In this study, the MIF were collected from the experimental field of Sichuan Academy of Agricultural Sciences (Chengdu, China). The MIF were dried at 37°C, and then 200 g of MIF were immersed in proportions of 1:20 (w/v) in ethanol at 60°C for 6 h. After centrifugation at 6,000 × g for 15 min, the sediment was discarded. Thereafter, the resulting MIF was dried at 60°C and stored at −20°C before use.

The molecular weight distribution of MIF was determined by high-performance gel permeation chromatography (HP-GPC). The operating procedures were Waters 515, high-performance liquid chromatography equipped with laser detector (LS), and differential refractive index (DRI); Shodex OHpak series SB-806 gel chromatographic column (300 mm × 7.8 mm); column temperature 40°C ± 0.1°C. The mobile phase was 0.05 M NaH₂PO₄-NaH₂PO₄ buffer (pH 6.7, with 0.02% NaN₃). The flow rate was 0.5 ml/min. The loading amount was 500 μl. Then, 0.05 M NaH₂PO₄-NaH₂PO₄ buffer (pH 6.7, with 0.02% NaN₃) is used to dissolve polysaccharide standards with the molecular weights of 738, 5,800, 1.22 × 10⁴, 2.37 × 10⁴, 4.8 × 10⁴, 1.0 × 10⁵, 1.86 × 10⁵, 3.8 × 10⁵, and 8.53 × 10⁵ g/mol, respectively. After being filtered with 0.45-μm membrane, the determination was performed according to the above chromatographic conditions. According to the molecular weight and retention time of standards, the standard curve was drawn, and then the molecular weight was calculated according to the retention time.

A total of 144 C57BL/6J mice (initial mass 18.02 ± 0.36 g), obtained from Dashuo Experimental Animal Co., Ltd. (Chengdu, China), were divided into four treatments with six pens per treatment (six mice per pen): (1) Control (fed a normal diet); (2) DSS treatment (fed a normal diet); (3) DSS treatment + 100 mg/kg MIF (LMIF; fed a normal diet + 100 mg/kg MIF); (4) DSS treatment + 200 mg/kg MIF (HMIF; fed a normal diet + 200 mg/kg MIF). On days 8–14, mice in the challenged groups were orally administered 3.5% DSS in drinking water, while other mice were administered normal saline (Bassaganya-Riera and Hontecillas, 2006). Moreover, all mice were individually caged under a controlled environment room.

At the end of the experiment, after 12-h starvation and ether anesthesia, blood samples from six mice with the average body weight in each group were collected, centrifuged at 1,500 × g (15 min) to obtain serum, and then stored at −20°C. Subsequently, the same mice were sacrificed, about 2-cm segments of the colon were isolated, gently flushed with normal saline, and then fixed in paraformaldehyde solution (4%) for morphological analysis. Finally, about 5-cm colonic tissues were collected and stored at −80°C until analyses.

The serum diamine oxidase (DAO) activity and D-lactate concentration were assessed using commercial kits purchased from Jiancheng Bioengineering Institute (Nanjing, China). All measurements were performed according to the manufacturer’s instructions.

After a 48-h fixation, the colonic segments were dehydrated using a graded series of alcohol and cleaned with xylene, embedded in paraffin, cut into cross sections of 5-μm thickness, and then stained with H&E (Fang et al., 2017). Then, the villus height and crypt depth were measured, and the ratio of villus height to crypt depth (VCR) was calculated from the value obtained above.

The colonic mucosa was homogenized with normal saline (1:9), and the homogenate was centrifuged at 1,500 × g (15 min) to attain supernatant. Then, the concentrations of interleukin-1β (IL-1β), IL-6, IL-10, tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ) in the colonic mucosal supernatant were assayed by ELISA kits (Zhuo Cai Biotechnology Co., Ltd., Shanghai, China).

Superoxide dismutase (SOD) activity, catalase (CAT) activity, malondialdehyde (MDA) content, and total antioxidant capacity (T-AOC) in the colonic homogenates were measured. Measurements were performed by the spectrophotometric method using commercially available kits (Nanjing Jiancheng Bioengineering Institute).

