AA was purchased from Sigma-Aldrich (Merck KGaA; Darmstadt, Germany). Pentobarbital was purchased from Chengdu XiYa Chemical Technology Co., Ltd. (Chengdu, China). Progesterone injections were obtained from Zhejiang Xianju Pharmaceutical Co., Ltd. (Taizhou, China). Absorbable gelatin sponges were purchased from Jinling Pharmaceutical Co., Ltd. (Nanjing, China). The pathogenic Escherichia coli strain and Ureaplasma urealyticum strain (t-strain mycoplasma) were obtained from Nanjing Bianzhen Biology Science and Technology Co., Ltd. (Nanjing, China).

A total of 75 female specific pathogen-free Sprague Dawley rats (aged 9 weeks; weighing 220–240 g; Shanghai SLAC Laboratory Animal Co., Ltd., Shanghai, China), which were maintained under the controlled conditions at 23°C with a 12-h light/dark cycle (60% humidity) and access to food and water ad libitum, were assigned randomly to the following five equal groups: A control group, consisting of rats that received vehicle only (propylene glycol, 1.0 mg/kg) via intragastric gavage once daily; a PID group consisting of rats that underwent the model of PID and received an equal volume of vehicle (propylene glycol, 1.0 mg/kg) via intragastric gavage once daily; a PID + AA 5 group consisting of rats that underwent the model of PID and received 5 mg/kg AA in vehicle (1.5 ml) via intragastric gavage once daily; a PID + AA 35 group consisting of rats that underwent the model of PID and received 35 mg/kg AA in vehicle via intragastric gavage once daily; and a PID + AA 75 group consisting of rats that underwent the model of PID and received 75 mg/kg AA in vehicle via intragastric gavage once daily. All procedures were approved by the Animal Ethics Committee of Zhejiang Chinese Medical University (Hangzhou, China).

A PID model in rats was established based on previously published protocols (13,14). Rats in the study were immediately administered intramuscular injections of buprenorphine (0.01 mg/kg; bid; Tianjin Medicine Research Institute Pharmaceutical Co., Ltd., Tianjin, China) to relieve pain following infection (17). All rats were acclimated for 7 days and subcutaneously injected with 45 mg/kg progesterone (Zhejiang Xianju Pharmaceutical Co., Ltd., Taizhou, China) prior to infection. An absorbable gelatin sponge was saturated with microbe-mixing solution (Nanjing Bianzhen Biology Science and Technology Co., Ltd., Nanjing, China) with U. urealyticum (1×10⁸ cfu/ml) and pathogenic Escherichia coli (1×10⁸ cfu/ml). The upper genital tract of each rat in the PID group was then implanted with a microbe-containing gelatin sponge and rats were inverted for 3 min. The cervixes of rats in the control group were implanted with microbe-free gelatin sponges. A total of four infections were performed every 2 days. Following the first infection, the experimental groups were administrated with AA via intragastric gavage and the PID and control groups were gavaged with an equal volume of vehicle. At 8 days following the first infection, rats were intravenously anesthetized with pentobarbital at a dose of 30 mg/kg. Following anesthesia, the right fallopian tube and uterus were harvested and stored at −80°C. When rats exhibited severe symptoms e.g. convulsion for a period of >10 min, they were euthanized with via intravenous administration of pentobarbital at a dose of 140 mg/kg as previously described (20).

The right fallopian tube and right uterine horn (n=5 from each group) were used for western blotting. Samples were homogenized and extracted using radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology, Nanjing, China), and then centrifuged at 5,000 × g at 4°C for 30 min. The protein concentration in the supernatant was measured using a bicinchoninic acid assay kit. Soluble lysates (30 µg/lane) were resolved using 10% SDS-PAGE and transferred onto polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). Membranes were blocked with 5% non-fat dry milk at 25°C for 2 h and incubation was performed with the following specific primary antibodies: Anti-NLRP3 (1:1,000; sc-34410), anti-caspase-1 (1:1,000; sc-1597 all Santa Cruz Biotechnology, Inc., Dallas, TX, USA), anti-p-NF-κB p65 (1:500; ab10859369), anti-p-inhibitor of NF-κB (IκB)-α (1:500; ab331284), anti-caspase-3 (1:1,000; ab9664; all Abcam, Cambridge, UK) and anti-β-actin (1:1,000; sc-1616; Santa Cruz Biotechnology, Inc.) at 4°C overnight. Membranes were then rinsed with Tris-buffered saline with Tween and further incubated with the secondary antibody (goat anti-mouse IgG; 1:500; bs12478; Bioworld, Biogottechnology, Co., Ltd., Nanjing, China) for 1 h at room temperature. Detection of specific proteins was performed with an enhanced chemiluminescence kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The levels of protein were analyzed using ImageJ (v2.1.4.7, National Institutes of Health, Bethesda, MD, USA) software.

