Aspirin

Ticagrelor (LKT Laboratories, Inc., Saint Paul, Minnesota, USA) and aspirin (Cayman Chemical, Ann Arbor, Michigan, USA) were purchased. Mice were randomly divided into two ticagrelor treatment groups (10 mg/kg and 30 mg/kg), an aspirin treatment group (60 mg/kg), and a vehicle treatment group. Drugs were dissolved in 0.4 mL 1.0% carboxymethylcellulose and administrated by oral gavage at 3 and 24 hours before MCAO and 0 and 6 hours after reperfusion. Mice in vehicle treatment group were administered the same amount of carboxymethylcellulose at same time point.

Caco-2 cells were plated in black 96-well plates at 3 × 10⁴ cells per well and incubated for 48 hours. After 48 hours, cell culture medium was refreshed, and cells were treated with indomethacin, ibuprofen (Cayman), naproxen (Cayman), aspirin, R2PPA, valeroyl silyciate (Cayman), celecoxib (Cayman), PGE₂ (Cayman), or vehicle control [dimethyl sulfoxide (DMSO)] for 16 hours overnight. The next day, cells were intoxicated with filter-sterilized C. difficile VPI10463 cultures at a final dilution of 1:5 or 1:15 or mock-infected (BHI) and incubated for 8 hours. Cell viability was indicated by measuring ATP levels using the CellTiter Glo Reagent (Promega). Data were normalized to ATP levels of vehicle-treated and mock-infected cells.

p53 (#2524), Bcl‐xL (#2762), and GAPDH (#2118) antibodies were purchased from Cell Signaling Technology. COX2 (#160106) antibody was purchased from Cayman Chemical. Doxorubicin was purchased from Sigma. Aspirin and celecoxib were purchased from Cayman Chemical.

All mouse procedures were approved by the Children’s Hospital of Philadelphia Institutional Animal Care and Use Committee. Mice were obtained from The Jackson Laboratory and were C57BL6/J males that were 4 weeks of age at arrival. Mice were acclimated for 7 days, and bedding was mixed upon arrival. After acclimation, mice were given cefoperazone at 0.5 g/liter in the drinking water for 5 days, with changes in antibiotic water every 2 days. After 5 days, water was changed to untreated water.
After the 5-day antibiotic treatment, mice were orally gavaged with indomethacin (Cayman) at 10 mg/kg, aspirin (Cayman) at 10 mg/kg, or R2PPA (Sigma-Aldrich) at 100 mg/kg of body weight or vehicle phosphate-buffered saline (PBS) for two consecutive days. Then, mice were infected with 10⁵ spores of C. difficile ribotype Cd196. Mice were monitored for survival and were euthanized after reaching a terminal endpoint of appearing moribund or experiencing weight loss >20% from baseline. C. difficile colony-forming units were quantified daily from fecal samples. Samples were diluted and homogenized in PBS, serially diluted, and plated onto taurocholate cycloserine cefoxitin fructose agar. All animals were confirmed C. difficile culture negative upon arrival to the animal facility and on the day of infection.