Neomycin

Broad-spectrum antibiotic treatment was performed according to previous publications.^{35 (link),36 (link)} Mice were fed by way of gavage with 0.2 ml broad-spectrum antibiotics at a concentration of 10 mg/mice/day including ampicillin (Solarbio), neomycin (Solarbio), metronidazole (Solarbio) and vancomycin (Lilly, Inc.) for 5 days to ensure equal doses taken by each mouse, then the antibiotics administered in drinking water (ampicillin, neomycin, and metronidazole: 1 g/L; vancomycin: 500 mg/L) until the end of the experiment to avoid too much stress which would have been induced by long-term gavage. Drinking water was exchanged twice a week. For the control group, saline was administrated in the same way as the antibiotics.

S-amlodipine was purchased from Beijing DehangWuzhou Technology Co., Ltd. Metronidazole, vancomycin, ampicillin, and neomycin were obtained from Beijing Solarbio Biotechnology Co. Ltd. HepG2 and BRL cells were obtained from Procell Life Science & Technology Co. Ltd. LPS derived from E. coli O55:B5 (L2880; Sigma) was purchased from Sigma-Aldrich Co., Ltd.
At weekly intervals, the rats were kept in an empty cage without bedding for 20 min to collect fresh stool samples into sterile 4 ml centrifuge tubes, which were rapidly transferred to lab and stored at −80°C until analysis.

Cells were treated by the procedures described in “Inhibition of NF-κB signaling pathway”，IKK inhibitor was used instead of neomycin (Solarbio, Beijing, China) and the nuclear protein was extracted.

We used the following materials: Con A was acquired from Sigma-Aldrich (St. Louis, MO). BBR, alcian blue stain kit, methylprednisolone (MP), ampicillin, neomycin, metronidazole, and vancomycin were afforded by Solarbio (Beijing, China). Anti-Caspase-3, anti-Bax, and anti-F4/80 antibodies were offered by cell signaling technology (Boston, MA). Anti-TNF-α, anti-interferon-γ, anti-IL-1β, anti-IL-17A, and anti-IL-10 antibodies were purchased from Bioss (Beijing, China). Rabbit anti-mouse primary antibodies, including zonula occludens-1 (ZO-1), occludin, TLR4, NF-κB P65, inhibitor of NF-κB (IκB), phospho-inhibitor of NF-κB (p-IκB), and β-actin, were obtained from Abcam (Cambridge, UK). Allophycocyanin-conjugated anti-CD4, fluorescein isothiocyanate–conjugated anti-IL-17A, and phycoerythrin-conjugated anti-Foxp3 antibodies were purchased from BD (Franklin Lakes, NJ). LPS ELISA kits were supplied by Cloud-clone (Hubei, China).

Eighteen recipient rats were treated with 200 mg/kg of the antibiotics: bacitracin (No. B8181, Solarbio), neomycin (No. N8090, Solarbio), and streptomycin (No. S8290, Solarbio) (abbreviated as ABX forthwith). ABX were administered for 6 days to eliminate intestinal microbiota and construct a pseudo-germ-free rat model (Wichmann et al., 2013 (link); Fan et al., 2018 (link); Liao et al., 2021 (link)). To confirm the successful creation of this model, 16S rRNA sequencing was performed on rats that had been treated with the broad-spectrum ABX.
Based on pseudo-germ-free rats, the STC model was established by administering loperamide hydrochloride (ABX + STC) for 1 week (Schuijt et al., 2016 (link); Zeyue et al., 2022 (link)). After successful modeling, based on the different sources of donor rat microbiota, the ABX + STC rats were randomly divided into three groups: Control → ABX + STC, STC → ABX + STC, and STC + TBD → ABX + STC group. The fecal suspension of donor microflora was transplanted into recipient rats by fecal microbiota transplantation (FMT) at a concentration of 1 mL/100 g body weight by intragastric administration over a 6-day period (Yano et al., 2015 (link)).