Am251

AM251, which is a selective CB1 receptor antagonist (Sigma-Aldrich Co. LLC, St. Louis, MO, USA), was dissolved in dimethyl sulfoxide (DMSO) and further diluted in saline (0.9% NaCl). DMSO concentration was under 10%. This DMSO-saline solution was used as the vehicle. AM251 was administered 3 minutes before the first LFS stimulation each day. The dose of AM251 (0.1 μg/rat) was selected based on the previous studies [20 (link), 21 (link)]. WIN55-212-2 (WIN) was used as the CB1 agonist in our study. Its dose (5 mg/kg) was chosen based on the previous studies [22 (link)], and the preparation process was similar to that of AM251.

The HT22 cell line was a gift from Xuzhou Medical College (Xuzhou, China). The primary anti-CB1 antibody and primary anti-Nox2 antibody were purchased from Abcam Ltd. (Cambridge, UK), the primary anti-cleaved caspase-3 antibody was obtained from Santa Cruz (USA), and bovine serum albumin (BSA) and the cy3-labeled secondary antibody were purchased from Beijing Cowin Bioscience Co., Ltd. (Beijing, China). The AEA, AM251, Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS), apocynin, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-tetrazolium bromide (MTT), and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The 4′,6-diamidino-2-phenylindole (DAPI) and ROS Reagent kit were obtained from Beyotime (Nantong, China). The lactate dehydrogenase (LDH), superoxide dismutase (SOD), and reduced glutathione (GSH) and oxidized glutathione (GSSG) kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

Young rat myocardial I/R injury models were used. Before ischemia, the rats were preconditioned with or without propofol. We further tested the role of receptor signaling in propofol conditioning by intravenous injection of selective CB1R antagonists AM251 (Sigma, 1 mg/kg) or selective CB2R antagonists AM630 (Sigma, 1 mg/kg) 1.5 hours prior to ischemia irrespective of propofol conditioning or not. The dosage and timing of AM251 or AM630 were taken from the study by Hajrasouliha et al. [17 (link)]. The two drugs were used separately in our study which comprised seven groups with six rats for each group. 24 hours after reperfusion, infarct area was evaluated using Evans blue plus TTC staining. Serum cTnI levels were measured 2 hours after ischemia. Oxidative-redox state indicators including serum MDA, MPO, and SOD were also measured at 24 hours of reperfusion.

Nicotine and capsazepine were obtained from Sigma (St Louis, MO, USA), AM251 (1-[2,4-dichlorophenyl]-5-[4-iodophenyl]-4-methyl-N-1-piperidinyl-1H-pyrazole-3-carboxamide) and AM630 ([6-iodo-2-methyl-1-[2-(4-morpholinyl-) ethyl]-1H-indol-3-yl] (4-methoxyphenyl) methanone) were obtained from Tocris (Ellisville, MO, USA).
AM251, AM630, and capsazepine were dissolved in DMSO. Stock solutions of the other drugs were dissolved in distilled water. Solutions were stored at −20 °C until use. The drugs were diluted in distilled water to the required final concentration on the day of use.

DO34—3-(Phenylmethyl)−4-[[4-[4-(trifluoromethoxy) phenyl]−1H-1,2,3-triazol-1-yl] carbonyl]−1-piperazinecarboxylic acid 1,1-dimethylethyl ester (Glixx Laboratories Inc, CAS #:1848233-58-8), AM251 (Tocris), alcohol (Sigma, St. Louis, MO), and tetrodotoxin (TTX, Biotium, Fremont, CA) were diluted to their final concentration in aCSF for acute slice recordings. For behavioral experiments, alcohol, sucrose (Sigma, St. Louis, MO), and quinine (Alfa Aesar, Tewksbury, MA) were diluted in water.