Extraction of SOCG
Herbal drug components of SOCG were obtained from Dongkyung Pharmaceutical Company (Seoul, Korea). Preparation procedure and chemical profile of SOCG have been described in our previous report [8]. Briefly, for water extraction, a total of 11 g of SOCG, which is composed of 4 g of Cyperi Rhizoma (Cyperus rotundus L.), 2 g each of Linderae Radix (Lindera aggregata (Sims) Kosterm.), Platycodi Radix (Platycodon grandiflorum (Jacq.) A. DC.), and Aurantii Fructus (Citrus aurantium L.), and 0.5 g each of Aucklandiae Radix (Aucklandia lappa DC.) and Glycyrrhizae Radix (Glycyrrhiza uralensis Fisch.) was boiled for 2 h, filtered, and concentrated under a reduced pressure by using the rotary vacuum evaporator. After freezing-frying, 1.6 g of the SOCG powder was obtained from 11 g of initial raw materials (14.6% of extraction ratio). SOCG powder was dissolved in purified water, filtered by using 0.22 μm Whatman filter paper (Cole-Parmer, Vernon Hills, USA) and stored at -20 °C as a stock solution (10 mg/ml) which was further diluted with saline solution (0.9% NaCl in water) for oral administration. Voucher specimens (No. 194A079–85) of collected herb samples were deposited in the herbarium of Han Kook Shin Yak Co., Ltd. (Nonsan, Korea).
Experimental animals
C57/BL6 mice (male, 18–22 g, Samtako, Seoul, Korea) were maintained in an animal room with regulated temperature (22 °C), 60% humidity, and a 12-h light/dark cycle (light on 7 am to 7 pm). Before the experiments, animals were acclimatized for 7 days in an animal room and were allowed to eat commercial pellet chow (Samyang Co., Seoul, Korea) and drink water ad libitum. Animals were randomly divided into four groups: an intact animal control group (CTL), a chronic restraint stress with saline injection as a SOCG vehicle (CRS), and CRS plus 100 mg/kg and 300 mg/kg of SOCG-treated groups (CRS + SOCG100 and CRS + SOCG300). To induce a depressive-like state, individual animals were subject to CRS by placing in a well-ventilated 50 ml conical tube for 6 h each day for 14 consecutive days. SOCG (100 and 300 mg/kg) or an equivalent volume of saline was orally administered using a 22-gauge oral needle 2 h before CRS on a daily basis for a 2 week period. A recommended daily dose of SOCG for human is 3.2 g (Han Kook Shin Yak Co., Ltd.), which is then calculated 658 mg/kg for the use of experimental animals according to a procedure of human equivalent dose calculation (Guideline by the Food and Drug Administration, USA). However, to determine drug efficacy, we adopted a range of lower dose as much as 50% or less for a current investigation. Our recent study also indicates that SOCG doses at 100 mg/kg and 300 mg/kg are optimal for the induction of depressive-like behavior [8]. All procedures were in strict accordance with the NIH guide for the care and use of laboratory animals and approved by the Committee on Use of Live Animals for Teaching and Research at Daejeon University (Protocol number: DJUARB2014–036, Daejeon, Korea).
