Morphine sulphate

LY2456302 (Diaz-Buezo et al., 2009 ; Mitch et al., 2011 (link)) and GR103545 were synthesized at Lilly Research Laboratories (Indianapolis, IN). Naloxone HCl, naltrexone HCl, naltriben methanesulfonate hydrate (naltriben), and morphine sulphate (morphine) were purchased from Sigma Aldrich (St. Louis, MO). LY2456302 was dissolved in sterile water with the drop-wise addition of lactic acid and was orally dosed (3mL/kg). Naloxone and morphine were dissolved in 0.9% saline and dosed subcutaneously or intraperitoneally, respectively (1mL/kg). For in vivo receptor occupancy studies, nonlabeled naltrexone (10 µg/kg), naltriben (10 µg/kg), and GR103545 (1.5 µg/kg) were dissolved in saline and dosed IV, as tracers for mu, delta, and kappa receptors, respectively, in a single solution (0.5mL/kg).

Cyproheptadine (Sigma-Aldrich, Saint Louis, MO, USA), Acetic acid (Cromato, Diadema, Brazil); Formaldehyde (Dinamica, Diadema, Brazil); Croton oil (Sigma-Aldrich, Saint Louis, MO, USA); Lambda-carrageenan type IV (Sigma-Aldrich, Saint Louis, MO, USA); Morphine sulphate (Cristália, Itapira, Brazil); Indomethacin (Sigma-Aldrich, Saint Louis, MO, USA); Ethanol (Tedia, Fairfield, OH, USA); hexane (Tedia, Fairfield, OH, USA); ethyl acetate (Tedia, Fairfield, OH, USA); acetonitrile (Tedia, Fairfield, OH, USA); and mEthanol (Tedia, Fairfield, OH, USA).

Diclofenac sodium, morphine sulphate, naloxone, carrageenan, histamine phosphate, indomethacin, gallic acid, quercetin, pentobarbitone, and Folin-Ciocalteu phenol reagent were collected from Sigma Chemical Co. (St. Louis, MO, USA). Ethanol and formalin were purchased from E. Merck, Germany. Analytical grade Tween 80, sodium carbonate, aluminum chloride, and potassium acetate were bought from E. Merck, India, Ltd.

After baseline nociceptive testing, morphine sulphate (Sigma) was administered to mice subcutaneously (s.c.) 20 mg/kg twice per day on days 1–3 and 40 mg/kg twice per day on day 4 in 50–100 μl volumes of 0.9 % NaCl. This protocol has been used to demonstrate opioid tolerance, dependence, hyperalgesia and other maladaptive phenotypes in mice in previous experiments [13 (link), 18 (link)].

YHS was dissolved in a vehicle solution of cremophor EL: ethanol: saline (2:1:17). Morphine sulphate (Sigma-Aldrich) was dissolved in saline. Dehydrocorybulbine (DHCB) was synthesized as previously reported [16 (link)] and dissolved in saline. L-tetrahydropalmatine (l-THP, Xi,an Xiaocao Botianical Development Co.,LTD) was dissolved in saline. YHS (100, 200, 250, 500 mg/kg, i.p., 5 ml/kg) and morphine (10 mg/kg, i.p., 5 ml/kg) were administered at different time points depending on the assays described in detail below. We also compared YHS efficacy to that of a mixture of l-THP and DHCB. YHS contains approximately 0.25% of l-THP and 0.18% of DHCB [16 (link)]. Therefore, a 500 mg/kg YHS administration (i.p., 5 ml/kg) was compared to the administration of a mixture of 1mg/kg l-THP (i.p., 5 ml/kg) and 1mg/kg DHCB (i.p., 5 ml/kg) in the tail flick assay. Both Solutions were administered 30 minutes before the assay.

16HBE14o cells were cultured at 37 °C with 5% CO₂ in 2% fibronectin coated culture-ware in Bronchialife supplemented media (Lifeline Cell Technology, Frederick, MD). For transfection of different plasmids (all constructs from Genecopoea, Rockville, MD) X-tremegene HP transfection reagent (Roche, Indianapolis, IN) was used according to the manufacturer’s specifications. For in vitro IL-17 measurements, the cells were preconditioned with 1 μM morphine sulphate (Sigma, St. Louis, MO) or PBS for 24 hours and treated with 2.5 μg/ml LTA (Sigma, St. Louis, MO) for 0, 0.5, 1, 2, 3 and 6 hours. At each time point, the culture supernatant was saved and ELISA was performed using IL-17 easy-set-go ELISA kit (ebiosciences, San Diego, CA) as per manufacturer’s instructions. For in vitro luciferase assays, 16HBE14o cells were transfected with IL-17 reporter construct (firefly luciferase) and control (renilla luciferase), pre-conditioned with saline or 1 μM morphine and treated with 2.5 μg/ml LTA for 0, 0.5, 6, 12 and 24 hours. Luciferase activity, as a surrogate for IL-17 synthesis, was performed using dual luciferase kit (Promega, Madison, WI).