Steigerwald Arzneimittelwerk GmbH (Darmstadt, Germany) provided the SJW extract STW3-VI; the methods of extraction and characterization were described in previous publications (Breyer et al., 2007; Grundmann et al., 2010; Jungke et al., 2011). Briefly, the phytochemical composition of the St. John's Wort dried extract contained 0.10–0.3% total hypericins (as hypericin, naphthodianthrone), maximum 6.0% hyperforin (Phloroglucinderivat), and minimum 6.0% flavonoids (as rutin) (European Pharmacopoeia, 2008). The drug-extract ratio (DER) was 3–6:1 and the extractant was 80 Vol.-% Ethanol.

The immortalized mouse hippocampal neuronal HT-22 cells were cultured in Dulbecco's modified Eagle's medium (Capricorn Scientific GmbH, Ebsdorfergrund, Germany) supplied with 10% fetal bovine serum (FBS), 100 U/ml penicillin, 0.1 mg/ml streptomycin (PAA GmbH). 2.5 × 10³ HT-22 cells were seeded in 96-well plates (BD Falcon™ Becton Dickinson GmbH, Heidelberg, Germany), and after 24 h culture another 24 h differentiation as described afterwards, following 48 h pre-treatment with or without 0.1–1.0 μg/ml STW3-VI or 0.1 μM (0.03 μg/ml), −1.0 μM (0.3 μg/ml) desipramine (Sigma-Aldrich, Saint Louis, USA). Differentiation was performed in modified, serum-free medium (Dulbecco's modified Eagle's medium, 1x N2 supplement [Life Technologies AG, Carlsbad, USA]), 50 ng/ml nerve growth factor-β (NGF-β), 100 μM phorbol 12,13-dibutyrate (PDBu; Santa Cruz Biotechnology Inc., Dallas, USA), 100 μM dibutyryl cAMP (Santa Cruz Biotechnology Inc.), 100 U/ml penicillin, as well as 0.1 mg/ml streptomycin (PAA GmbH, Cölbe, Germany) (Suo et al., 2003). All treatments were performed in pre-warmed (37°C) differentiation medium with reduced NGF-β content (5 ng/ml) or with buffered Hank's solution (Capricorn Scientific GmbH, 5.26 mM KCl, 0.43 mM KH₂PO₄, 134.2 mM NaCl, 4.09 mM NaHCO₃, 0.33 mM Na₂HPO₄, 5.44 mM glucose, 2 mM CaCl₂, and 20 mM HEPES, pH 7.4; Chiricozzi et al., 2010). The human acute monocytic leukemia cell line THP-1 (DSMZ GmbH, Braunschweig, Germany) was cultured in RPMI 1640 (PAA GmbH); with 10% FBS (PAA GmbH); 100 U/ml penicillin, as well as 0.1 mg/ml streptomycin (PAA GmbH). 3.0 × 10⁴ THP-1 cells were seeded in 96-well plates (BD Falcon™). After 24 h differentiation with 160 nM phorbol-12-myristate-13-acetate (PMA, Sigma-Aldrich, St. Louis, MO, USA), the medium was changed, and the macrophages were pre-treated during 24 or 48 h with different concentrations of STW3-VI (40–100 μg/ml) and afterwards 3 h with or without 0.1 or 0.01 μg/ml LPS-EB from E. coli O111:B4 (Cayla-InvivoGen Europe, Toulouse, France). The antagonist LPS from Rhodobacter sphaeroides (LPS-RS, Cayla-InvivoGen) was used as control of the TLR4 specific binding by competitive inhibition at 100-fold excess of the agonist LPS-EB.