Reverse transcription-polymerase chain reaction (RT-PCR)
For RT-PCR, a total 16 animals were used and equal number of animals assigned into 4 experimental groups. Hippocampus was isolated and immediately used to extract total RNA by using Easy-BLUE reagent (Intron, Sungnam, Korea). A reaction for cDNA synthesis was carried out in 30 μl using total RNA (1 μg) as a template, 1X reaction buffer (50 mM Tris-HCl, 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 104 μM dNTP), RNasin (30 U), random primer (16 μM Promega, Madison, USA), and MMLV reverse transcriptase (200 U, Promega) for 2 h at 37 °C. For RT-PCR analysis, 30 cycles of amplification were optimal for a quantitative comparison of the target mRNA expression. The primer sequences used for PCR were the forward primers (5′-ACTCGACTTTCGGCGCTTT-3′, 5′-GCTTTGTGAACACCGACCAC-3′, 5′-GGTGTGCCTTTCCCCATCATT-3′, 5′-GGCAAGGAAGCTGGTGGTGATTT-3′, 5′-AAGAGCAGTGGAAGGACAGC-3′) and the reverse primers (5′-CTGCAAAAAGCACTGTCCCC-3′, 5′-GAGCCCGGGAGTTAATGGAG-3′, 5′-CAACATGTAGGTGATGCCCAG-3′, 5′-GGCGTGGTGGTCCTGCCAGGG-3′, 5′-TGGTATCGCCTTTGCCCATT-3′) for 5-HT1AR, 5-HT1B receptor, corticotropin releasing hormone receptor type 1 and type 2, also known as corticotropin releasing factor 1 and 2 (CRF1 and CRF2) respectively, and glucocorticoid receptor, and the forward and reverse primers (5′-TACGGATGTCAACGTCACAC-3′, 5′-CACACTGTCCCCATCTATGA-3′) for actin. PCR-amplified DNA was analyzed on agarose gels, and the band intensities were quantified using i-Solution software (Image & Microscope Technology, Burnaby, Canada).
Immunohistochemistry
A total of 8 animals, each group consisting of 2 animals, were used for immunohistochemistry. Animals were deeply anesthetized with ketamine and xylazine, and transcardially perfused with 4% paraformaldehyde in phosphate-buffered saline (PBS). Brain was removed and postfixed for 1 h and kept overnight in 15% sucrose in PBS. Coronal brain sections of 20 μm were collected on positively charged slides and were kept at -70 °C until use. For immunofluorescence staining, tissues on the slides were fixed with 4% paraformaldehyde, 4% sucrose in PBS at room temperature for 40 min, permeablized with 0.5% nonidet P-40 in PBS, and blocked with 2.5% horse serum and 2.5% bovine serum albumin for 4 h at room temperature. Tissues were incubated with primary antibody, washed with PBST (PBS plus 0.1% triton X-100) three times for 10 min each, and incubated with fluorescein-goat anti-mouse (1:400, Molecular Probes, cat no. f2761) or rhodamine-goat anti-rabbit secondary antibodies (1:400, Molecular Probes, cat no. r6394) in 2.5% horse serum and 2.5% bovine serum albumin for 1 h at room temperature and cover-slipped with gelatin mount medium. For some experimental purpose, Hoechst staining reaction for nuclear visualization was performed between washing steps after secondary antibody reaction. Tissue sections were treated with 25 μg/ml of Hoechst 33,258 in 0.1% PBST for 10 min. The secondary antibody reaction was performed in a dark place. Images from immunofluorescence staining were captured and transferred to the computer software (ACT-1). The merged images were produced by layer blending mode options of the Adobe Photoshop (version 7.0). The primary antibodies used were anti-5-HT1AR (1:300, Abcam, cat no. ab85615), anti-phospho-Erk1/2 kinase (1:800, Sigma, cat no. 9101 L), anti-GAP-43 (1:800, Santa Cruz Biotech, cat no. ab16054) and anti-NF-200 (1:400, Sigma, cat no. N0142) antibodies.
