Herbs for KXS (ginseng, hoelen, polygala, and acorus) were purchased from LvYe Medicinal Material Company (Beijing, China), and the quality met the requirements outlined in the Chinese Pharmacopoeia (2010) (19). The voucher specimen was registered and deposited in the Herbarium of the Traditional Chinese Medicinal pharmacy. KXS was supplied in powder form derived from a mixture of the aqueous extract as described previously (20). Briefly, 1.5 kg ginseng, 1.5 kg hoelen, 1 kg polygala and 1 kg acorus gramineus was soaked in 50 l water for 3 h and was subsequently extracted three times using a circumfluence extraction method. The aqueous extracts were filtered and evaporated under reduced pressure. The obtained concentrates were freeze-dried to powder form and stored at 4°C. Average yield was 1 g KXS powder/4.83 g total herbs. KXS powder was standardized using a high-performance liquid chromatography-fingerprint method, as previously described (21).

In total, 24 male Wistar rats, weighing 180±10 g at 6 weeks, were obtained from the Animal Breeding Center of the People's Liberation Army general hospital (Beijing, China). All rats were housed in a temperature- (23±2°C) and humidity-controlled (60±10%) facility with a 12-h light/dark cycle with free access to food and water. All animal experimental protocols were approved by the Animal Experimentation Ethics Committee of General Hospital of Chinese PLA (X5-2013-04). All animal handling procedures were performed in accordance with the Principles of Laboratory Animal Care and Chinese legislation for the use and care of laboratory animals.

CMS was induced in 16 rats following the established protocol (22). Rats received 4 weeks of stress stimulation, which included high-speed agitation (10 min), deprivation of food or water (24 h), immobilization (2 h), continuous illumination (24 h), tilted cage (12 h) and forced swimming in ice water (5 min). Rats were randomly assigned one stimulation daily at 3–5 p.m. for four weeks. The remaining eight rats were housed undisturbed without contact with the stressed animals as the control group.

The 16 CMS-induced rats were randomly divided into two groups treated with either water (n=8; CMS group) or KXS at 370 mg/kg orally at 9–10 am daily for three weeks (n=8; KXS group).

Sucrose-preference test was performed to define anhedonia prior to surgery (baseline) and after CMS induction. Rats were food-and water-deprived for 18 h, and subsequently fed with pre-weighted bottles containing 1% sucrose solution and water for 1 h. Intake was measured by weighing the bottles before and after each test. All tests were carried out in the home cage to minimize extraneous novelty and disturbance. Sucrose preference was calculated as: Sucrose preference = sucrose intake / (sucrose intake + water intake). Anhedonia was defined as a reduction in sucrose preference relative to baseline levels.

Locomotor activity was assessed to detect immobility or changes in motor activity in an open-field test. A metallic cubic open field arena (80×80×80 cm) was used. The floor of the box was 25 squares (5 squares long; 5 squares wide). Rats were individually placed into the center of the arena and allowed to explore freely for 3 min. The floor of the open-field apparatus was wiped cleaned with 70% ethanol between tests. The behavior, including the number of square lines crossed with all four paws, and rearing, including the number of times each rat stood on its hind limbs, were recorded.

At the end of experiment, all rats were sacrificed with 10% chloral hydrate solution (3.5 ml/kg; i.p.). Hippocampus and prefrontal cortex tissues were collected and lysed with radioimmunoprecipitation assay buffer (Applygen Technologies, Inc., Beijing, China), schizolysised for 20 min on ice and centrifuged at 12,000 × g for 10 min at 4°C. Protein samples were heated at 95°C for 8 min, separated by 12% SDS-PAGE and transferred to nitrocellulose membranes. Membrane were blocked for 2 h in TBST (25 mM Tris, 140 mM NaCl, 27 mM KCl and 0.02% Tween 20) containing 5% bovine serum albumin and incubated with primary antibodies specific for BDNF (ab108319, 1:200)(Abcam, Cambridge, MA, USA), TrkB (BS1431, 1:500), ERK (AP0491, 1:500), pERK (BS4621, 1:500), Akt (BS1502, 1:500), PI3K (BS3678, 1:500), pGSK3β (BS4084, 1:500) and GSK3β (BS1402, 1:500; Bioworld Technology, Inc., St. Louis Park, MN, USA), pCREB (#9198, 1:1,000) and CREB (#9197, 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA) at 4°C over night. Following three washes with TBST, membranes were incubated for 1 h at room temperature with horseradish peroxidase-labeled secondary antibodies (BS13278, 1:5,000; Bioworld Technology, Inc.), washed with TBST three times. Blots were developed using a electrochemiluminescence system (UVP LLC, Upland, CA, USA).

