MeOH extract of Pedicularis resupinata (PRE, voucher specimen No. KPM032-051) was purchased from the Korea Plant Extract Bank of the Korea Research Institute of Bioscience and Biotechnology (Daejeon, South Korea). PRE was dissolved in 50% HPLC-grade MeOH and analyzed by HPLC (Jasco, Tokyo, Japan) using a Waters Symmetry ® C18 column (4.6 × 250 mm, 5 μm). The mobile phase was established using 0.2% formic acid (A) and ACN/MeOH (60:40, v/v) (B), by gradient elution. The chromatographic separation was processed with a solvent (A) gradient that decreased from 80% to 50 % within 40 min of running time, and then increased to 80% with a 45 min running time. The injection volume of PRE was 10 μL with a flow rate of 1 mL/min, and detected at 280 nm using a PDA detector. The concentration of acteoside was 0.78 ± 1.15 (mean ± SD) mg/g. Representative chromatograms of acteoside and PRE are shown in Fig. 1
Animal and treatments
ICR mice (male, 7 weeks old, weighing 25–28 g) were purchased from KOATECH Animal Inc. (Pyeongteak, South Korea). The mice had ad libitum access to food and water in a climate-controlled chamber (temperature, 21 ± 2 °C and light–dark cycle, 24 h, lights on at 07:00, and lights off at 19:00). All mice (n = 4 per cage) were habituated to the facility for 1 week prior to starting the experiments. All animal experiments were approved by the Institutional Animal Care and Use Committee of the Korea Food Research Institute (KFRI-M-19016). To induce depression-like behavior, a high-dose of CORT was intraperitoneally (i.p.) injected according to a previous study. CORT (Sigma-Aldrich, MO, USA) was dissolved in 0.9% (w/v) saline containing 0.1% dimethyl sulfoxide (DMSO; Sigma-Aldrich) and 1% Tween-80. St. John’s wort (80% MeOH-extract dried powder of Hypericum japonicum), which was the positive control, and PRE was dissolved in distilled water. The mice were randomly assigned to six groups (n = 8): (1) sham, (2) control, (3) St. John’s wort 300 mg/kg, (4) PRE 30 mg/kg, (5) PRE 100 mg/kg, and (6) PRE 300 mg/kg. Mice in the control and sample-treated groups were orally administered CORT (40 mg/kg, i.p.) once daily, while those in the sham group were treated with an equal volume of vehicle. After 3 weeks of treatment, the mice tested in depression-related behavioral tasks, beginning 1 h after sample administration, as per the experimental scheme (Fig. 2), and were then sacrificed for western blot analysis.
Open filed test (OFT)
The mice were placed in the center of an open field maze (50 × 50 × 50 cm), and their movements were recorded using a video camera; behaviors, total distance, and time in the center and periphery zones were tracked for 5 min using the SMART video tracking sys-tem (SMART v3.0, Panlab SL, Barcelona, Spain).
Sucrose preference test (SPT)
The SPT was performed 24 hours after OFT, following a previously established protocol (Lim et al., 2019a). The two bottles contained the 1% sucrose solutions were located in the cage for 24 h. Next, one of the bottle of the sucrose solution was located with water for 24 h. The mice then were placed in cages individually with free access to the two bottles for 24 h, and the consumed volumes were recorded. Sucrose preference was calculated as follows: consumption (%) = [sucrose intake/(sucrose intake + water intake)] × 100.
Passive avoidance test (PAT)
The PAT was performed using the passive avoidance apparatus (GEMINI, SD instruments, San Diego, CA, USA), as previously described. The PAT experiment was conducted across 2 days. In the training trial, each mouse was placed in a safe zone with a closed door. After acclimatization for 1 min, the door opened automatically, and the mice were allowed to enter the dark zone. When the mice entered the dark zone, an electrical foot shock of 0.5 mA for 3 s, was elicited. On the next day, the mice were again placed in the safe zone, and the latency to enter the dark zone was recorded.
Tail suspension test (TST) and forced swim test (FST)
In the case of the TST, each mouse was suspended by its tail using adhesive tape and attached to a hook that automatically measured their movement. The immobility time during the 6-min test was measured using an automated TST apparatus (BioSeb, Chaville, France). In the FST, the mice were forced to swim for 6 min in a transparent Plexiglas cylinder (height, 13 cm; diameter, 24 cm) filled with water to a depth of 10 cm (temperature, 22–24°C). The immobility time during the last 4 min was measured using a SMART video tracking system (SMART v3.0, Panlab SL, Barcelona, Spain).
Cell viability assay
Cell viability was analyzed using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide MTT assay, as previously described. SH-SY5Y cells (ATCC, Manassas, VA, USA) were cultured in Dulbecco’s modified Eagle’s medium (Gibco, NY, USA) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin-streptomycin-glutamine (Gibco). The cells were maintained in a humidified atmosphere containing 5% CO2 at 37 °C. The cells were seeded in 96-well plates (2 × 104 cells/well) and incubated for 24 h. To assess the effect of PRE on cell viability, a MTT assay was used to determine the viability of cells treated with CORT. Cells were seeded at a density of 2 × 104 cells/well in 96-well plates and incubated overnight. The next day, the cells were co-treated with 10, 50, or 100 µg/mL of PRE and CORT (200 μm) for 24 h. MTT solution (thiazolyl blue tetrazolium bromide, M5655, Sigma-Aldrich, MO, USA) was added to each well at a final concentration of 0.5 mg/mL, and the plates were incubated at 37 °C for 4 h. The medium was then discarded, and the formazan product was dissolved in DMSO (Sigma-Aldrich). Cell viability was determined by measuring the optical density at 570 nm wavelength.
Hippocampal brain tissues were homogenized in RIPA buffer containing a protease and phosphatase inhibitor cocktail. The quantified proteins (20 μg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 4% skim milk in Trisbuffered saline with 1% Tween-20 for 40 min, and subsequently probed with the following primary antibodies: BDNF mouse monoclonal antibody (1:1,000 dilution, sc-65514, Santa Cruz Biotechnology, CA, USA), glucocorticoid receptor (1:1,000 dilution, sc-393232, Santa Cruz Biotechnology, CA, USA; GR phosphorylation at serine 211 (Ser211, 1:1,000 dilution, #4161, Cell Signaling, MA, USA), and an-ti-β-actin rabbit polyclonal antibody (1:1,000 dilution, #4967, Cell Signaling, MA, USA) overnight at 4 °C in 3% skim milk in TBST. After incubation with the horseradish peroxidase linked secondary antibody for 2 h, immunoreactive proteins were detected using a chemiluminescence detection system (LI-COR Biosciences, Lincoln, NE, USA) and then analyzed using ImageJ software (NIH, Bethesda, MD, USA).
Statistical analyses were performed using Student’s t-test for two-sample comparisons and one-way analysis of variance, followed by Tukey’s post-hoc test using Prism 8 (GraphPad Software, Inc., San Diego, CA, USA) for multigroup comparisons. All data are presented as the mean ± SEM. Differences were considered significant at p < 0.05.