Animal model

Male C57BL/6 mice (8 weeks old) were provided by SPF Biotechnology Co. Ltd (Beijing, China). All procedures were approved by the Institutional Animal Care and Use Committee of Beijing Stomatological Hospital (Ethical code: No. KQYY-202210-001). Twenty-four mice were randomly separated into 4 groups (Ctrl group, MYDGF group, DSS group, and DSS+MYDGF group) after one week of adaptive feeding. Distilled water was used to configure DSS (9011-18-1, MP Biomedicals, CA, USA) into 3% solution as drinking water for the experimental groups. MYDGF protein (HY-P73303, MCE, NJ, USA) was diluted with distilled water to a concentration of 500 μg/mL. MYDGF and DSS+MYDGF groups were injected with MYDGF via tail vein on 1d, 3d, and 5d after the experiment began (5 μg/mouse). The same volume of distilled water was injected into the tail vein of DSS group.

The weight of the mice and the shape and consistency of stools were measured daily. Disease activity index (DAI) was used to evaluate the severity of colitis. Based on the references, DAI scores were assessed for each mouse includes weight loss, stool shape and bloody stool [35]. DAI rating from day 1 of DSS processing to day 7. On day 7, blood was collected from eyes after anesthesia, and all mice were sacrificed for cervical dislocation. The complete colon from the epityphlon to the anus was collected and the length of the colon was measured. Photographs of the colon were obtained immediately after the sample is collected.

Blood routine examination

Mice blood were collected using blood collection vessels containing EDTA anticoagulant. White blood cells, red blood cells and other indicators were detected by automatic blood routine analyzer (DxH800, Beckman, CA, USA).

Hematoxylin and eosin (H&E) staining and histological activity index (HAI)

Colon tissue samples were collected and fixed in 4% formalin for 48 h. After dehydration, waxdip and embedding, samples were sectioned at 5-µm thickness. Sections were rehydrated with graded ethanol and vitrified by dimethylbenzene. H&E staining was done to observe the histological changes. Five different views in each section were captured. The HAI was calculated based on mucosal inflammation and the degree of epithelial [35].

Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay

Paraffinized samples were sectioned at 5-μm thickness. TUNEL staining was performed with a One Step TUNEL Apoptosis Assay Kit (C1090, Beyotime, Shanghai, China) according to the manufacturer’s instructions. Following the TUNEL reaction, slides were treated with DAPI (F6057, Sigma, MO, USA). Images were captured under a confocal microscope and the number of TUNEL-positive cells was calculated in six random fields per slide.

Immunohistochemistry (IHC) and immunofluorescence (IF) staining

Paraffin sections for IHC staining after dewaxing were antigen retrieval using microwave heating. Then, 3% Bovine Serum Albumin (BSA) were used to block the non-specific antigen. Sections were incubated with primer antibody (Caspase 3, A21677, Abclonal, Wuhan, China; E-Cad, A20798, Abclonal; COX2, A3560, Abclonal; Occludin, 27260-1-AP, Proteintech, Rosemont, USA; CD86, A1199, Abclonal; Arg1, 16001-1-AP, Proteintech; P-P65, AP1294, Abclonal; P-ERK, AP0886, Abclonal) overnight at 4 ℃. The next day, different second antibodies (PV-6001 and PV-60002, ZSGB-Bio, Beijing, China) were incubated for 1 h at 25 ℃. IHC was performed DAB kit (SP-9000, ZSGB-Bio). IF was used TSA amplification fluorescence kit (abs50012, Absin, Shanghai, China). Five different views in each section were captured. The integral optical density (IOD) and positive area/cells were calculated by ImageJ Pro Plus.

Western blot

The colonic tissues were lysed in RIPA lysis buffer containing protease inhibitor. Proteins were extracted and quantified using BCA assay (P0011, Beyotime, China). The total protein of each sample was 15 μg. The primary antibody (Bax, 60267-1-Ig, Proteintech; Bcl-2, 68103-1-Ig, Proteintech; Caspase 3, A21677, Abclonal; E-Cad, A20798, Abclonal; COX2, A3560, Abclonal; Occludin, 27260-1-AP, Proteintech; iNOS, 18985-1-AP, Proteintech; TNF-α, 17590-1-AP, Proteintech; IL-4, 66142-1-Ig, Proteintech; CD80, 66406-1-Ig, Proteintech; CD68, 28058-1-AP, Proteintech; Arg-1, 16001-1-AP, Proteintech; P-IKKα, AP1066, Abclonal, IKKα, A19694, Abclonal; P-P65, AP1294, Abclonal; P65, 66535-1-Ig, Proteintech; P-IκBα, AP0999, Abclonal; IκBα, 10268-1-AP, Proteintech; P-ERK, AP0886, Abclonal; ERK, 11257-1-AP, Proteintech; P-JNK, 80024-1-RR, Proteintech; JNK, 24164-1-AP, Proteintech; P-P38, 28796-1-AP, Proteintech; P38, 14064-1-AP, Proteintech; β-action, AC026, Abclonal) were incubated overnight at 4 ℃ after proteins were transferred to Polyvinylidene Fluoride membrane. Next day, the membranes were incubated with different secondary antibodies (AS003 and AS014, Abclonal). Each primary antibody was incubated in the whole membrane. The experiment was repeated three times independently. The repeats were performed with lysates from different mice. Optical densities (OD) of blots were determined in the Image J software. The result is expressed as the ratio of the protein OD to the actin OD or other protein OD.

Enzyme-linked immunosorbent assay (ELISA)

The levels of inflammatory cytokines IL-6, TNF-α and IL-1β were measured using ELISA kits as described [36]. The serum of mice was obtained by centrifugation of 1500 g and 15 min. ELISA was performed using commercial kits with antibodies against IL-6 (RK00008, Abclonal), TNF-α (430901, BioLegend, CA, USA) and IL-1β (432601, BioLegend). The results were assessed using a microplate reader at 450 nm and corrected at 570 nm.

Statistical analysis

Results are presented as mean ± standard deviation (SD). All graphs were generated using GraphPad Prism 9.3 software (GraphPad Software, San Diego, USA). All data were analyzed using SPSS 22.0. Two-way analysis of variance (ANOVA) was used to compare the differences among two groups with multiple columns such as weight and DAI. One-way analysis of variance (ANOVA) was used to compare the differences among multiple groups and Bonferroni’s multiple comparisons test was used for post-hoc analysis of the ANOVA results. The unpaired non-parametric t-test was used to compare two groups for western blot. P-values < 0.05 were considered statistically significant.