After 48 h, differentiated mouse hippocampal neuronal HT-22 cells were immediately fixed (10 min) with 4% v/v paraformaldehyde (PFA/phosphate-buffered saline [PBS], pH 7.4), and blocked with 1% rabbit serum (PAA Laboratories GmbH, Pasching, Austria) in PBS. Thereafter the cells were incubated overnight at 5–7°C with goat anti murine NMDAζ1, C-20 (Santa Cruz Biotechnology Inc.), afterwards visualization was performed with anti-goat Cy3 conjugated antibody (Dianova Vertriebs-GmbH, Hamburg, Deutschland) and nuclei were counterstained with DAPI (Thermo Fisher Scientific, St. Leon-Rot, Germany). Photos of neurons were taken using a Zeiss Axiovert 135 microscope and processed with the software AxioVision Release 4.8.2 (Carl Zeiss GmbH, Göttingen, Germany).

2.5 × 10³ HT-22 cells were seeded in 96-well plates (BD Falcon™) and differentiated as described. Previously the viability at different concentrations of STW3-VI (0.1, 0.5, and 1.0 μg/ml) or desipramine [0.1 μM (0.03 μg/ml), 0.5 μM (0.15 μg/ml), and 1.0 μM (0.3 μg/ml)] was determined as described below. After treatment with 0.5 μg/ml of STW3-VI or 1.0 μM desipramine hydrochloride (Sigma-Aldrich) the differentiation and neurites growth was followed and photographs were taken at 0, 24, and 48 h, using an inverted microscope (Zeiss Axiovert 135) equipped with a X–Y motorized stage and AxioVision 4 Modul Mark & Find 2 (Carl Zeiss GmbH). The total number of neurites per cell—as described by others (Xu et al., 2011)—was determined by counting the number of neurites directly stemming from the soma using Image J 1.46r software (National Institutes of Health, Bethesda, USA).

Cell viability was measured using PrestoBlue™ reagent (Invitrogen-Life Technologies GmbH, Darmstadt, Germany), according to manufacturer's protocol. PrestoBlue™ was directly added to the cells in the culture medium at a final concentration of 10%. Thereafter the optical density (OD) was measured at 570 and 600 nm (as reference) using a Sunrise ELISA-reader (Tecan, Salzburg, Austria). Results were expressed in % of cytotoxicity [100 − (OD_570/600nm of samples × 100/OD_570/600nm of control without substances)]. Afterwards, the cells were stained with crystal violet solution (0.04% crystal violet in 4% [v/v]) (Merck KGaA, Darmstadt, Germany), solubilized with 1% SDS/PBS and the absorbance was measured at 595 and 660 nm (as reference). The PrestoBlue™ absorbance was normalized against crystal violet.

The 25 × 10⁴ HT-22 cells were differentiated as described above and after differentiation were treated 24 h with STW3-VI and afterwards 6 h with glutamate or NMDA together with glycine (5 mM). The cells were washed three times with PBS, mixed with 2.5% sulfosalicylic acid (SSA), sonicated, and centrifuged. Thereafter, the clear supernatants were used to determine total glutathione (tGSH), oxidized glutathione/glutathione disulfide (GSSG), and reduced glutathione (red. GSH) according to the method of Tietze (1969), as described previously (Kinscherf et al., 1998). Red. GSH was calculated by subtraction of GSSG from tGSH. The protein concentration was determined spectrophotometrically using the Pierce BCA (bicinchoninic acid) Protein Assay (Thermo Scientific, Rockford, USA). The values of tGSH and GSSG were normalized against protein concentration.

