Rice bran (RB) feed
Corn, wheat, soybean meal, and other ingredients make up the standard diet, which was sufficient to meet the needs of the test mice. The RB was purchased by Jiangsu Xietong Pharmaceutical Bio-engineering Co., Ltd (Nanjing, China). The process of preparing RB was as follows: firstly, the shell of the rice (japonica rice) was removed to obtain brown rice. Then, the brown rice's outer skin and part of the endosperm were removed to obtain RB. Referring to the additive amount of Yue Sun et al., 20% of the RB was added to the regular diet to create the RB feed, this additive amount is in line with the human consumption amount [19]. To meet the needs of specific pathogen-free experimental mice and eradicate harmful bacteria, both regular feed and RB feed must be sterilised and exposed to radiation. Supplementary Table S1 displays the nutrient content of ordinary feed and RB.
Animal experiments and methods
The animal experiments complied with the relevant regulations of the Animal Ethics Committee of Nanjing Agricultural University (SYXK 2017-0007). In the tests, male C57BL/6J mice (Charles River Laboratory Animal Technology Co., Ltd, Beijing, China) weighing 18–10g and aged 6–8 weeks were employed. The temperature and humidity levels of the habitat where the animals were kept ranged from 20–23°C to 40–70%. The mice were first divided into four groups of eight mice each at random: control (CN) group、DSS (DSS) group、sulfasalazine + DSS (SU + DSS) group and rice bran + DSS (RB + DSS) group. After that, mice were kept in the animal room and given unlimited access to food and water for a week to aid in their acclimation. After one week of acclimatization, the gavage samples and feed for each group of mice were : (1) CN group: oral 0.4 mL 0.85% normal saline and common feed; (2) DSS group: oral 0.4 mL 0.85% normal saline and common feed ; (3) SU + DSS: oral dose of 50 mg/kg sulfasalazine [20] and common feed. (4) RB + DSS: oral 0.4 mL 0.85% normal saline and RB feed. For the final seven days of the experiment, all groups—aside from the control group—had their drinking water replaced with a 2% DSS solution. The experimental flowchart is shown in Supplementary Fig. S1.
Beginning on day fourteen, each mouse's weight was noted and their faecal was gathered. The disease activity index (DAI) score [21] and organ Indices [22] were computed using Supplementary Table S2's DAI scoring criteria. At the end of the experiment, blood samples were collected from the eyes of anaesthetized mice. Immediately after dissecting the mice, the livers, spleens, and kidneys were removed, treated with cold PBS (0.85%), blotted dry using filter paper sheets, and weighed. The colon of each mouse was removed, placed in 4% paraformaldehyde tissue fixative, and stored at -80°C for histopathological staining. The 200 mg of colon tissue and 1.8 mL of sterile saline were ground using an MB-48L tissue grinder (Meibi Instruments Co Ltd., Hangzhou, China) at -4°C, 60 HZ, and ground for 45 s. Then, the colon tissue was centrifuged by Micro 21R centrifuge (Thermo, Waltham, Massachusetts, USA) at 12,000 xg for 10 min, and the colon tissue supernatant was aspirated for antioxidant assay. The contents of the cecum were collected and immediately frozen in liquid nitrogen for the determination of SCFA.
Histological analysis
Histopathological examination was performed using the previously described techniques. The colon tissue samples were dried using ethanol, fixed in 4% paraformaldehyde for 24 h, and then rendered transparent using xylene. After paraffin embedding, the sample was cut into 4 µm pieces. The tissue was then sealed with slightly dried neutral gum after being dehydrated with ethanol and rendered transparent with xylene. Based on earlier research, the histopathology grading method was used to score the histology [23].
Determination of oxidative stress and inflammatory cytokines
Inflammatory cytokines in serum were identified using enzyme-linked immunosorbent assay (ELISA) kits (Solarbio Science & Technology Co., Ltd., Beijing, China). Oxidative stress levels (MDA, GSH, and GSH-Px) in colon homogenates were determined using the appropriate kits (Jiancheng Technology Co., Ltd., Nanjing, China).
Quantification of SCFAs
Using the protocol described in earlier research, the amount of SCFAs was determined [24, 25]. To eliminate solid debris, a 50 mg sample of the cecal contents was obtained and homogenized with saturated NaCl. After acidifying the material with 10% sulfuric acid, it was homogenized for 30 s. The supernatant was collected in a centrifuge tube containing Na2SO4 following the addition of ether and centrifuging at 10000 xg for 15 min. After a ten-minute interval, the supernatant was centrifuged once again for fifteen min at 14,000 xg. After that, the resultant supernatant was put through a membrane filter and 7890 B GC-MS (Agilent Technologies, California, USA) analysis was performed according to the methods given by [26].
RT-qPCR
Colon tissue (10 mg) was ground using a tissue grinder. Reagents for RNA extraction and RT-qPCR process were provided by Vazyme Biotechnology, Nanjing, China [26]. The primer sequences are listed in Supplementary Table S3. Use of GAPDH as an internal reference gene.
Analysis of gut microbiota
DNA was extracted from mouse faeces using a DNA extraction kit (kits were provided by Vazyme Biotechnology, Nanjing, China). The DNA samples were transferred to Majorbio Biopharm Technology Co., Ltd. (Shanghai, China) for high-throughput sequencing after they had been purified. PCR amplification of the V3-V4 region of bacterial 16S rDNA.