Avidin peroxidase, bicinchoninic acid (BCA), and other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-mouse interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α purified antibody (Ab), anti-mouse IL-6, TNF-α and IL-1β biotin-conjugated Ab, and recombinant mouse (rm) IL-1β, IL-6 and TNF-α Ab were purchased from BD Pharmingen (San Diego, CA, USA). Abs for BDNF, GAPDH, ERK and phosphorylated ERK (pERK) were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

A sample of IW was obtained from an oriental drug store, Noa Pharmacy (Seoul, South Korea), and then authenticated by Professor HM Kim, College of Korean Medicine, Kyung Hee University (Seoul, South Korea). A voucher specimen was deposited at the College of Korean Medicine, Kyung Hee University (IW voucher no. 304047). IW was extracted by decocting the dried herbs (total 58.125 g) with boiling distilled water (D.W; 1 liter) for ~2.5 h. The decoction was then filtered using 3MM chromatography paper, lyophilized in a freeze-dryer (Operon Co., Ltd., Kimpo, South Korea) and stored at 4°C. An IW yield of 9.7% was obtained by freeze-drying. IW was dissolved in D.W. IW was filtered through a 0.22 µm syringe filter and then diluted with D.W. (1 and 10 mg/ml). The dose range of 0.01, 0.1 and 1 g/kg was selected to determine whether dose dependency was apparent, in accordance with a previous report (9).

Male ICR mice (3 weeks old, 10–12 g) were purchased from the Dae-Han Experimental Animal Center (Daejon, South Korea), and subsequently housed at the College of Korean Medicine, Kyung Hee University. The animals were maintained at a temperature of 22±1°C at a relative humidity of 55±10% under a 12:12 light/dark cycle, lights on at 7:00 throughout the study. Food and water were available ad libitum. All manipulations were carried out between 9:00 and 16:00, and no animal was used more than once. All protocols were approved by the institutional animal care and use committee of Kyung Hee University (Seoul, South Korea). Following the first measurement of immobility times, the mice were randomly separated into control, fluoxetine and IW (0.01, 0.1 and 1 g/kg) groups, based on the documented immobility times. IW (0.01, 0.1, and 1 g/kg) was orally administered to mice once per day for 2 weeks using an atraumatic feeding needle. Fluoxetine (10 mg/kg), an antidepressant of the selective serotonin reuptake inhibitor class, was used as a positive control. The FST was performed at the end of the 2-week administration period. During the 6 min FST, the times of immobility was measured. The instrument comprised two Plexiglas cylinders (height, 25 cm; diameter, 10 cm; Deoksan Lab, Seoul, South Korea), placed alongside a Makrolon cage (Dae-Han Experimental Animal Center) filled with water (10 cm height) at a temperature of 23–25°C. For the FST, two mice (one from each experimental group) were assessed concurrently for a 6 min period of time inside the vertical Plexiglas cylinders. An opaque screen was placed between the two cylinders to prevent the mice from seeing each other. The total duration of immobility, following a stabilizing duration of 2 min, was measured for a period of 4 min. Each mouse was considered to be immobile when it ceased struggling and remained floating motionless in the water, making only those movements necessary to maintain its head above the water surface. Each group contained five mice. Mice were anesthetized using an intraperitoneal injection of ketamine (80 mg/kg) and xylazine (4 mg/kg). Following anesthetization, blood was withdrawn from the hearts of forced swimming-tested mice. Then, the serum was prepared by centrifugation at 1,900 x g at 4°C for 10 min. The hippocampus was dissected out of the brain and homogenized with a homogenization buffer (20 mM HEPES pH 7.5, 1.5 mM MgCl₂, 0.2 mM EDTA, 0.1 M NaCl, 0.2 mM DTT). The protein extracts were prepared by centrifugation at 12,000 x g for 10 min at 4°C.

The 5HT levels were measured, according to the manufacturer protocol using a Mouse 5HT/ST ELISA kit (MyBiosource, San Diego, CA, USA).

The NA levels were measured according to manufacturer protocol using an NA Urine ELISA kit (Labor Diagnostika Nord GmbH & Co. KG, Nordhorn, Germany).

Total RNA was isolated from tissue according to the manufacturer's specification using easy-BLUE RNA extraction kit (iNtRON Biotechnology, Inc., Kyungki-Do, South Korea). The concentration of total RNA in the final elutes was determined by NanoDrop (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Total RNA (2.5 µg) was heated at 65°C for 10 min and then chilled on ice. Each sample was reverse-transcribed to cDNA for 90 min at 37°C using First-Strand cDNA Synthesis kit (GE Healthcare Life Sciences, Chalfont, UK). RT-qPCR was performed using SYBR Green master mix (Applied Biosystems; Thermo Fisher Scientific, Inc.), and the detection of mRNA was analyzed using an ABI Step One real time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). Primer sequences for the reference gene, GAPDH, and the genes of interest were as follows: GAPDH, forward 5′-TCGACAGTCAGCCGCATCTTCTTT-3′ and reverse 5′-ACCAAATCCGTTGACTCCGACCTT-3′; estrogen receptor (ER)-β, forward 5′-GACTGTAGAACGGTGTGGTC-3′ and reverse 5′-CCTGTGAGGTAGGAATGCGA 3′); IL-6, forward 5′-AAATTCGGTACATCCTCGACGGCA-3′ and reverse 5′-AGTGCCTCTTTGCTGCTTTCACAC-3′); TNF-α, forward 5′-AGGACGAACATCCAACCTTCCCAA-3′ and reverse 5′-TTTGAGCCAGAAGAGGTTGAGGGT-3′); IL-1β, forward 5′-AAACAGATGAAGTGCTCCTT-3′ and reverse 5′-TGGAGAACACCACTTGTTGC-3′. These were obtained from the Bioneer Corporation, Daejeon, South Korea. The thermocycling profile used was as follows: Initial step of 95°C for 10 min, followed by 95°C for 15 sec and 60°C for 30 sec, for 40 cycles, prior to melting curve analysis. The levels of target mRNA were normalized to the level of GAPDH and compared with the control. Data were analyzed using the 2^−ΔΔCq method (14).

