune-active peptide RHPYFYAPELLYF, which can inhibit the secretion of inflammatory factors and promote the secretion of anti-inflammatory factorsinvivoandinvitro, was screened in ZCWP. The peptide can inhibit inflammation through the STAT5 signaling pathway[3], and hedgehog skin extract can reduce nitric oxide production and regulate inflammation-related genes such asIL-6 through the MAPK signaling pathway[4], providing a mechanistic explanation for the anti-inflammatory effects of ZCWP. Nevertheless, the potential molecular mechanism by which ZCWP water extracts treat DSS-induced UC remains unknown. The results showed that the water extracts of ZCWP can downregulate the expression of the inflammatory factors IL-1β, IL-6, CSF2, TNF, IFN-γ, CXCL1, CXCL2, CXCL5, CXCL9, and CXCL10 and relieve colonic injury caused by DSS. In fact, HE staining showed that DSS treatment disrupted colonic villi and crypt structures, with rounded crypts and increased muscularis mucosa thickness. However, these morphological changes were significantly reversed after treatment with ZCWP water extracts. Macrophages are major phagocytic cells that play an important role in defending against microbial infections and maintaining tissue balance in inflammatory diseases[5-6]. DSS modeling induces massive infiltration of M1 macrophages in the colon, and the administration of ZCWP water extracts can inhibit the effect of DSS, suggesting that the protective effect of ZCWP water extracts against DSS-induced colonic injury may be related to its immunomodulatory and anti-inflammatory activity.

Bioinformatics analysis methods have been used to conduct relevant explorations to gain insight into the potential physiopathological mechanisms of diseases. Transcriptome sequencing is an effective technique to study gene expression, analyze differentially expressed genes, and perform functional enrichment[7]. The differentially enriched gene pathways mainly included the IL-17 signaling pathway, TNF signaling pathway, and Toll-like receptor signaling pathway. It was found that autophagy, α-kinase-1, and IL-17 signaling pathways were consistently suppressed in active and stationary ulcerative colitis models[8]. Qing Chang Suppository Powder inhibits the DSS-induced IL-17 signaling pathway in HT-29 cells and UC model mice[9]. HMM targets five pivotal genes (RELA,MAPK14,MAPK1,JUN, andAKT1) through four active ingredients (quercetin, kaempferol, formononetin, and isorhamnetin) to modulate the PI3K-AKT1 and TNF signaling pathways for the treatment of UC[10]. In the CLP-induced sepsis mouse model and LPS-induced cellular inflammation model, PTX3 inhibited the inflammatory response and attenuated intestinal mucosal barrier damage through the TLR signaling pathway[11]. The above studies are consistent with the pathway enrichment analyses conducted in this study.

The GO results showed that the differentially expressed genes were significantly enriched in a series of biological processes related to the development of inflammation, such as response to cytokine and response to cytokine, and that these differentially expressed genes were mainly distributed in the cytoplasm and intrinsic membrane components. In addition, the enriched molecular functions of the differentially expressed genes were mainly related to protein binding and chemokine activity.

The interactions of individual proteins with each other are crucial for various life processes, so it is important to learn about the response mechanisms of energy substance metabolism and biological signals in special physiological states such as diseases, as well as to learn about the functional connections between proteins by systematically analyzing the interactions of a large number of proteins in biological systems by constructing PPI networks. According to the analysis, the top 10 key interacting proteins were CXCL1, CXCL2, and CXCL5, which suggests that ZCWP might treat DSS-induced UC by acting on these targets.

CIBERSORT is a bioinformatics analysis that allows the identification of immune cell type compositions[12]. Through immune infiltration analysis, we found that drug treatment was able to decrease the proportion of M1 macrophages and increase the proportion of M2 macrophages. Treatment with ZCWP water extracts may reduce M1 macrophage-mediated inflammation since polarized M1 macrophages secrete a variety of inflammatory mediators, including TNF-α, IL-6, IL-12, and IL-1β. Moreover, these overexpressed inflammatory mediators were involved in the process of the inflammatory response[13-14]. M2 macrophages play a role in tissue repair and remodeling, angiogenesis, and tumor formation[15], and these results suggest that pharmacological treatment can reduce the inflammatory response at the pathological site and promote tissue repair.