Primary hippocampal neuronal cultures were prepared as described previously (23), with a few modifications. A total of 36 Newborn Wistar rats (age, <24 h) were purchased from the Peking University Health Science Center (Beijing, China). Hippocampi were dissected on ice, minced and digested with 0.25% trypsin for 15 min at 37°C. Susequently, the samples were supplemented with 5 ml fetal bovine serum (FBS) to terminate the digestion procedure. Cell suspension was passed through a 200 mesh (diameter, 74 µm) cell strainer and separated by density gradient centrifugation for 20 min at 300 × g at room temperature. The suspension was centrifuged for 5 min at 225 × g at room temperature and the pellet was resuspended in 10 ml Dulbecco's modified Eagle's medium (DMEM). Following the measurement of cell density using a hemacytometer, the neuronal cells were plated into 6-well plates at 5×10⁵ cell/ml for culture in DMEM supplemented with 10% FBS, 1 mmol/l glutamine, 20 mmol/l sodium pyruvate, and 10 ng/ml nerve growth factor (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA). 200 nM K252a (Sigma Aldrich; Merck Millipore, Darmstadt, Germany) was added to cells 30 min before the exposure to KXS as an inhibitor of TrkB. Cells were used for assay analysis at day 7 when the percentage was ~85%, as assessed by microtubule-associated protein 2 (MAP-2) immunostaining.

Cell viability was evaluated via MTT assay (24). Briefly, 20 µl MTT solution (2 mg/ml in PBS) was added at a final concentration of 0.5 mg/ml and incubated at 37°C for 4 h. Following removal of the medium, 150 µl DMSO was added. Optical densities were subsequently read using a microplate reader at 570 nm (1420 Vitor 3; Perkin-Elmer, Waltham, MA, USA). Viability was expressed as a percentage of the KXS-treated cells according to the control cells.

All data were expressed as the mean ± standard error. Differences between groups were analyzed by one-way analysis of variance followed by Dunnett's test. Statistically analyses were performed using SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

Table I shows the results from the open-field and sucrose-preference tests. The index of sucrose preference in the CMS group was significantly lower than that of the control group (P<0.01) and the KXS group (P<0.05). Similarly, the number of lines crossed and rearing values in the CMS group were significantly lower than that of the control group (P<0.01) and the KXS group (P<0.01, P<0.05), suggested that KXS could improve the depressive symptom in rats induced by CMS.

Western blot analysis demonstrated that the protein expression levels of BDNF, pCREB and TrkB in the hippocampus and TrkB in the prefrontal cortex were significantly reduced in the CMS group compared with the control group (P<0.05). Furthermore, the protein expression levels of BDNF, pCREB and TrkB in the hippocampus and prefrontal cortex were increased in the KXS group compared with the CMS group (P<0.01; Fig. 1), suggested that the antidepressive effect of KXS might be involved in its regulating on BDNF and TrkB through promoting the pCREB level.

Fig. 2 demonstrates that the protein expression levels of pERK/ERK (P<0.01), PI3K (hippocampus, P<0.01; prefrontal cortex, P<0.05), and Akt (hippocampus, P<0.01; prefrontal cortex, P<0.05) in the hippocampus and prefrontal cortex, and pGSK3β/GSK3β (P<0.05) in the hippocampus of the KXS group were significantly elevated, compared with the CMS group. The results suggest that the regulation effect of KXS on BDNF and pCREB might be related to its activation on the upstream signaling pathway of CREB.

Fig. 3 shows MAP-2 immunostaining of neuronal cells. The morphous and purity of the neuronal cells was suitable for the experiment. In cells treated with KXS, cell viability was significantly increased, as compared with the control cells (P<0.01). The level of BDNF release increased in a time-dependent manner, peaking at 6 h post-treatment (Fig. 4). KXS could increase the nerve cell viability which might be involved in the increasing level of BDNF.

Fig. 5 indicates that the protein expression levels of BDNF, pERK and pTrkB in the KXS group were significantly higher than that of the control group (P<0.01). Such an increase was not observed when cells were treated with K252a, which is an inhibitor of TrkB. The effects of KXS on the pathway components in cells coincided with those of animal experiments.