Samples of the right uterus and fallopian tube were collected and homogenized, and then centrifuged at 5,000 × g at 4°C for 30 min. The concentration of IL-1β (ERC007.96; Neobioscience, Beijing, China), IL-6 (ERC003.96; Neobioscience), TNF-α (ERC102a.96; Neobioscience), MCP-1 (ERC113.48; Neobioscience), chemokine C-X-C motif ligand 1 (CXCL-1; ab219044, Abcam) and chemokine C-C motif ligand 5 (RANTES; ERC105.96; Neobioscience,) supernatants was determined using ELISA kits according to the manufacturer's protocol. Malondialdehyde (MDA; S0131) production and superoxide dismutase (SOD; S0060) activity was measured by commercial kit (Beyotime Institute of Biotechnology) following the manufacturer's protocol. Myeloperoxidase (MPO) activity was assessed using an MPO assay kit (A044; Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

Data are expressed as the mean + standard error of the mean and were analyzed through SPSS software (version 16.0, SPSS Inc., Chicago, IL, USA). Statistical analysis was performed using a one-way analysis of variance followed by Dunnett's post hoc test. P<0.05 was determined to indicate a statistically significant difference.

Levels of proinflammatory cytokines IL-1β (Fig. 1A), IL-6 (Fig. 1B) and TNF-α (Fig. 1C), as well as chemokines MCP-1 (Fig. 1D), RANTES (Fig. 1E) and CXCL-1 (Fig. 1F) were detected. The results indicate that IL-1β, IL-6, TNF-α, CXCL-1, MCP-1 and RANTES expression was significantly increased following infection in the PID group compared with the control group, whereas AA significantly decreased the expression of inflammatory cytokines and chemokines in the PID + AA 35 and PID + AA 75 groups compared with the PID group (Fig. 1). No significant differences were observed between the PID and PID + AA 5 groups, PID + AA 35 and PID + AA 75 groups, PID + AA 35 and control groups, and PID + AA 75 and control groups.

MPO activity was assessed, as it is an oxidative enzyme in neutrophils and acts as a specific marker of neutrophil infiltration (16,17,21) (Fig. 2). The results demonstrated that MPO activity was significantly increased in the PID group compared with the control group, whereas AA significantly inhibited MPO activity in the PID + AA 35 and PID + AA 75 groups compared with the PID group. The differences observed between the PID and PID + AA 5 groups, PID + AA 35 and PID + AA 75 groups, PID + AA 35 and control groups, and PID + AA 75 and control groups were not significant.

To determine the potential mechanisms of AA, the expression of NLRP3, caspase-1 p-NF-κB p65, p-IκB-α, and cleaved caspase-3 were measured. PID induced a significantly higher expression of NLRP3, active caspase-1, p-NF-κB p65, p-IκB-α and cleaved caspase-3 protein in the PID group compared with the control group (Fig. 3). By contrast, these changes were significantly reversed in the PID + AA 35 and PID + AA 75 groups compared with the PID group. However, the levels of active caspase-1, p-NF-κB p65, p-IκB-α and cleaved caspase-3 (only in the PID + AA 35 group) were significantly increased in the PID + AA 35 and PID + AA 75 groups relative to control animals. Furthermore, the differences observed between PID + AA 35 and control groups, and PID + AA 75 and control group on the level of NLRP3 were not significant. No significant differences were observed between the PID and PID + AA 5 groups or the PID + AA 35 and PID + AA 75 groups.

The effects of AA on SOD activity and MDA production were assessed (Fig. 4). There was a significant decrease in SOD activity in the PID group compared with the control group, whereas the MDA level was significantly increased. However, treatment with AA in the PID + AA 35 and PID + AA 75 groups significantly increased the SOD activity and decreased the production of MDA compared with the PID group. However, the levels of MDA were significantly increased in the PID + AA 35 group relative to control animals. No significant differences were observed between the PID and PID + AA 5 groups, the PID + AA 35 and PID + AA 75 groups, and PID + AA 75 and control groups.