Neomycin

Ampicillin, metronidazole, neomycin, and vancomycin hydrochloride were purchased from MedChemExpress (Monmouth Junction, NJ, United States). Antibodies against PERK, p-PERK, and p-eIF2α were purchased from Cell Signaling (Danvers, MA, United States), and antibodies against eIF2α and β-tubulin were from Santa Cruz (Dallas, TX, United States). The enhanced chemiluminescence (ECL) kit was purchased from Thermo Fisher Scientific (Waltham, MA, United States). Mouse TMAO Elisa kit was purchased from Kisong Biotechnology (Beijing, China). Hs-CRP Elisa kit was purchased from Elabscience Biotechnology (Wuhan, China), mouse LPS Elisa kit from Cusabio (Wuhan, China), and 3,3-dimethyl-1-butanol (DMB) from Sigma-Aldrich (St. Louis, MO, United States).

The FMT materials were obtained from the rats in the sixth week of Experiment 2. The rats were stimulated to defecate by gentle means, such as grasping and rectal massage. The fecal pellets were collected directly in a sterile cryotube, quick-frozen in liquid nitrogen immediately, and subsequently stored at −80°C until use. The recipient rats were then given an antibiotic cocktail by gavage daily for 2 weeks until 48 h before FMT. The antibiotic cocktail comprised ampicillin (MedChemExpress, Monmouth Junction, NJ, USA; 110 mg/kg), vancomycin (MedChemExpress; 55 mg/kg), metronidazole (MedChemExpress; 110 mg/kg), and neomycin (MedChemExpress; 110 mg/kg) as previously reported (Yan et al., 2020 (link)). The gavage volume was 2.5 ml/kg, and the antibiotic cocktail solution was thoroughly mixed before each gavage. The fecal pellets collected in phase 1 were added with sterile phosphate-buffered saline (PBS) buffer (10 ml/g, containing 20% sterile glycerol). The fecal samples of each group were fully mixed and resuspended. After the fecal mixture suspension solution was centrifugated at 2,000 rpm at 4°C for 10 min, the supernatants were collected and quickly stored at −80°C until use. The above operations were conducted under sterile conditions. The recipient rats were orally inoculated with 1 ml of prepared fecal contents from donor rats once a day.

Mice were treated with a mixture of four antibiotics in drinking water for 4 weeks^{59 (link)}. The antibiotic mixture consists of ampicillin (200 mg/L), metronidazole (200 mg/L), neomycin (200 mg/L), and vancomycin (100 mg/L), which were purchased from MedChemExpress, China. The presence of microbes in the feces of mice was determined by 16S rRNA.