A total of 36 female Sprague-Dawley rats, (~8 weeks old), were purchased from Shanghai Xipuer-Bikai Laboratory Animal Science (Shanghai, China).

Following 3 days of acclimatization, an endometriosis-inducing surgery was performed. Then all rats were randomly divided into two groups: the model group (n=27) and the sham group (n=9). At 4 weeks following surgery, model rats were further randomly divided into three groups (n=9): ENDO group, BCTC group and vehicle group. BCTC (3875, Tocris Bioscience, Bristol, UK) dissolved in 25% cyclodextrin (332593; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was administered to rats at 10 mg/kg body weight, as previously reported (15–17). The vehicle group were administered with 0.5 ml/100 g cyclodextrin. A tail-flick test was performed prior to surgery, 1 h before and after treatment of BCTC or vehicle groups. Finally, all rats were sacrificed by cervical dislocation and decapitation, and the L1-L6 DRG were rapidly excised. Half of the DRG were fixed in 4% paraformaldehyde for immunohistochemistry. The other half was stored at −80°C for subsequent western blotting and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). L1-L6 DRG were used in the present study for the reason that the tail and pelvic visceral pain afferents project to this area (18). The flow chart of the experiment is illustrated in Fig. 1. All the experimental procedures were approved by the institutional experimental animals review board of Shanghai Gynaecology and Obstetrics Hospital, Fudan University (Shanghai, China).

The rats were anesthetized with 300 mg/kg chloral hydrate. For the model group, following laparotomy, left uterine horns were ligated using 7/0 silk suture and excised. Tubular segment was incised to expose the endometrium, then cut into two small pieces (~3 mm in diameter). For the sham group, same size of fat tissues were excised from each abdomen. Two pieces of uterine or fat tissues were sutured onto the peritoneum with a 7/0 silk suture. At the end, the midline incision was closed with a 4/0 silk suture. In order to stimulate the growth of implanted endometrium, all rats received 0.2 mg/kg 17β-estradiol (E2758; Sigma-Aldrich; Merck KGaA) in subcutaneous injection every 2 days for the next 2 weeks. Meanwhile, all rats were given penicillin (40,000 U/d) intramuscularly for 3 days to avoid intraperitoneal infection (19).

A beam of high-intensity light of tail-flick Meter (Anhui Zhenghua Biological Instrument E quipment Co., Ltd., Anhui, China) was given focused on the tail 2 cm distal to the tip while gently restraining the animal by hand. The time (in sec) from the start of the lighting to the flick of tail from the heat source was evaluated. The average of three readings was used as the tail-flick response latency for each animal. The basal response latency was set as 2 sec, and 10 sec was set as the cut-off time to avoid tissue damage. Tail flick latency was presented as the percentage of maximal possible effect (MPE). MPE%=[(test response time-basal response time)/(cut-off time-basal response time)]x100%.

Following tissue collection, DRG tissues were fixed in 4% paraformaldehyde overnight at room temperature, prior to sectioning into 4-µm thick sections, which was performed by Servicebio (Wuhan, China). The paraffin sections were dried at 65°C for 2 h, following routine deparaffinization and rehydration procedures. Tissue slides were immersed in citric acid (0.1 mol/l) and boiled at 98°C for 20 min before left cooling naturally at room temperature. The slides were incubated with 10% goat serum in order to block nonspecific binding agents. The rabbit polyclonal antibodies against CGRP (ab47027; Abcam, Cambridge, MA, USA), TRPV1 (ab63083; Abcam), SP (sc9758; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) using as primary antibodies, were diluted to 1:200, 1:100, 1:50, respectively. Sections were incubated with antibody overnight at 4°C in a humidified chamber. The biotinylated secondary antibody (1:300; MR-M100, MingRui BioTech, Shanghai, China) was incubated for 30 min at room temperature. The streptavidin-peroxidase system was used. DAB was stained until appropriate for microscopic examination. For negative control slides, the primary antibody was replaced with PBS.

