Iκb α

Western blotting assay was performed in accordance with the procedure in a previous study (Dai et al., 2021). The primary antibodies were Nrf2 (Proteintech, 1:1000), HO-1 (Wanleibio, 1:1000), NF-κB P65 (Bioss, 1:1000), phospho-NF-κB (Bioss, 1:1000), IκB-α (Wanleibio, 1:1000), phospho-IκB-α (Bioss, 1:1000), and GAPDH (1:8000; Bioss, China).

Deer antler base powder was purchased from Jilin Zhenyuan Deer Industry Co., Ltd. (Changchun, China) and was identified by Professor Zhao Yan of Jilin Agricultural University. Lipopolysaccharide from Escherichia coli O127:B8 and d-GalN were purchased from Sigma (St. Louis, MO). The aspartate transaminase (AST), alanine aminotransferase (ALT), malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH) detection kits were provided by Nanjing Jiancheng Bioengineering Research Institute (Nanjing, China). The ELISA kits for tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL-1β) were purchased from R&D systems (Minneapolis, MN). Antibodies used for immunohistochemistry and immunofluorescence including COX-2 (ab88522, 1:200), iNOS (ab1789451:200) and 4-HNE (ab46545, 1:100) were purchased from Abcam (Cambridge, UK). Western blot analysis of antibodies ERK (WL01770,1:750), p-ERK (WLP1512,1:750), JNK (WL01295,1:500), p-JNK (WL01813,1:500), p38 (WL00764,1:750), p-p38 (WLP1576,1:750), p-65 (WL01273b,1:750), p-p65 (WL02169,1:750), IκB-α (WL01936,1:500), p-IκB-α (WL02495,1:500) and β-actin (WL01372,1:5000) were all purchased from Shenyang Wanlei Biotechnology Co., Ltd. (Shenyang, China).

An ice-cold radio-immunoprecipitation assay (RIPA) buffer with 1% protease inhibitor and 1% phosphatase inhibitor (Beyotime Institute of Biotechnology, China) was used to homogenize colonic tissues obtained from the control, DSS and FMT groups. Homogenate was centrifuged at 12,000 g at 4°C for 15 min to obtain total protein and its concentrations were measured with the bicinchoninic acid protein assay (BCA, Beyotime, China) method. Sample electrophoresis was carried out on SDS-PAGE gel and then placed on 0.45 μm polyvinylidene fluoride membranes. Membranes were kept for 2 h with 5% fresh nonfat milk at room temperature before incubation with the primary antibodies at 4°C overnight as follows: IκBα (1:400, Wanleibio, China), p-IκBα (1:400, Wanleibio, China), p65 (1:500, Wanleibio), p-p65 (1:1,000, Wanleibio), and β-actin (1:500, Wanleibio,). After washing thrice with saline buffer and Tween, blots were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature, and the immune complexes were detected by using an enhanced chemiluminescence kit (Beyotime Biotechnology). Lastly, the Gel-Pro Analyzer was used to quantify protein levels which appeared as luminescent bands.