The commercially available formulation Dan-zhi-xiao-yao-san was obtained from E-FONG Pharmaceutical Corp. (Guangzhou, China). The manufacturer performed quality control testing of individual formulation components to ensure their uniformity. The formulation contained the following 10 medicinal herbs: Atractylodis macrocephale rhizoma, Bupleuri radix, Angelicae sinensis, poria, Glycyrrihizae radix, tree peony bark, Gardenia jasminoides, Paeonia lactiflora Pall, mint and roasted ginger. The powder was dissolved in 0.9% NaCl prior to use. Mifepristone was purchased from Sigma-Aldrich (St. Louis, MO, USA). Monoclonal mouse anti-α-synuclein (ab27766), polyclonal rabbit anti-PP2A (ab137825) and monoclonal mouse anti-glyc-eraldehyde-3-phosphate dehydrogenase (GAPDH; ab8245) were obtained from (Abcam, Cambridge, UK). Unless otherwise indicated, all other chemicals were purchased from Sigma-Aldrich.

Female Sprague-Dawley rats (n=50; aged, 2–3 months; weight, 200–250 g) were bred in the Laboratory Animal Care Facility at Guangzhou University of Chinese Medicine (Guangzhou, China) and maintained under constant conditions of 22±1°C, 60±10% relative humidity and a 12 h light/dark cycle. All animal studies were performed in accordance with international ethical standards, and the study protocols were approved by the Animal Care and Use Committee of Guangzhou University of Chinese Medicine (Guangzhou, China). The rats were randomly assigned to one of five groups (n=10/group), which included a control group, chronic stress group (Model), chronic stress + low-dose Dan-zhi-xiao-yao-san group (Low; 5.256 g/kg Dan-zhi-xiao-yao-san), chronic stress + high-dose Dan-zhi-xiao-yao-san group (High; 52.56 g/kg Dan-zhi-xiao-yao-san), and a chronic stress + mifepristone group (25 mg/kg mifepristone as a positive control). The chronic stress model was generated, as previously described with certain modifications (14). Briefly, the rats were subjected to a stress-inducing condition once daily over a period of 21 days. The order of stressor used was as follows: i) Food deprivation for 24 h, ii) water deprivation for 24 h; iii) electric foot shocks for 30 min (1 mA; duration, 1 sec; average frequency, 1 shock/min), iv) forced swimming at 4°C for 5 min and v) immobilization for 6 h. The rats in the drug treatment groups were administered the specified dose of Dan-zhi-xiao-yao-san or mifepristone by intragastric administration each day, 4 h prior to exposure to the stressful condition, whereas rats in the control group were administered the same volume of distilled water. The rats in all five groups were sacrificed 24 h after their final exposure to stressful conditions.

The elevated plus maze test was performed, as previously described (15). Briefly, the elevated plus maze consisted of two closed arms, each 49 cm long, 10 cm wide and 30 cm high, and two open arms. The arms of the apparatus are elevated 50 cm above the ground. Each rat is placed at the junction of the open and closed arms with the head pointed towards one open arm. The data regarding the rat performance in the maze test were automatically recorded using the Any-maze video tracking system (Stoelting Company; Wood Dale, IL, USA). Any-Maze can be programmed to automatically record all possible indices in each task and digitally records a video of a rat's performance in each test. The number of entries and the duration spent on the open arms are recorded for periods of 5 min. An increase in open arm activity (duration and/or number of entries) reflects anti-anxiety behavior.

The neuroprotective effects of Dan-zhi-xiao-yao-san were examined by observing the histological integrity and damage to tissue in the hippocampus of each rat brain following staining with HE. Briefly, each rat brain was isolated and cut into 5 µm sections. The sections were subsequently stained using a combination of Mayer's hematoxylin and 0.5% aqueous eosin and observed under a FV1000 light microscope (Olympus, Tokyo, Japan). When using this method, the nucleus and other acidic structures in the cells were stained blue, while the cytoplasm was stained red.

Expression levels of α-synuclein and PP2A in the hippocampus region of the sections of rat midbrain were examined by immunohistochemical staining, as previously described (16). Briefly, the paraffin sections of rat midbrain were deparaffinized and hydrated. The antigenic sites were exposed by incubation in 10 mM citrate buffer (pH 6.0) at 90°C, and endogenous peroxidase activity was quenched. Following blocking with 5% bovine serum albumen in phosphate-buffered saline with Tween-20, the tissue sections were incubated with primary antibodies, followed by incubations with goat anti-rabbit IgG conjugated to horseradish peroxidase (HRP; ab97051, Abcam)and 3,3-N-diaminobenzidine tertrahydrochloride (Solarbio, Beijing, China) to reveal α-synuclein and PP2A expression. The sections were subsequently re-stained with HE and observed under a microscope (FV1000; Olympus).

