Quercetin and VPA (VPA sodium salt, cat. no. P4543) were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). All of these drugs were dissolved in 2% dimethyl sulphoxide in 0.1 M Na₂SO₄ adjusted to pH 5.0. Tamoxifen citrate was purchased from Fudan Forward Pharmaceutical Company (Shanghai, China).

Newborn ICR mice (day 1 after birth; n=43) and their birth mother mice (n=10) were purchased from Shanghai Laboratory Animal Corporation (Shanghai, China). Each mother mouse and her offspring were housed in one cage under controlled conditions (~20°C, 12:12 light/dark cycle) and had free access to food and fresh water.

Following the protocol of Green et al (11) and Parrott et al (12), female neonatal mice were orally administered 1 mg/kg tamoxifen suspended in peanut oil/lecithin/condensed milk mixture (2:0.2:3, by volume) at a dose volume of 5 µl/g body weight from day 2 to day 5 after birth. Female control neonatal mice (n=9), designated randomly, were fed with the equivalent amount of solvent, without tamoxifen. When mice were 3 weeks old, they were weaned and separated from their mothers, and female mice continued as part of the study.

All experiments were performed under the guidelines of the National Research Council's Guide for the Care and Use of Laboratory Animals (13) and approved by the institutional experimental animals review board of Shanghai Obstetrics and Gynecology Hospital, Fudan University (Shanghai, China).

Neonatal mice dosed with tamoxifen as described (n=34) and 9 control mice were dosed with solvent only. Starting from 4 weeks after birth, a hotplate test was administrated to all mice every 4 weeks, and their body weight was also measured and recorded. At 16 weeks after birth, all mice dosed with tamoxifen were randomly divided into 4 groups, each receiving a certain treatment by daily intragastric administration for 3 weeks as follows: Group L (n=9) received a low-dose (25 mg/kg body weight) quercetin treatment; Group H (n=9) received a high-dose (50 mg/kg body weight) quercetin treatment; Group V (n=8) received VPA treatment (160 mg/kg body weight); Group U (n=8), or untreated group, received the vehicle only. The group of mice (n=9) that were neonatally administrated with the solvent only, were designated as group C, received no treatment at all and served as a blank control. The doses of these drugs were determined based on our previous studies and other studies (10,14–18).

After the 3 weeks of drug treatment, the final hotplate test was performed for all mice with induced adenomyosis and the body weight was also recorded. All mice were anaesthetized and perfused via the ascending aorta with warm (37°C) saline followed by warm solution composed of 4% paraformaldehyde and 0.2% picric acid in 0.1 M phosphate buffer. The perfusion was then immediately followed by the same fixative (4°C). The uteruses were then excised and were post-fixed overnight at room temperature in the same fixation solution, then embedded in paraffin for histopathological examination and immunohistochemical analysis. L6 and S1 dorsal root ganglions (DRGs) were dissected out and the tissues were post-fixed in the same fixative for 90 min at 4°C, and were then immersed in 10% sucrose in 0.1 M phosphate buffer overnight.

The depth of myometrial infiltration of ectopic endometrium was evaluated following the criteria described by Bird et al (19). Briefly, Grade 1 was defined to be the case when penetration of ectopic endometrium to superficial myometrial, Grade 2 as penetration to mid-myometrium and Grade 3 as penetration beyond mid-myometrium. For ease of statistical analysis, Grade 0 of depth was recorded in the complete absence of any ectopic endometrium in the myometrium.

For histological examination, serial 4-mm sections were obtained from each paraffin-embedded tissue block, and then three randomly selected sections were chosen for hematoxylin and eosin (H&E) staining to confirm pathological diagnosis. Sections were stained with 0.6% hematoxylin and 0.5% eosin for 5 min at room temperature. If endometrial glands and stroma were observed to be displaced in myometrium, a diagnosis of adenomyosis was made.

The hotplate test is a commonly used method for measuring nociception in rodents (20), and a test of the response threshold to thermal stimuli, representing a form of acute thermal pain (21). In the current study, the hotplate test was performed using a Hot Plate Analgesia Meter (BME-480; Institute of Biomedical Engineering, Chinese Academy of Medical Sciences, Tianjin, China) consisting of a metal plate of 25×25 cm in size. The surface of the plate can be heated to a constant temperature of 55.0±0.1°C as measured by a built-in digital thermometer, and a plastic cylinder (20 cm in diameter, 18 cm in height) was placed on the hotplate. Mice were brought to the testing room and allowed to acclimatize for 10 min prior to the test. The latency to respond to thermal stimuli, defined to be the time (in sec) elapsed from the moment when the mouse was inserted inside the cylinder to the point when it licked its hind paws. Each mouse was tested only once in one session. The latency was calculated as the mean of two readings recorded at intervals of 24 h.

