Prostaglandin D2

Compound UK4b was synthesized, purified, and characterized as previously reported^{27 (link)}. The final sample of the compound had a purity of > 95%. Oxycodone hydrochloride was ordered from Sigma-Aldrich (St. Louis, MO). 1% λ-carrageenan (Sigma-Aldrich, St. Louis, MO) was prepared in saline (0.9% sodium chloride)^{47 (link)}. Complete Freund’s Adjuvant (CFA) was purchased from Chondrex, Inc. (Woodinville, WA). Male CD-1 mice (28–32 g) and Sprague–Dawley rats (200–275 g) were ordered from Harlan (Envigo, Indianapolis, IN). All the animal experiments were conducted in our animal laboratories within the University of Kentucky’s Division of Laboratory Animal Resources (DLAR) facility (PHS assurance number A3336-01; USDA number 61-R-0002; AAALAC, Intl. Unit # 13). Veterinary care and animal husbandry were provided and supervised by the staff of the DLAR facility. All animals were housed in clean, adequately-sized, plastic cages at 21–22 °C and were allowed ad libitum access to food and water for one week before experiments. They were monitored daily by the study staff and by members of the veterinary staff for general health to detect signs of discomfort due to testing and/or the administration of drugs. All animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the National Institutes of Health (NIH), and were in fact also consistent with the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines (https://arriveguidelines.org). The animal procedures used in this study had been approved by the University of Kentucky’s Institutional Animal Care and Use Committee (IACUC).The Ugo Basile Thermal Plantar Instrument (Stoelting, Wood Dale, IL) was used to measure the paw withdrawal latency (PWL) to a noxious heat source. Paw edema of rats was measured with the Digital Water Plethysmometer (Harvard Apparatus, Cambridge, MA). Mechanical thresholds were measured with von Frey filaments (Health Products for You, Brookfield, CT) using the Up-Down Method. Pharmacokinetics (PK) of UK4b were measured in serum using an Agilent HPLC system 1200 Series G1311A Quaternary Pump, 1100 Series G1329A ALS Autosampler, and 1260 Series G1314B Variable Wavelength Detector. Pharmacodynamic (PD) effects of UK4b on prostaglandin E2 (PGE₂), prostaglandin I2 (PGI₂), thromboxane A2 (TXA₂), prostaglandin F2α (PGF_2α), and prostaglandin D2 (PGD₂) were measured using commercially available enzyme-linked immunosorbent assay (ELISA) kits from Cayman Chemical (Ann Arbor, MI): Catalog #514531 for PGE₂, #512011 for PGD₂, #515211 for PGF_2α, #515211 for PGI₂, and #501020 for TXA₂ in rat tissue samples; catalog #514531 for PGE₂ in mouse tissue samples.

Additional rats (n ≥ 4 per group for each prostanoid) were used to collect necessary paw tissue samples in the model of carrageenan-induced paw edema and hyperalgesia. The collected paw tissue samples were analyzed using the ELISA kits (as noted above) to determine the PGE₂, PGD₂, PGF_2α, PGI₂, and TXA₂ levels according to the vendor’s instructions. While each kit requires different steps to purify the sample, the timing of administrating Uk4b and carrageenan, timing of sacrifice, and method of tissue collection remain the same across all five kits. The carrageenan groups receive a subcutaneous (SC) injection of 100 μl 1% λ-carrageenan in saline to the plantar surface of one hind paw at time 0 h (h). In the pre-treatment with UK4b + carrageenan group, rats received an IP injection of UK4b (10 mg/kg) 4 h before carrageenan administration (at − 4 h). In the negative control group, no treatment was administered, but the animals followed the same timing as the other groups. All rats were sacrificed at 26 h, then the skin, muscle, and connective tissue were excised from the plantar and dorsal surface of the paw that received carrageenan.

The samples, mixed with eight females or males per repeat, were crushed and homogenized in the Phosphate Buffered Saline (PBS) solution (0.01 M, pH 7.4). Three repeat samples were used for each treatment. The homogenate was centrifuged at 5000 rpm/min for 10 min and the supernatant was separated. The collected supernatant was then stored at −80 °C. The levels of the total PGs, PGE2, PGD2, 11β-13,14-dihydro-15-keto PGF2α (PGFM), 5-iPF2a-VI, and ROS were detected by commercial ELISA kits (Shanghai Hengyuan Biological Technology Co., Ltd., Shanghai, China) according to the manufacturer’s instructions. These kits employed HRP-conjugate mouse antibodies to quantify PGs and ROS levels in samples. Statistical differences were subjected to one-way analysis of variance (ANOVA), followed by Tukey’s multiple comparison test. The statistical significance level was set at p < 0.05.