Hippocampus tissue extracts were used for Western blot analysis. Western blotting was performed, as previously described. Samples were heated at 95°C for 5 min and briefly cooled on ice. Following the centrifugation, protein was estimated using a Pierce BCA Protein Assay kit (Thermo Fisher Scientific, Inc.). The 50 µg aliquots were resolved by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis at 120 V for 2 h. The resolved proteins were electro-transferred overnight to nitrocellulose membranes in 25 mM Tris, pH 8.5, 200 mM glycerin, and 20% methanol at 20 V. Blots were blocked for at least 2 h with 6% bovine serum albumin then incubated with rabbit polyclonal anti-mouse BDNF (1:500, sc-546), mouse monoclonal anti-mouse GAPDH (1:500, sc-32233), rabbit polyclonal anti-mouse ERK (1:500, sc-94), and mouse monoclonal anti-mouse pERK (1:500, sc-7383) Abs for 1 h at room temperature. The membranes were washed three times with phosphate-buffered saline containing 0.05% Tween-20. Blots were developed by peroxidase-conjugated rabbit/mouse secondary Abs (1:5,000, Santa Cruz, CA, USA) for 30 min, and proteins were visualized by enhanced chemi-luminescence procedures (GE Healthcare Life Sciences), according to the manufacturer's protocol.

The levels of cytokines in the serum and in the hippocampal tissues were analyzed using an ELISA. The ELISA was performed, as described previously (10). The 96-well plates used were coated with 100 µl aliquots of anti-mouse IL-1β/IL-6/TNF-α monoclonal antibodies at a concentration of 1.0 µg/ml in PBS, respectively and incubated overnight at 4°C. Following 3 washes with PBS containing 0.05% Tween (PBST), 100 µl of samples or IL-1β/IL-6/TNF-α standards were added and incubated at 37°C for 2 h. The wells were washed 3 times with PBST and biotinylated anti-mouse IL-1β/IL-6/TNF-α antibodies (1 µg/ml) were added and incubated at 37°C for an additional 2 h. Next, the wells were washed 3 times with PBST and the avidinperoxidase was added and incubated for 30 min at 37°C. The wells were then washed with PBST, and a substrate solution was added. The plates were read at 405 nm. Cytokine levels in the hippocampus were divided according to the total protein levels, which were estimated using a Pierce BCA protein assay kit (Thermo Fisher Scientific, Inc.).

Data are expressed as the mean ± standard error of the mean. The analyses were performed using SPSS v. 11.5 (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference. Comparison between the effects of different treatments were analyzed using an independent t-test and one-way analysis of variance, followed by Tukey's multiple range tests

The IW (0.01, 0.1 and 1 g/kg) was orally administered for 2 weeks, following which, the durations of immobility were determined in a FST. The immobility durations determined in the IW groups (0.01, 0.1 and 1 g/kg) were significantly decreased, compared with the durations in the D.W. groups (Fig. 1A; P<0.05). The fluoxetine group also showed significantly decreased immobility durations, compared with the D.W. groups (Fig. 1A; P<0.05).

Monoamine systems have wide-ranging effects on animal behavior, and noradrenergic and serotonergic systems have long been implicated in depression (2). The reduced levels of 5HT or NA are considered to be involved in the underlying pathophysiology of clinical depression (15). Antidepressants elevate extracellular levels of monoamines by inhibiting their degradation or reuptake (3). Thus, the present study analyzed the levels of 5HT and NA in the brain following FST. As shown in Fig. 1B and C, the levels of 5HT and NA in the IW-administered group (1 g/kg) were significantly increased, compared with those in the D.W.-administered group (Fig. 1B and C; P<0.05).

ER-β has a beneficial effect on depression (16), therefore, the present study investigated whether the antidepressant effect of IW was associated with the expression of ER-β. The administration of IW (0.01 g/kg) significantly increased the mRNA expression of ER-β, compared with the D.W. group. (Fig. 2, P<0.05).

Reduced the levels of BDNF and pERK in response to stress can lead to the impaired neurogenesis and depressive symptoms (17–20). The protein levels of BDNF in the IW groups were higher than those of the control groups (Fig. 3A and B; P<0.05). The protein levels of pERK in the IW groups were also higher than the levels in the control groups (Fig. 3C and D, P<0.05).

The overexpression of pro-inflammatory cytokines, including IL-6, TNF-α, and IL-1β, results in autoimmune or inflammatory reactions in depression (9). The protein levels of IL-6, TNF-α and IL-1β were significantly decreased by IW administration in the hippocampus (Fig. 4A–C; P<0.05). The present study also measured the mRNA levels of IL-6, TNF-α, and IL-1β in the hippocampus. As the shown in Fig. 4D–F, IW administration significantly decreased the mRNA levels of IL-6, TNF-α and IL-1β (P<0.05).

Pro-inflammatory cytokines are important in the pathophysiology of depression (9). Therefore, the present study analyzed the protein levels of IL-6, TNF-α, and IL-1β in the serum. The levels of IL-6 in the serum were significantly decreased by IW administration, however, no significant differences were observed in the levels of TNF-α and IL-1β (Fig. 5A–C; P<0.05).