Protein was extracted using radioimmuniprecipitation assay buffer (Beyotime Institute of Biotechnology, Shanghai, China) and centrifuged at 1,000 × g for 5 min at 4°C. The protein concentration in the resulting supernatant was quantified using a bicinchoninic acid assay (Beyotime Institute of Biotechnology). Samples of denatured protein (20 µg) were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane (EMD Millipore, Billerica, MA, USA). Following blocking with non-fat milk for 2 h at room temperature, the membrane was incubated overnight at 4°C with antibodies against α-synuclein, PP2A, and GAPDH (1:500). Following primary antibody incubation, the membrane was incubated with the goat anti-rabbit HRP-conjugated secondary antibody (ab97051, Abcam) for 1 h at room temperature. Enhanced chemiluminescence Advance Western blotting detection reagents (GE Healthcare, Buckinghamshire, UK) were used for detection. The relative expression of α-synuclein and PP2A, relative to GAPDH, were quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Samples of brain tissue from the hippocampus region were analyzed for the mRNA expression of α-synuclein, PP2A and an internal control, β-actin, by RT-qPCR. Briefly, the total RNA was extracted using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). cDNA synthesis was performed using 2 µg RNA and a High-Capacity cDNA Reverse Transcription kit (Thermo Fisher Scientific, Inc.). The mRNA expression of α-synuclein, PP2A and β-actin were detected and amplified by real-time PCR using a Bestar™ Real time PCR Master Mix kit (Biomart, Beijing, China). The final expression levels were calculated following normalization against the expression of β-actin. A total of three independent experiments were performed, and each analysis was performed in triplicate. The forward and reverse primers (Sangon Biotech, Shanghai, China) used are shown in Table I.

Plasma samples were analyzed for corticosterone levels using an ELISA kit (cat. no. K014-H1; Arbor Assays, Ann Arbor, MI, USA) and following instructions provided by the manufacturer. All analyses were performed in triplicate.

The data are expressed as the mean ± standard deviation. Statistically significant differences between the groups were identified by one-way analysis of variance, followed by the paired Student's t-test. P<0.05 was considered to indicate a statistically significant difference.

The elevated plus maze test is a behavioral assay widely used to measure the effects of antianxiety agents in rodents (15), and the percentage of time spent in open arms and the number of entries into open arms in this test are sensitive biomarkers for levels of anxiety (15). In the present study, both open arm durations and open arm entries were significantly decreased in the chronic stress rat model (Fig. 1), suggesting that the model can be used to produce an anxiety-like effect. Rats treated with either Dan-zhi-xiao-yao-san or mifepristone for 21 consecutive days by intragastric administration exhibited significantly decreased levels of anxiety as evidenced by their lesser degrees of decrease in open arm durations and open arm entries (Fig. 1), indicating that both Dan-zhi-xiao-yao-san and mifepristone exerted dose-dependent antianxiety effects.

Microscopic examinations of rat mid-brain areas stained with HE revealed that chronic stress induced degenerative changes in the hippocampus region of stressed rats when compared with non-stressed rats (Fig. 2). Rats in the model group exhibited neuronal cells with pyknotic nuclei, which were clearly distinguishable from viable neuronal cells in the control group that displayed round and pale stained nuclei. However, following intragastric administration of either Dan-zhi-xiao-yao-san or mifepristone for 21 consecutive days, neuronal cell death was significantly inhibited. These results demonstrated that Dan-zhi-xiao-yao-san exerted a neuroprotective effect in animals subjected to chronic stress.

The effects of our chronic stress model on the expression of α-synuclein and PP2A proteins were assessed in the hippocampus region by immunohistochemical methods. As shown in Fig. 3A and B, chronic stress induced increased expression of α-synuclein and decreased expression of PP2A, when compared with these expression levels in the control group. Next, the protein expression levels of α-synuclein and PP2A were assessed by western blotting. Similar to the immunohistochemical staining, it was revealed that chronic stress significantly upregulated the expression of α-synuclein and downregulated the expression of PP2A in the hippocampus region when compared with these expression levels in the control group (Fig. 4). Whether the induced changes in the protein expression levels of α-synuclein and PP2A were modulated via expression of their respective mRNAs was next determined. RT-qPCR revealed that exposure to chronic stress induced increased expression of α-synuclein and decreased expression of PP2A, by modulating their respective mRNA levels (Fig. 5). The effects of Dan-zhi-xiao-yao-san and mifepristone on chronic stress-induced changes in α-synuclein and PP2A were determined by immunohistochemical, western blotting and RT-qPCR methods. As shown in Figs. 3–5, Dan-zhi-xiao-yao-san markedly inhibited chronic stress-induced upregulation of α-synuclein. Additionally, Dan-zhi-xiao-yao-san markedly inhibited chronic stress-induced downregulated of PP2A. Finally, as a positive control, mifepristone reversed the chronic stress-induced changes in α-synuclein and PP2A expression. Collectively, these results suggested that Dan-zhi-xiao-yao-san modulated changes in the expression levels of α-synuclein and PP2A, induced by chronic stress.

The present study next investigated the effects of Dan-zhi-xiao-yao-san on chronic stress-induced expression of corticosterone using ELISA to determine the levels of corticosterone in rat serum. As shown in Fig. 6, the levels of corticosterone in serum samples from the model group were significantly higher compared with those in the control group. By contrast, both Dan-zhi-xiao-yao-san and mifepristone significantly reversed the chronic stress-induced changes in corticosterone levels.