All animal experiments were conducted according to the ethical guidelines of the Ethics Committee of Shandong University of Traditional Chinese Medicine (Jinan, China).

A total of 50 non-pregnant, specific pathogen-free female Wistar rats, weighing 150–180 g and aged 5–6 weeks, were purchased from the Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China; lot no. 2012052307409). These animals were maintained in cages, with a reversed 12-h light/dark cycle (lights on at 9:00 p.m.; lights off at 9:00 a.m.), with free access to water prior to the experiments. The rats were maintained at a temperature of 22–26°C and at a relative humidity of 50–70%.

The rats were randomly divided into control, PMS liver-qi stagnation model, and Shu-Yu treatment groups (n=10 per group). Shu-Yu capsules, which mainly consisted of white peony root, bupleurum, cyperus tuber and licorice root, were purchased from Yangguang Haichuan Pharmaceutical Science and Technology Development Co., Ltd. (investigational new drug approval no. 2008L11169; Qingdao, China). An in vivo model of PMS liver-qi stagnation was established in the PMS liver qi stagnation method rats and the Shu-Yu treatment group using the chronic restraint stress method, as previously described (18). In the treatment group, model rats were subjected to daily administration (9:00 a.m.), of a Shu-Yu capsule via gavage, at a dose of 0.41 g/kg body weight (~8 times the clinical dosage), over the modeling period. Rats in the control and model groups received sterile water at a dose of 10 ml/kg body weight over the same period. In order to assess behavioral alterations, rats were stimulated by glass rod pricking and behavior was judged subjectively.

Primary hippocampal neurons for culture were obtained from ~100 neonatal Wistar rats (Experimental Animal Center, Shandong University of Traditional Chinese Medicine; male:female, 1:1; weight, 5–7 g) within 24 h of birth. The rats were maintained in the conditions described above until they were sacrificed using decollation and their brains were removed. Briefly, the hippocampi were separated under sterile conditions and digested with 0.25% trypsin at 37°C for 20 min. Following filtering with a 200-mesh filter, the cells were seeded into poly-lysine-coated 6-well plates at a density of 4×10⁵/ml, then cultured in a 37°C, 5% CO₂ incubator. After 24 h, the cells were incubated at 37°C with serum-free Neurobasal-A medium (10888–022; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), which was replenished every 3 days. After 7 days, the cells were cultured with Neurobasal-A medium (10888–022; Gibco; Thermo Fisher Scientific, Inc.) containing 10% fetal bovine serum (FBS; 10099-133; Gibco; Thermo Fisher Scientific, Inc.) and B-27 supplement (17504–044; Gibco; Thermo Fisher Scientific, Inc.) for 24 h at 37°C.

To prepare serum from the model rats, the rats were sacrificed via decapitation following modeling and blood serum samples were collected. The serum was inactivated in a 56°C water bath for 30 min and sterilized with a 0.45-mm membrane filter. Then, 200 µl doses of serum (10% V/V) from the control, model and treatment groups were added to each well to incubate the cultured primary hippocampal neurons for 24 h at 37°C. An additional blank group of cells received no treatment.

For the preparation of drug-containing serum, 30 normal male Wistar rats (Experimental Animal Center, Shandong University of Traditional Chinese Medicine), weighing 140–160 g, were administered with sterile water (10 ml/kg body weight/day), a Shu-Yu capsule (0.41 g/kg body weight/day) and naloxone (10 g/kg body weight/day; lot no. H20064789; Beijing Kawin Biotech, Beijing, China) for 5 days. The rats were maintained in the same conditions described above. Following the last drug administration (50 min), a 6-ml blood sample was collected via the inferior vena cava following an intraperitoneal injection of 1% sodium pentobarbital and the rats were then sacrificed by decollation. Serum was obtained by centrifugation at 1,680 × g (4°C) for 15 min. Then, 200 µl serum (10% V/V) was added to each well for incubation of the primary neurons at 37°C for 24 h.

