Occludin

The harvested colon was fixed in formalin (4%), and paraffin was used for embedding samples. Serial sections were used for immunohistochemical analysis every 25 sections. For antigen retreat, Tissue sections were boiled in sodium citrate (10 mmol/L, pH 6.0) for 10 min at 100°C and sections were blocked with BSA (Sigma, St. Louis Missouri, United States). The sections were then incubated with primary antibody solutions for ZO-1 (Affinity, Jiangsu, China), Claudin-1 (Bioss, Beijing, China), and Occludin (Affinity, Jiangsu, China). Anti-rabbit antibodies conjugated with fluorescence dyes (Alexa 594 and Alexa 488) were used as the secondary antibody (Boster, Wuhan, China).

The paraffin-embedded colon sections were subjected to hematoxylin and eosin (H&E) as described earlier.^{26 (link)} For Alcian blue-periodic acid-Schiff (AB-PAS) stainings, the paraffin sections were dewaxed with xylene and rehydrated with different concentrations of ethanol. Then, the sections were incubated with Alcian blue solution dye for 5 min, washed under running tap water for 2 min, rinsed with distilled water, and stained with periodic acid solution for 5 min. These were treated with Schiff dye solution dye for 30 min. After washing with running tap water, hematoxylin was used for re-dying for about 1 min, followed by differentiation, back blue, and dehydration. Finally, the stained sections were covered with a cover slide. Following deparaffinization and dehydration of sections, immunohistochemical analyses were performed involving antigen retrieval and application of anti-TRADD, FADD, RIPK1, cCASP3, ZO-1, Occludin, and Claudin-1 primary antibodies (1:200, Affinity, USA). This was followed by incubation with secondary antibodies conjugated with biotin and horseradish peroxidase conjugated streptavidin (Zhongshang Goldenbridge). Post DAB staining, proteins were observed microscopically (Olympus, Japan).

Inulin was obtained from Yuanyebio Biotechnology Co., Ltd, (S11143, Shanghai, China). Ampicillin (Cat# A5354), neomycin (Cat# N6386), metronidazole (Cat# 16677), vancomycin (Cat# V2002) were bought from Sigma Aldrich (St. Louis, MO, USA). Specific antibodies including ZO-1 (1:1000; #AF5145; RRID: AB_2837631), Occludin (1:1000; #DF7504; RRID: AB_2841004), Claudin-3 (1:1000; #AF0129; RRID: AB_2833313) and β-actin (1:1000; #AF7018; RRID: AB_2839420) were purchased from Affinity Biosciences (OH, USA). S100A8 (#70802) and HDAC3 (#60538) antibody were bought from Cell Signaling Technology (Boston, USA). Acetylated-H3 antibody (Cat# ab47915; RRID: AB_873860) was purchased from Abcam (Cambridge, England). Tumor necrosis factor (TNF)-α (Cat# 430915) and interleukin (IL)-1β (Cat# 432615) enzyme-linked immunosorbent assay (ELISA) kits were obtained from Biolegend (San Diego, California, USA). Myeloperoxidase (MPO) (A044-1-1) assay kit was bought from Nanjing Jiancheng Bioengineering Institute (Nanjing, China,).

Dextran sulfate sodium (DSS, MW 36000-50000) was obtained from MP Biomedicals (CA, USA). The myeloperoxidase (MPO) assay kit was obtained from Jiancheng Bioengineering Institute (Nanjing, China). Zonula occludens protein 1 (ZO-1) and Occludin antibodies were obtained from Affinity (Liyang, China). FITC anti-CD4, PE anti-Foxp3, and PE anti-IL-17A antibodies were obtained from Biolegend (CA, USA). CD3e antibody, APC anti-CD25 antibody, Foxp3/transcription factor staining buffer set and IC fixation buffer were obtained from eBioscience™ (CA, USA). CD16/CD32 antibody was obtained from BD Biosciences (CA, USA). STAT3 and phospho-STAT3 (p-STAT3) antibodies were obtained from Cell Signaling Technology (MA, USA). The QuantiCyto^® Mouse TNF-α enzyme-linked immunosorbent assay (ELISA) kit was obtained from NeoBioscience (Shenzhen, China).

Deoxycholic acid (DCA), cholic acid (CA), and vancomycin were bought from Sigma Aldrich (St. Louis, MO, USA). SBI-115, MDL-12330A, and H89 were purchased from MedChemExpress (MCE, USA). The primary antibodies, including phosphorylation-p65 (p-p65, #AF2006; RRID: AB_2834435), p-65 (#AF5006; RRID: AB_2834847), p-IκB (#AF2002; RRID: AB_2834433), IκB (#AF5002; RRID: AB_2834792), Occludin (#DF7504; RRID: AB_2841004), ZO-1 (#AF5145; RRID: AB_2837631), Claudin-3 (#AF0129; RRID: AB_2833313) and β-actin (#AF7018; RRID: AB_2839420) were obtained from Affinity Biosciences (OH, USA). NLRP3 (#15101), ASC (#67824), and IL-1β (#12242) were bought from Cell Signaling Technology (CST, Boston, USA). Goat anti-rabbit or Rabbit anti-mouse secondary antibodies were bought from ImmunoWay Biotechnology Company. Mouse TNF-α (Cat #430915) and IL-1β (Cat #432615) ELISA assay kits were obtained from Biolegend (CA, USA). Myeloperoxidase (MPO) assay kit was bought from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

Tissue protein extraction reagent (78510, Thermo Fisher Scientific) was added to tissues or cells to extract the total proteins, and the protein content was determined using a BCA Protein Assay Kit (23225, Thermo Fisher Scientific, California, USA). The protein samples were separated by 8%, 12%, and 15% SDS‒PAGE and then transferred to PVDF (IPVH00010, Thermo Fisher Scientific) membranes using the wet transfer method. After blocking at room temperature for 3 h with 5% skimmed milk powder, specific primary antibodies were added and samples were incubated at 4 °C overnight. After washing with TBST the next day, blots were incubated with murine or rabbit secondary antibodies for 2 h at room temperature. Finally, the membranes were washed again with TBST, and a Western blot detection system (Tanon 4500, Shanghai, China) was used to measure protein expression. The specific primary antibodies used in this experiment included β-actin (4967S), AMPK (2532S), p-AMPK (2535S), mTOR (2972S), p-mTOR (2971S), p65 (8242S), p-p65 (3033S), p38 (9212S), p-p38 (9211S), p62 (5114S), LC3 (2775S), GFP (ZY60501M), ZO-1 (AF5145), Occludin (DF7504), Claudin-3 (AF0129). Anti-GFP antibody were purchased from Zeye Biotechnology (Shanghai, China), ZO-1, Occludin and Claudin-3 were purchased from Affinity Biosciences, and all other antibodies were purchased from Cell Signaling Technology.