Total gDNA from digesta samples was extracted using a Stool DNA Isolation Kit (Tiangen Biotech Co., Ltd., Beijing, China), following the manufacturer’s directions. The genes of bacterial 16S rRNA in the region of V4 were amplified by using PCR with primers (515F/806R). The PCR products were subjected to electrophoresis on 2% agarose gel, and the mixed PCR products were purified with AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, United States) for sequencing on an Illumina MiSeq system. All 16S rRNA gene sequencing data were saved in the National Center for Biotechnology Information and can be accessed in the Short Read Archive under the accession number PRJNA679459.¹

Quality filtering on the raw reads was performed under specific filtering conditions to obtain the high-quality clean reads according to the Cutadapt quality-controlled process (Martin, 2011). The reads were compared with the reference database using UCHIME algorithm (Edgar et al., 2011), to detect chimera sequences, and then removed to get the clean reads (Haas et al., 2011). Clustered into operational taxonomic units (OTUs) utilizing Uparse v7.0.1001 at 97% sequence similarity (Edgar, 2013). Species annotation was carried out on the OTU representative sequences. For colonic bacteria, α-diversity index was assessed using QIIME 1.7.0. Principal coordinate analysis (PCoA) tools in R language were used for PCoA.

Protein samples were extracted from colonic tissues using lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China). The lysates were centrifuged at 12,000 × g for 10 min at 4°C, and the supernatant was collected. A bicinchoninic acid (BCA) protein assay kit (Beyotime Institute of Biotechnology) was used to determine the protein concentration in the supernatant. Thereafter, 30 μg of protein extractions were separated by 10% sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) and then transferred to a polyvinyldifluoride (PVDF) membrane (Merck Millipore Ltd., Tullagreen, Ireland) using wet Trans-Blot System (Bio-Rad Laboratories, Inc., Hercules, CA, United States). After blocking with Tris-buffered saline Tween 20 (TBS/T) containing 5% bovine serum albumin (BSA) at room temperature for 1 h, the membranes were incubated with primary antibodies at 4°C overnight against phospho-Nrf2 (Sigma-Aldrich, St. Louis, MO, United States), Keap1 (Sigma-Aldrich), heme oxygenase-1 (HO-1; Sigma-Aldrich), NAD(P)H dehydrogenase (quinone 1) (NQO-1; Sigma-Aldrich), occludin (Sigma-Aldrich), claudin-1 (Proteintech Group, Inc., Wuhan, China), zonula occludens-1 (ZO-1; Sigma-Aldrich), ZO-2 (Sigma-Aldrich), Toll-like receptor 4 (TLR4; Proteintech Group, Inc.), MyD88 (Proteintech Group, Inc.), IL-1 receptor-associated kinase 1 (IRAK1; Proteintech Group, Inc.), TNF receptor-associated factor 6 (TRAF6; Proteintech Group, Inc.), phospho-NF-κB (Proteintech Group, Inc.), or β-actin (Proteintech Group, Inc.). The polyvinylidene fluoride (PVDF) membranes were washed thrice with TBS/T, then incubated with second antibodies at room temperature for 2 h, and washed thrice with TBS/T again. BeyoECL Moon (Beyotime Institute of Biotechnology) was used to visualize signals. The Image Lab software (Bio-Rad Laboratories, Inc.) was utilized to quantify protein abundance.

Individual rat was used as the experimental unit, and all data were analyzed by SPSS 20.0 (SPSS, Inc., Chicago, IL, United States). Statistical differences between groups were determined by Student’s t-test, while among groups, differences were determined by Tukey’s multiple-range test. Results were presented as means ± standard deviations. Differences were taken to indicate significance when p < 0.05.

From the results of HP-GPC detection, the mass average molar mass (Mw) of MIF was 6.666 × 10⁵ g/mol, the number average Molecular Weight (Mn) was 6.118 × 10⁵ g/mol, and the D value (Mw/Mn) was 1.09. The dispersity ratio was close to 1, and the molecular weight distribution was narrow, indicating that the MIF was relatively pure (Table 1).