In addition, for the treatment of the MOR activator, DAMGO (E7384; Sigma-Aldrich; Merck Millipore, Darmstadt, Germany), primary neurons were incubated, in triplicates, with the above-mentioned drug-containing serum at 37°C for 24 h, then treated with 1 µmol/l DAMGO per well for 0, 5, 15, 30, 45 and 60 min.

The expression levels of MOR and brain-derived neurotrophic factor (BDNF) and the phosphorylation levels of extracellular signal-regulated kinases (ERK) and cyclic adenosine monophosphate response element-binding protein (CREB) were detected via western blot analysis. Total protein was obtained from the hippocampi of the neonatal rats following sacrifice and the primary neuron culture. Protein concentration was determined using an enhanced BCA protein concentration assessment kit (P0010; Beyotime Institute of Biotechnology, Haimen, China), according to the manufacturer's instructions. A total of 150 µl pre-iced RIPA lysis buffer (P0013B) containing 1% PMSF (ST506; both Beyotime Institute of Biotechnology) was added into each well to lyse the cells on ice for 30 min. Then, the solution was transferred into a centrifuge tube, which was subjected to the centrifugation at 10,800 × g (4°C) for 5 min. The supernatant was kept and stored until further use. A total of 50 µg protein was loaded per lane and separated with 10% SDS-PAGE and then transferred onto a nitrocellulose membrane. The membrane was blocked with Tris-buffered saline with Tween (TBST) containing 5% fat-free milk at room temperature for 1 h, then incubated with rabbit anti-rat anti-MOR polyclonal antibody (1:200 dilution; ab10275; Abcam, Cambridge, MA, USA), anti-BDNF polyclonal antibody (1:1,000 dilution; ab46176; Abcam), anti-CREB monoclonal antibody (1:800 dilution; 9197; Cell Signaling Technology, Inc., Danvers, MA, USA), anti-phospho-CREB monoclonal antibody (1:800 dilution; 9198; Cell Signaling Technology, Inc.), anti-ERK polyclonal antibody (1:900 dilution; 9102s; Cell Signaling Technology, Inc.), anti-phospho-ERK polyclonal antibody (1:800 dilution; 9101s; Cell Signaling Technology, Inc.) and anti-β-actin monoclonal antibody, as an internal control (1:3,000 dilution; A1978; Sigma-Aldrich; Merck Millipore), at room temperature for 2 h. The membrane was then incubated with goat anti-rabbit secondary antibody (1:1,800 dilution; SB-200; Sungene Biotech, Tianjin, China) at room temperature for 1 h and washed with TBST. The membrane was developed using electrochemiluminescence (ECL kit; P90720; Merck Millipore), according to the manufacturer's instructions, and the bands were analyzed with ImageJ software v.1.48u (National Institutes of Health, Bethesda, MA, USA).

The cyclic adenosine monophosphate (cAMP) level in the culture supernatant was detected with an ELISA kit (F-15181; Xitang Biotechnology Co., Ltd., Shanghai, China), according to the manufacturer's instructions.

Data are presented as the mean ± standard deviation. Statistical analysis was performed with GraphPad Prism 5.0 software (GraphPad Software, Inc., La Jolla, CA USA) and the one-way analysis of variance test was used for comparison of the means. P<0.05 was considered to indicate a statistically significant difference.

A PMS liver-qi stagnation rat model was established using a chronic restraint stress method. The behavior observations indicated that, compared with control rats, PMS liver-qi stagnation rats were lethargic, and had coarse hair and dull eyes. According to subjective assessment, when stimulated with glass rod pricking electric current, the rats in the control group reacted swiftly, while the model rats' response was slower. From the open-field test, it was found that the scores of rats in the model group were significantly decreased relative to the control group (P<0.01). Following treatment with the Shu-Yu capsule, the treatment group scores were significantly increased relative to the untreated model group (P<0.05; Fig. 1).

