Thirty healthy female nonpregnant Kunming mice (18–22 g) were purchased from Hunan Slack Jingda Lab Animal Co., Ltd. [Hunan, China, SYXK (Xiang) 2013-0004] All animals were kept on natural circadian rhythms at optimal temperature (20°C–25°C) and humidity (50%–70%), with free access to water and food. After a week, the 30 mice were divided into three groups (Saline group, PD group, and MCC950 group), with 10 mice in each group. All experimental procedures were approved by the Instructive Notions with Respect to Caring for Laboratory Animals promulgated by the Ministry of Science and Technology of China in 2006.
Pharmaceutical and main reagents
Oxytocin was purchased from Shanghai Hefeng Pharmaceutical Co., Ltd. (No. H31020850, Shanghai, China), and estradiol (No. J20130009, Bayer, Germany) was purchased from Bayer. Both are approved by the State Food and Drug Administration (SFDA). Other reagents and materials included MCC950 (CP-456773, Selleck, USA; CAS number: 210826-40-7; formula: C20H24N2O5S; molecular weight: 404.48 g/mol), mouse PGF2α kit (production lot number: 516011, Cayman Chemical, USA), mouse PGE2 kit (production batch number: 514010, Cayman Chemical, USA), BCA protein quantification kit (production lot number: A53225, Thermo, USA), NLRP3 antibody (production batch number: NBP2-12446, Novus Biological, USA), phospho-NF-κB-p65 antibody (production batch number: #3033, Cell Signaling Technology, USA), NF-κB-p65 antibody (production lot number: #4746, Cell Signaling Technology, USA), IL-1β antibody (production batch number: ab9787, Abcam, UK), IL-18 antibody (production lot number: ab191860, Abcam, UK), caspase-1 antibody (production batch number: NBP1-45433, Novus Biological, USA), beta-actin mouse monoclonal antibody (production lot number: #4211, Cell Signaling Technology, USA), goat anti-rabbit secondary antibody (production batch number: AP132P, Merck Millipore, Germany), and goat anti-mouse secondary antibody (production lot number: AP124P, Merck Millipore, Germany).
Construction of the PD mouse model
Mice in the Saline group were given 0.2 ml saline by gavage at 18:00 for 10 days and intraperitoneally injected with 0.2 ml saline on the 11th day. To mimic PD in vitro, mice in the PD group and MCC950 group were given estradiol (0.2 mg/ml, 0.2 ml/day) by gastric gavage at 18:00 for 11 days. Intraperitoneal injection of 0.2 ml MCC950 (2 mg/ml) was given to mice in the MCC950 group. On the 11th day, both groups were given estradiol via intraperitoneal injection and 0.2 ml oxytocin (2 U/ml) within an hour . All mice were fasted for 12 h before gavage. After daily intervention, they had free access to water and food.
Measurement of writhing times
To assess the writhing response, we recorded 20-min writhing times and the writhing latency of mice. (Writhing response: the abdomen of the mouse was contracted and concave with straight hind limbs; writhing latency: from intraperitoneal injection of oxytocin to the first writhing reaction .)
Thermal tail-flick test
The thermal tail-flick test of acute pain was performed on the 11th day after pharmaceutical injection. Each experimental mouse was placed in a fixator with the tail exposed. When it was stable, 1/3rd of the tail was placed in hot water at 52°C. The tail-flick latency (from the tail entering the water to exiting) was measured with a stopwatch (accuracy: 0.01 s) for 4 consecutive times each with an interval of 1 min . Each tail was measured in triplicate, and the average value was calculated.
Enzyme-Linked Immunosorbent Assay (ELISA)
The levels of PGF2α and PGE2 in uterine tissues were determined with an ELISA. The supernatant including total protein (TP) was obtained by homogenization and centrifugation from the same segment of ipsilateral uteruses of 5 mice randomly selected in each group. Each sample was incubated overnight with diluent and standard solution at 4°C. Subsequently, the plate was washed and Ellman's reagent was added, it was reacted at 4°C in the dark for 60 min, and the absorbance value of each hole at 412 nm was measured. The concentration of PGF2α was determined by the standard curve according to absorbance value of the standard hole and its corresponding concentration. The TP in collected supernatant was quantified by a BCA protein quantitative kit. Each sample was added with prepared standard compound based on the gradient method and incubated with working solution at 37°C for 30 min, then the absorbance value of each hole at 412 nm was measured. The concentration of TP was determined by the standard curve according to absorbance value of the standard hole and its corresponding concentration. The ratio between PGF2α/PGE2 and TP indicated the content of PGF2α in the supernatant of uterine homogenate. All procedures were according to the manufacturer’s protocol.
The supernatant containing TP of the collected mouse uterine tissues was separated by homogenization using liquid nitrogen and 1 ml cold lysis buffer for 10 min, followed by centrifugation at 4°C and 12,000 r/min for 10 min. To fully denature TP, the supernatant was added with sample buffer and heated in a boiling water bath for 10 min. The calculation and balance of protein concentration, together with the protein standard curve, was based on protein quantification via a BCA protein quantification kit. Samples were subjected to electrophoresis, membrane transfer, and blocking. Immunoblot analysis was performed with specific primary antibodies followed by secondary antibody: NLRP3 (1:1000), caspase-1 (1:500), IL-1β (1:1000), IL-18 (1:1000), phospho-NF-κB p65 (1:1000), NF-κB p65 (1:1000), COX-2 (1:1000), or β-actin (1:5000). The membrane was then stripped and incubated with β-actin as an internal control. The intensity of chemiluminescence exposed by ECL was measured by Quantity One gray analysis software.
All data were analyzed using the SPSS version 22.0 statistical analysis package. All data are expressed as means ± standard deviation (S.D.) and were analyzed by two-tailed, unpaired, Student’s t-test or ANOVA if appropriate. A value of p < 0.05 was considered statistically significant.