The release of hTNF was measured using enzyme-linked immuno sorbent assay (ELISA). Therefore, 3 × 10⁴ THP-1 cells were seeded (100 μl medium/well) in 96-well plates (BD Falcon™) and were differentiated into macrophages using 0.1 μg/ml phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich, St. Louis, USA) in RPMI 1640 medium for 5 days. Afterwards, the macrophages were pre-treated with STW3-VI (24 or 48 h) and post-treated with 0.01 μg/ml ultrapure lipopolysaccharide (LPS-EB) from E. coli O111:B4 (Cayla—InvivoGen Europe, Toulouse France) for 3 h, that is exclusively recognized by toll-like receptor 4 (TLR4). The LPS antagonist from R. sphaeroides (LPS-RS) was used as control for specific binding to TLR4 by competitive inhibition at 100-fold excess of the agonist LPS-EB. After LPS treatment the culture medium was harvested, centrifuged at 500 × g (5 min) and the supernatant was stored at −20°C. Human TNF was determined in the culture medium using the assay DuoSet ELISA Development kit (R&D Systems Europe, Ltd., Abingdon, UK) according to the manufacturer's instructions. Microplates NUNC MaxiSorp™ (Thermo Fisher Scientific) 96 wells were used. The peroxidase reaction was visualized with 50 μl peroxidase substrate Sigma Fast™ o-phenylenediamine dihydrochloride (OPD) (Sigma-Aldrich, St. Louis, USA) (30 min; room temperature [RT]), and stopped with 25 μl 3 N HCl. The absorbance was measured at 490 nm, reference 655 nm. The adherent cells were fixed with 4% PFA-PBS and afterwards stained with crystal violet solution. The amount of hTNF released into the medium was normalized against crystal violet (0.04% crystal violet in 4% [v/v]). The results are expressed as percentage of the released hTNF after stimulation with LPS-EB, which was considered as 100%.

RNA was extracted using peq-GOLD Isolation Systems TriFast™ (PEQLAB Biotechnologie GmbH, Erlangen, Germany) according to the manufacturer's instructions. RNA concentration was measured using a NanoDrop 2000c Spectrophotometer (Thermo Scientific, Schwerte, Germany). RNA integrity was analyzed using the RNA 6000 NanoChip kit and an Agilent 2100 Bioanalyzer (Agilent Technologies GmbH., Waldbronn, Germany). An aliquot of 0.5 μg total RNA was treated with 1 unit of RNAse (Thermo Fisher Scientific) (30 min; 37°C). Reverse transcription was performed with 500 ng oligo (dT)_12–18 primer, 20 unit of the Affinity Script multiple temperature cDNA synthesis (Agilent Technologies) and 24 units of Ribo Lock™ RNAse inhibitor (Thermo Fisher Scientific) (1 h; 42°C). The cDNA was used for qRT-PCR using the QuantiTect primer assays (QIAGEN GmbH, Hilden, Germany). Data analyses and qPCR were performed using the Mx3005P™ QPCR System (Stratagene) and the relative standard curve method. The standard curve was generated from a pool of cDNA. For each sample, the relative amount was calculated by linear regression analysis from their respective standard curves. Amplified product was confirmed by melting point curve analysis (55–95°C).The mean expression of human ACTB (QT0009531, 146 bp) and RPLPO (QT01839887, 170 bp) or murine ACTB (QT01136772, 77 bp) and RPLPO (QT00249375, 125 bp) sequences were used as reference genes (housekeeping genes) to normalize the mRNA input. The mRNA expressions of human IL-6 (QT00083720, 107 bp), TNF-α (QT01079561, 104 bp), or murine IL-6 (QT00098875, 128 bp), TNF-α (QT00104006, 112 bp) were analyzed.

The SigmaPlot®-12 software (Systat Software GmbH, Erkrath, Germany) was used to carry out statistical analyses by the unpaired Student's t-test or Mann-Whitney U-test. Data are shown as mean + SEM. p ≤ 0.05 was considered as statistically significant.

Evidence suggests that the glutamatergic system, especially the NMDA receptors, contribute to the pathophysiology of depression (Serafini et al., 2013). Thus, we decided to use differentiated mouse hippocampal neurons that express the NMDA receptor. After 48 h culture in differentiation medium as previously described by others (Suo et al., 2003), the immortalized mouse hippocampal neuronal HT-22 cells were transformed from an immature (Figure 1A) to a more mature neuronal state (Figure 1B). Undifferentiated mouse hippocampal neuronal cells had a more rounded morphology with few neurites or apparent synapses (Figure 1A). Upon exposure to differentiation conditions, the hippocampal cells changed to a neuron-like triangular shape and some of these cells were with extended neurites (Figure 1B). Besides morphological characteristics, using immunofluorescence-staining techniques, we also found functional indicators of neuronal differentiation, including NMDA receptor expression (Figures 1C–F).