To investigate the expression levels of MOR in the hippocampus of PMS liver-qi stagnation rats, a western blot analysis was performed. The results indicated that, relative to the control group, the expression of MOR in the hippocampus was significantly increased in the PMS liver-qi stagnation model group (P<0.01). In addition, hippocampal MOR expression was significantly decreased in model rats following treatment with the Shu-Yu capsule (P<0.01; Fig. 2). These results indicate that Shu-Yu capsule treatment significantly decreased the expression of MOR in the hippocampus of PMS liver-qi stagnation model rats.

The effects of model rat serum on the expression of MOR and BDNF in the primary hippocampal neurons and on cAMP levels in the culture supernatant were evaluated by western blot analysis (Fig. 3). Primary hippocampal neurons were treated with rat serum from the control, model and treatment groups, while cells in the blank group received no treatment. Results from western blotting indicated that there was no significant difference in MOR and BDNF expression between the blank cells and primary neurons treated with normal rat serum (P>0.05). For the primary neurons treated with model rat serum, levels of MOR were significantly elevated (P<0.05), while levels of BDNF were significantly lower (P<0.05) than in primary neurons treated with normal rat serum. These effects appeared to be reversed by treatment with the Shu-Yu capsule treatment group serum (P<0.05; Fig. 3A and B). Furthermore, results of the ELISA indicated that there was no significant difference in the cAMP level of culture supernatant between the blank cells and the cells treated with normal rat serum (P>0.05). Meanwhile, the level of cAMP in the culture supernatant from cells treated with model rat serum was significantly lower than that in cells treated with normal rat serum (P<0.05), with this level significantly elevated for cells treated with the Shu-Yu capsule treatment group serum relative to the model rat serum-treated rats (P<0.05; Fig. 3C). Collectively, these results suggest that, when cultured with the serum from model rats, MOR expression is increased and BDNF expression decreased in primary hippocampal neurons, while the cAMP level in the culture supernatant is decreased. However, these effects were reversed by incubation with the serum from the Shu-Yu capsule treatment group.

To evaluate the role of MOR in PMS liver-qi stagnation pathophysiology, primary neurons were treated with the MOR activator, DAMGO. A western blot analysis was then performed to detect the phosphorylation of CREB and ERK, the expression of BDNF in the cells and the cAMP level in the culture supernatant. In order to determine the appropriate treatment period, primary neurons were treated with 1 µmol/l DAMGO for 0, 5, 15, 30, 45 and 60 min, and the phosphorylation level of ERK was detected. The results indicated that 15–30 min was the most effective treatment duration for DAMGO (Fig. 4); therefore, a 20 min treatment duration was used in the following experiments. It was observed via western blotting that cells treated with the activator alone and the cells treated with activator plus normal rat serum had significantly decreased phosphorylation levels of CREB and ERK, and decreased levels of BDNF expression, relative to blank control cells (P<0.05). By contrast, in cells treated with the activator and Shu-Yu-containing serum, the phosphorylation levels of CREB and ERK, and BDNF expression, were significantly increased relative to cells treated with the activator plus normal rat serum (P<0.05). Similar effects were observed in cells treated with the activator and naloxone-containing serum (P<0.05; Fig. 5A-C).

In the evaluation of cAMP level, it was observed that cAMP levels in the culture supernatant from cells treated with DAMGO or DAMGO plus normal rat serum was significantly decreased relative to the control cells (P<0.01). In turn, cAMP was significantly increased in cells treated with DAMGO plus Shu-Yu- or naloxone-containing serum (P<0.01 and P<0.05, respectively; Fig. 5D). Collectively, these results suggest that MOR activation may decrease hippocampal CREB and ERK phosphorylation, hippocampal BDNF expression and cAMP level in hippocampal culture supernatant, and that these effects were reversed by the administration of serum from Shu-Yu capsule-treated rats.