DSS challenge enhanced (p < 0.05) the DAO activity and increased the concentration of D-lactate in C57BL/6J mice (Table 2). Dietary 200 mg/kg MIF inclusion reduced (p < 0.05) the serum D-lactate concentration in DSS-challenged mice.

Relative to the control mice, DSS challenge was found to reduce (p < 0.05) the colonic villus height without affecting crypt depth and VCR (Figure 1). Between the DSS-challenged mice, 100 and 200 mg/kg MIF supplementation increased (p < 0.05) the colonic villus height, and 200 mg/kg MIF supplementation additionally increased colonic VCR.

According to Table 3, it is found that DSS challenge decreased (p < 0.05) the SOD, CAT, and T-AOC activities and increased the MDA content in the colon of C57BL/6J mice. Supplementation with 100 and 200 mg/kg MIF increased (p < 0.05) the colonic SOD and CAT activities in DSS-challenged mice.

Dietary 200 mg/kg MIF ingestion reduced (p < 0.05) the contents of the IL-1β, TNF-α, and IFN-γ and increased (p < 0.05) the IL-10 content in colonic mucosa of DSS-challenged mice (Table 4). Moreover, 100 mg/kg MIF supplementation increased (p < 0.05) the colonic mucosal IL-10 concentration in DSS-challenged mice.

Figure 2 shows the effects of MIF on tight junction protein (occludin, claudin-1, ZO-1, and ZO-2) abundances in DSS-challenged mice. DSS challenge decreased (p < 0.05) the abundances of occludin, claudin-1, and ZO-1 proteins. Dietary supplementation with 100 and 200 mg/kg MIF elevated (p < 0.05) the abundance of claudin-1 protein, and 200 mg/kg MIF also increased (p < 0.05) the abundances of occludin and ZO-1 proteins in DSS-challenged mice.

The differences in colonic Nrf2 pathway-related protein abundances among the four groups are shown in Figure 3. The colonic protein abundances of p-Nrf2 and HO-1 were lower in the DSS group (p < 0.05) than that in the control group. However, supplementation with 100 and 200 mg/kg MIF increased (p < 0.05) the colonic protein abundances of p-Nrf2 and HO-1 in DSS-challenged mice. Neither DSS nor MIF affected (p > 0.05) the Keap1 and NQO-1 protein abundances in C57BL/6J mice.

Figure 4 shows that the DSS challenge elevated (p < 0.05) the TLR4, MyD88, IRAK1, TRAF6, and p-NF-κB protein abundances, whereas supplementation with 200 mg/kg MIF reduced (p < 0.05) the TLR4, MyD88, IRAK1, TRAF6, and p-NF-κB p65 protein abundances in DSS-challenged mice. Moreover, 100 mg/kg MIF downregulated (p < 0.05) the TLR4 protein abundance in DSS-challenged mice.

According to Table 5, it is found that DSS treatment decreased (p < 0.05) the Shannon index and Simpson index of bacteria in C57BL/6J mice. Supplementation with 200 mg/kg MIF increased (p < 0.05) the Shannon index and Simpson index of bacteria in DSS-challenged mice. Neither DSS nor MIF affected (p > 0.05) the Chao1 index or abundance-based coverage estimators (ACE) index of bacteria in C57BL/6J mice.

As shown in Figure 5, the PCoA revealed that microbial community was significantly altered after DSS challenge or MIF supplementation, with an evident separation (p < 0.05) among the three groups.

The bacterial composition was assessed at different taxonomic levels (Figure 6 and Supplementary Table 1). At the phylum level, the dominant bacterial groups were Bacteroidetes, Firmicutes, and Proteobacteria; these were followed by the bacteria from phyla Verrucomicrobia, Fusobacteria, Actinobacteria, Deferribacteres, Tenericutes, and Melainabacteria. DSS challenge decreased (p < 0.05) the abundances of Bacteroidetes and Verrucomicrobia, increased (p < 0.05) the abundances of Firmicutes, Proteobacteria, Deferribacteres, and Melainabacteria. However, 200 mg/kg MIF supplementation increased (p < 0.05) the abundances of Proteobacteria, Deferribacteres, and Melainabacteria.