Single-cell sequencing analysis verified that the cell subpopulation in colitis tissues was dominated by M1 macrophages. Interestingly, we found that M1 macrophages and fibroblasts have strong cellular communication, that fibroblasts are involved in the repair, remodeling, and conditioning of injured tissues and that colonic mucosal fibroblasts play an important role in wound healing and the development of fibrosis. In the early stages of inflammation, the presence of large numbers of fibroblasts is one of the characteristics of wound healing, but their prolonged survival leads to the massive deposition of intestinal collagen and the development of intestinal fibrosis[16]. Tumor-specific FAP fibroblasts and SPP1 macrophages were found to be positively correlated in 14 independent colorectal cancer cohorts, and their tight localization was verified by immunofluorescence staining and spatial transcriptomics[17]. Colonic fibroblasts, but not tumor cells, are the major cellular component that recruits and determines the fate of infiltrating monocytes to specific macrophage populations, which are characterized by high CD163 expression and CCL2 production[18]. Cancer-associated fibroblasts enhance tumor-associated macrophage enrichment and inhibit NK cell function in colorectal cancer[19]. Through single-cell sequencing and cell communication analysis, we also found signaling pathways for communication between macrophages and fibroblasts, and the above findings suggest that ZCWP water extracts may also be effective in preventing and treating intestinal fibrosis. The experimental results indicate that the expression levels of TGF-1β, Smad2, Smad3, α-SMA and FN1 in the colon tissues of the model group were increased compared with those in the colonic tissues of the control group. The expression levels of TGF-1β, Smad2, Smad3, α-SMA and FN1 in colon tissues of the treatment group decreased compared with those in the colonic tissue of the model group. Epithelial mesenchymal transition (EMT) is a key pathway involved in the development of intestinal fibrosis, which is characterized by gradually decreasing levels of cytokeratin and increasing levels of the interstitial cell marker α-smooth muscle actin (α-SMA). The activation of TGF-β1 can induce EMT by changing the cell morphology, upregulating the expression of the interstitial cell marker α-SMA and downregulating the expression of the epithelial cell marker E-cadherin[20]. Studies have indicated that[21]a large number of fibroblasts can be found at the site of intestinal fibrosis, and the synthetic capability of type III collagen apparently increases. TGF-β1 is associated with the occurrence and development of fibrosis to some degree[22].

We performed a liquid-phase mass spectrometry analysis of ZCWP water extracts to determine its major components. Nine proteins, including COLA1 (collagen alpha-1(I) chain) were found to be in relatively high abundance. COLA1 is the alpha 1 chain of type I collagen and is involved in biological processes such as remodeling of the extracellular matrix and regulation of immune responses[23-25]. ALB (serum albumin) was found to be a significant differentially expressed gene in the ZCWP water extract treatment group[4]. These nine proteins were all found to have certain anti-inflammatory or immune-enhancing activities. ZCWP water extracts show immune enhancing and anti-inflammatory activity, probably due to its multiple active components.

Instead of performing a detailed dose-effect relationship study, this paper focused on investigating the protective effect of clinical doses of ZCWP on a mouse model of ulcerative colitis and further exploring its possible mode of action. The doses applied in this study were chosen based on those used in clinical practice. However, we failed to study the effects of multiple doses of the drug, which is a limitation of our study. In the future, an investigation of different doses will be added based on the present study. Due to specific reasons, a positive control drug was not adopted in the study. Currently, mesalazine is the commonly used drug for treating this disease, and it exerts its therapeutic effect by inhibiting the production of cyclocxygenase and prostaglandin in the colon and alleviating local inflammation. Nonetheless, we hypothesize that the water extract of ZCWP exerts a protective effect on DSS-induced ulcerative colitis by inhibiting the polarization of M1 macrophages, downregulating the expression of inflammatory factors, and blocking strong communication between M1 macrophages and fibroblasts instead of interfering with the pathways of cyclooxygenase and lipoxygenase. Given that no alternative control drug in the treatment of ulcerative colitis can ensure the results of actively inhibiting M1-type macrophage polarization, downregulating the expression of inflammatory factors, and blocking the strong communication between M1-type macrophages and fibroblasts, this study did not incorporate a traditional positive control group.

In summary, the water extracts of ZCWP can relieve DSS-induced UC and have a protective effect on DSS-induced UC in mice.