Western blot analysis
A total of 16 animals, each group consisting of 4 animals, was used to dissect hippocampal tissues. Isolated hippocampi were washed with ice-cold PBS, and sonicated under 50–200 μl of triton lysis buffer (20 mM Tris, pH 7.4, 137 mM NaCl, 25 mM β-glycerophosphate, pH 7.14, 2 mM sodium pyrophosphate, 2 mM EDTA, 1 mM Na3VO4, 1% triton X-100, 10% glycerol, 5 μg/ml leupeptin, 5 μg/ml aprotinin, 3 μM benzamidine, 0.5 mM DTT, 1 mM PMSF). Protein (15 μg) was resolved in 12% SDS polyacrylamide gel and transferred to immobilon polyvinylidenedifluoride (PVDF) membranes (Millipore, Bedford, USA). Blots were blocked with 5% nonfat dry milk in PBST (17 mM KH2PO4, 50 mM Na2HPO4, 1.5 mM NaCl, pH 7.4, and 0.05% Tween-20) for 1 h at room temperature and then incubated overnight at 4 °C in 0.1% triton X-100 in PBS plus 5% nonfat dry milk containing primary antibodies. Protein bands were detected by using the Amersham ECL kit (Amersham Pharmacia Biotech, Piscataway, USA), with horse radishperoxidase-conjugated goat anti-rabbit (1:400, Life Technologies, cat no. R6394) or goat anti-mouse secondary antibodies (1:400, Life Technologies, cat no. F2761). Transduction Laboratories, Lexington, USA). Primary antibodies used in the present study were anti-phospho-Erk1/2 kinase (1:4000, Cell Signaling, cat no. 9101 L), anti-GAP-43 (1:4000, Cell Signaling, cat no. ab16054), and anti-actin (1:10,000, MP Bio, cat no. A1978) antibodies.
DRG neuron culture and analysis of neurite outgrowth
Mice (C57BL/6) were subjected to a 2 week period of CRS and SOCG administration as described above, and 24 h after the last constraint, DRG at lumbar 4–5 levels were dissected for primary neuron culture as described previously [13]. A total of 16 animals, each group consisting of 4 animals, was used to prepare DRG neurons. DRG isolated from each experimental group were pooled to prepare neuron culture. We used DRG neurons for in vitro SOCG study because primary DRG neurons, unlike brain neurons including hippocampal neurons, can be prepared from adult animals and maintained in a stable condition for several days. Here we assumed that the extent of neurite outgrowth might reflect in vivo neural activity including depression-like brain activity. For the preparation of DRG neurons from mice given sciatic nerve injury (SNI), sciatic nerves on the left side were exposed on the middle thigh and a crush injury was given by holding a nerve with the forceps for 30 s twice as described previously [14], and the DRG at left lumbar 4–5 were prepared at 7 d post injury. A total of 8 animals, in which 4 animals were subjected to SNI and another 4 animals were non-surgery control (CTL), were used to prepare DRG. Dissociated cells were plated onto 12 mm coverslips (Marlenfeld GmbH & Co. KG, Lauda-Königshofen, Germany) precoated with poly-L-ornithine (0.1 mg/ml; Sigma) and laminin (0.02 mg/ml, BD Bioscience, San Diego, USA) and cultured for 24 h before the harvest. Immunofluorescence staining of cells was essentially same as those of brain sections on the slides. Antibodies used were anti-neurofilament-200 (NF-200) (1:400, Sigma, cat no. N0142) and anti-5-HT1AR (1:300, Abcam, cat no. ab85615) antibodies. Digital images of neuronal process were captured and transferred to the Adobe Photoshop (version 7.0). The length of neurite processes exhibiting clear outgrowth (longer than cell diameter) from the cell body was analyzed by using i-Solution software program (Image and Microscope Technology).
Behavioral tests
Animals were placed in a room for behavioral tests and subjected to preliminary swimming for 10 min. Three hours later, FST was performed in a transparent, cylindrical plexglas container (20 cm diameter, 46 cm height) filled with tap water (20 cm height, 20 °C). Animal movement was monitored for 6 min by video tracking software (SMART 3.0; Panlab, Barcelona, Spain) and the immobility time was measured for the last 4 min. For OFT, animals were placed in a room and adjusted overnight before the test. Animal was placed in a white-colored plexglas box (30 × 30 × 30 cm3) and its movement was monitored for 10 min following an initial 1 min of adjustment period. Immobility time was measured by using video tracking software (SMART 3.0; Panlab S.L.U., Barcelona, Spain). For both tests, 4 animals per experimental group were used and average immobility time was compared among experimental groups.
Statistical analysis
Data were presented as mean ± standard error of mean (SEM). The mean number data in individual groups were compared by one-way ANOVA followed by Tukey test (SPSS computer software version 12.0), and statistically significant differences were reported as *p < 0.05, **p < 0.01, ***p < 0.001.