Several theories attempt to explain the causes of depression, including alterations in neurotrophic factors, changes of monoaminergic neurotransmission and dysregulation of adult hippocampal neurogenesis and plasticity (Wainwright and Galea, 2013). In this context, according to the concept of neural plasticity, the development of synaptic adaptations as well as dendritic spines are included (Wainwright and Galea, 2013). Therefore, we investigated the effect of STW3-VI on differentiated mouse hippocampal neuronal HT-22 cells in vitro. Incubation of differentiated mouse hippocampal HT-22 neurons with SJW (0.1, 0.5, or 1 μg/ml) or desipramine (0.1, 0.5, or 1 μM) did not significantly affect the viability (Figure S1). However, the neurite outgrowth was significantly increased (25%; p ≤ 0.05) after 48 h STW3-VI (0.5 μg/ml) treatment in comparison with the control (medium alone), whereas treatment with 1 mM desipramine (Desi.) showed no effect (Figure 2A). Cell viability was not significantly affected after treatment, neither with STW3-VI nor with desipramine (Figure 2B and Figure S1).

Glutamate excitotoxicity has been shown to play a role in depression. Over-stimulation of NMDA receptors due to excessive glutamate concentrations result in excessive Ca²⁺ influx, which has been suggested to be implicated in the pathophysiology of many neurodegenerative diseases as well as in depression (Mody and MacDonald, 1995; Sattler and Tymianski, 2000). First we investigated the cytotoxic effects of various concentrations of glutamate and NMDA and found that 0.1 and 0.5 mM glutamate significantly (p < 0.01) inhibited the viability by 27.1 and 15.4% in comparison with the control (Figure S2A). NMDA significantly (p < 0.01) inhibited the viability by 39.0 and 26.2% after treatment with 0.1 and 0.5 mM, respectively (Figure S2B). To investigate the protective properties of STW3-VI in differentiated mouse hippocampal HT-22 neurons, we determined the viability after treatment with glutamate or NMDA as well as with 5 or 10 μg/ml or without STW3-VI pre-incubation (Figures 3A,B). Our data show that the cell viability was significantly decreased by 30.6% (p ≤ 0.001) and 46.2% (p ≤ 0.01) after treatment with 0.1 mM glutamate (Figure 3A) or 0.1 mM NMDA (Figure 3B), when compared to control. STW3-VI pre-treatment lead to a concentration-dependent abolishment (of glutamate/NMDA-induced decrease of viability) by 25% (p ≤ 0.01) and 50% (p ≤ 0.01) at 10 μg/ml STW3-VI in comparison to 0.1 mM glutamate (Figure 3A) or 0.1 mM NMDA (Figure 3B) treatment, indicating a cytoprotective effect of STW3-VI against glutamate- or NMDA-induced neurotoxicity.

Cytoprotective enzymes and antioxidants are able to limit the cellular damage and thus, to control the toxicity of free radicals. The neurotransmitter glutamate can induce oxidative stress in cells of the brain, where a deficiency of reduced glutathione (GSH) has been observed and therefore, has been suggested to be of pathophysiological significance in depression (Gawryluk et al., 2010). Moreover, low GSH levels increase cellular sensitivity toward oxidative stress by accumulating reactive oxygen species (ROS). Treatment of differentiated mouse hippocampal HT-22 neurons with 0.5 mM glutamate significantly decreased intracellular GSH levels by 3.4-fold as compared to control (p < 0.05; Figure 4A), whereas pre-treatment with STW3-VI successfully prevented this GSH depletion. Thus, intracellular GSH was 2.3-fold increased in STW3-VI pretreated cells than in neurons incubated with glutamate alone (p < 0.05; Figure 4A). On the other hand, treatment with NMDA showed only a non-significant tendency to decrease intracellular GSH (Figure 4B), which was also prevented by STW3-VI. Thus, STW3-VI showed a protective effect against glutamate-induced oxidative stress by activation of the glutathione defense system (Figure 4A).