For all experimental groups, 10 µm-thick sections of the fixed L6 and S1 DRGs were cut in series in a cryostat and mounted on gelatin-coated slides. Slides were stored at −20°C prior to immunostaining. Sections were quenched with 0.3% H₂O₂ for 10 min and then blocked in 0.3% Triton X-100 in 0.05 M Tris-NaCl with 10% normal goat serum (NGS) for 1 h at room temperature. Subsequently, sections were incubated with rabbit polyclonal anti-TRPV-1 (1:5,000; cat. no. acc-030; Alomone Labs, Ltd., Jerusalem, Israel), rabbit monoclonal anti-p-p38 MAPK (1:50; cat. no. 4631; Cell Signaling Technology, Inc., Danvers, MA, USA), or monoclonal anti-ERK1/2 (1:100; cat. no. 4376; Cell Signaling Technology, Inc.) in 0.3% Triton X-100 in 0.05 M Tris-NaCl, including 1% NGS at 4°C overnight. Subsequently, biotinylated secondary antibody and Supervision Universal (anti-rabbit) Detection Reagent (horseradish peroxidase) (1:100; cat. no. GK500705; Shanghai Gene Tech Company, Shanghai, China), was incubated at room temperature for 30 min. The bound antibody complexes were stained for 3–5 min or until appropriate for microscopic examination with 3,3′-diaminobenzidineand then counterstained with 0.6% hematoxylin for 50 sec at room temperature and mounted.

Quantitative analysis was performed according to previous studies (22). Briefly, at least 5 sections from each DRG at each time point were used for quantitative analysis. All mice were sacrificed at the end of the experimental period. The fields were randomly selected in every 3 sections under an Olympus BX51 light microscope (Olympus Corporation, Tokyo, Japan). The number of immunostained neuron profiles was divided by the total number of neuron profiles, and the percentage of labeled neuron profiles was determined. To determine the percentage of labeled neuron within a subset of DRG neurons, the neuron profiles with a clear nucleus were counted.

The computer package SPSS18.0 for Windows (SPSS, Inc., Chicago, IL, USA) was used for all the analyses. Means ± standard deviations of experiments are used to describe statistics. Differences between paired groups were tested using Student's t-test and differences between varied treated groups was analyzed using one-way analysis of variance followed by a post hoc least significant difference test for multiple comparisons. P<0.05 was considered to indicate a statistically significant difference.

The body weight of all the mice at 4, 8, 12 and 16 weeks after the induction of adenomyosis, and 3 weeks after quercetin and VPA treatment were recorded (Fig. 1). The results showed that the body weight of the mice dosed with tamoxifen appeared to be lower than that of the control mice at 4, 8, 12 and 16 weeks; however, no statistically significant difference was detected (Fig. 1A). Following treatment with either the high-and low-dose of quercetin or VPA for 3 weeks, the mice had lower body weight compared to the untreated group, but the difference was not significant (Fig. 1B). Additionally, the high-and low-dose of quercetin and VPA treatment caused a body weight loss compared to the control group, with no significant difference (Fig. 1B).

The effect of quercetin treatment on the myometrium infiltration in mice induced with adenomyosis was evaluated. As the box plots demonstrated in Fig. 2, compared with the control mice, mice dosed with tamoxifen had significantly more infiltration (P<0.01). Mice treated with either low- or high-dose quercetin or VPA had reduced infiltration compared with the untreated mice dosed with tamoxifen. There was no correlation between dosing with quercetin and the depth of myometrial infiltration (P=0.059).

In order to determine the response to of mice to noxious thermal stimuli at 4, 8, 12, 16 and 19 weeks after birth, a hotplate test was used to investigate changes in response latencies. As demonstrated in Fig. 3A-E, the mice dosed with tamoxifen had significantly decreased response latency compared with control mice (P<0.05). Before treatment, there was no significant difference in response latency among groups. Compared to group C, a significant difference at 16 weeks and after treatment of groups U, V, L and H was observed, as presented in Fig. 3D and E. The response latency of the mice of groups H, L and V, was prolonged following VPA and quercetin treatment at 19 weeks compared to the mice at 16 weeks (P<0.05; Fig. 3F). When compared to the untreated mice, the response latency of the mice either treated with high- or low- quercetin or VPA all exhibited significantly prolonged response latency (P<0.05; Fig. 3F).

Immunohistochemical method was used to detect the level of Trpv-1, p-p38 and p-ERK in DRG neurons as presented in Figs. 4–7. Compared with the control group, there was an obvious increase in Trpv-1, p-p38 and p-ERK-positive cells in L6 and S1 neurons of the untreated group (P<0.01; Fig. 4). In animals dosed with either high- or low-dose quercetin or VPA the number of Trpv-1, p-p38 and p-ERK-positive cells were all significantly lower than that of the untreated group (P<0.01; Fig. 4). There was no significant difference among the VPA/quercetin-treated groups and control group. Immunohistochemical staining demonstrated that the levels of Trpv-1, p-p38 and p-ERK present in DRG neurons were significantly higher in mice dosed with tamoxifen compared with the control group (Figs. 4–7). High and low-dose quercetin significantly reduced the expressions of Trpv1, p-p38 and p-ERK compared to untreated group (; P<0.01; Figs. 4–7). High dose quercetin reduced the levels of Trpv1, p-p38 and p-ERK compared to low dose quercetin, however the change was not statistically significant.