Following the manufacturer's instructions, total proteins were extracted using radioimmunoprecipitation assay reagent (Beyotime Institute of Biotechnology, Haimen, China), the bicinchoninic acid assay (Beyotime Institute of Biotechnology) was used to determine concentration of the protein lysate. Following denaturation in loading buffer, the protein lysate was separated by 15% SDS-PAGE gel and transferred onto nitrocellulose membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked with 5% non-fat milk for 1 h and then incubated with the primary antibody against TRPV1 (1:2,000), SP (1:500), CGRP (1:500) and β-actin (1:4,000; ab8229; Abcam) overnight at 4°C. Following washing in TBS containing 1% Tween-20 (TBST), the membranes were incubated with an anti-rabbit horseradish peroxidase-conjugated secondary antibody (1:5,000; sc2357; Santa Cruz Biotechnology, Inc.) for 1 h at room temperature. Following three washes with TBST, the blots were visualized using an electrochemiluminescence system (Image Quant LAS 4000 mini analyzer; General Electric, Fairfield, CT, USA) with Image Quant LAS 4000 mini control software (version 1.2; General Electric). The relative band intensities were analyzed using Image Lab software (version 4.0; Bio-Rad Laboratories, Inc., Hercules, CA, USA). The experiment was repeated three times.

Total RNA was extracted from L1-L6 DRG using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's protocols. First Strand cDNA was synthesized using Maxima First Strand cDNA Synthesis kit (Thermo Fisher Scientific, Inc.). The RT-qPCR was performed in an ABI PRISM 7000HT system (Applied Biosystems; Thermo Fisher Scientific, Inc.) in triplicates using the SYBR Green Master Mix (Takara Biotechnology Co., Ltd., Dalian, China). The primers used in RT-qPCR reactions are presented in Table I. The PCR conditions were as follows according to the protocol: 15 sec at 95°C, 1 cycle; 5 sec at 95°C and 34 sec at 60°C, for 40 cycles. β-actin served as the internal control. The 2^−ΔΔCq method was used to calculate the relative mRNA levels (20).

Data are expressed as the mean ± standard deviation, Student's t test was used to determine the significance of differences between two groups. The comparison among three groups was analyzed by one-way analysis of variance. P<0.05 was considered to indicate a statistically significant difference. All computations were made with SPSS software (version, 17.0; Chicago, IL, USA).

All rats under endometriosis surgery exhibited 1 or 2 cysts in transplanted sites (Fig. 2A) except one rat eliminated because of failing to form the cyst in the vehicle group. There was no formed cyst in the rat of sham group (Fig. 2B).

In tail-flick test 1, there was no significant difference in MPE% between the sham group and the model group (P=0.996) before sham or endometriosis surgery. In tail-flick test 2, four weeks following the surgery, the model group exhibited a significant decrease in MPE% compared to pre-surgical baseline (P<0.01). No significant changes were observed in MPE% of sham group (P=0.081; Fig. 3A). In tail-flick test 3, rats in the BCTC group had a significant improvement in tail-flick latency 1 h following BCTC intervention (P<0.01), while there were no significant changes for rats in the vehicle group compared with MPE% before intervention (P=0.069; Fig. 3B).

TRPV1 staining was primarily localized in the cytoplasm of DRG cells. Increased immunostaining of TRPV1 was observed in the DRG cells of rats with endometriosis compared with rats in sham group. Treatment with BCTC resulted in a reduction of immunoreactivity of TRPV1 in the DRG of rats with endometriosis (Fig. 4).

As is presented in the bar graph of Fig. 5, the relative expression levels of TRPV1, SP and CGRP protein in DRG were significantly higher in the ENDO group compared with sham group (all P<0.05). TRPV1, SP and CGRP expressions were decreased in the BCTC group compared with the ENDO group (P<0.01). There was no significant difference in the relative expression levels of TRPV1 (P=0.998), SP (P=0.841) and CGRP (P=0.801) in the vehicle group compared with the ENDO group.

The mRNA levels of TRPV1, SP and CGRP in DRG were significantly higher in the ENDO group compared with sham group (all P<0.01). Expressions of TRPV1, SP and CGRP mRNA were significantly lower in the BCTC group compared with the ENDO group (all P<0.01). There were no significant differences in TRPV1, SP and CGRP mRNA levels between vehicle group and ENDO groups (P=0.273, 0.629, 0.928, respectively) (Fig. 6A).