The measurement of the release of pro-inflammatory cytokines after treatment with a pro-inflammatory stimulus, such as LPS, is a method to analyse inflammatory processes in vitro; inhibition inhibition of this release by pharmacological intervention indicates anti-inflammatory properties of the substances properties. We used ultrapure-LPS-EB that is only recognized by TLR4, and activates pro-inflammatory signaling. LPS-EB (0.01 or 0.1 μg/ml) induced a significant 54-fold (p < 0.01) and 80-fold (p < 0.01) TNF release compared to the control (Figure 5A). Furthermore, as a control of the TLR4 specific binding, a competitive inhibition assay was performed with LPS of R. sphaeroides (LPS-RS). LPS-RS is a potent antagonist of LPS from pathogenic bacteria and binds with high affinity to the TLR4 but does not induce TLR4 signaling. Incubation of differentiated human THP-1 macrophages with the LPS-EB-antagonist—at 100-fold higher concentration than LPS-EB—abolished the hTNF release almost completely (Figure 5A). Additionally, incubation (24 or 48 h) of differentiated human THP-1 macrophages with various concentrations (20–100 μg/ml) of STW3-VI extracts showed no cytotoxic effect when compared with the control (Figure S3). Activation with 0.01 μg/ml LPS-EB induced a significant 90.9-fold (p < 0.001) or 10.3-fold (p < 0.001) TNF release, after 24 h (Figure 5B) or 48 h treatment (Figure 5C) when compared to the control (without LPS). Pre-incubation (24 h) of differentiated human THP-1 macrophages with STW3-VI (80 or 100 μg/ml) significantly (p ≤ 0.01) reduced the LPS-EB (0.01 μg/ml)-induced TNF release by 47.3 and 53.8% (Figure 5B). Prolonged (48 h) pre-incubation with STW3-VI (40–100 μg/ml) was even more effective in reducing LPS-EB-induced TNF release by 25.0–64.8% (Figure 5C). STW3-VI alone had no significant effect on the hTNF release in differentiated human THP-1 macrophages (Figures 5B,C). These results suggest an anti-inflammatory activity of STW3-VI through its inhibitory effect on the specific TLR4 signaling pathway.

To further explore the anti-inflammatory effect of STW3-VI on human THP-1 macrophages, we examined the expression of IL-6 and TNF mRNA after STW3-VI pre-treatment, followed by LPS-EB activation (3 h). There was a significant (p ≤ 0.001) 111.9-fold increase of IL-6 mRNA level after LPS treatment in comparison to the untreated control (medium alone; Figure 6A). However, pre-incubation with STW3-VI (60 or 80 μg/ml) significantly (p ≤ 0.01) attenuated the LPS-mediated IL-6 mRNA expression to 45- and 20-fold of control values (Figure 6A). TNF mRNA expression was also significantly increased by 87-fold (p ≤ 0.01) after stimulation with LPS compared to control (Figure 6B). Pre-incubation with 80 μg/ml, but not 60 μg/ml STW3-VI significantly (p ≤ 0.05) attenuated this increase to 63.9-fold (Figure 6B). Pre-treatment with STW3-VI alone had no significant influence on IL-6 or TNF mRNA expression (Figures 6A,B).

Neurons, astrocytes, microglia and endothelial cells are the sources of IL-6 in the CNS, upon stimulation such as injury; abundant amounts of IL-6 are expressed and secreted (Erta et al., 2012). We found a significant increase of 58.0 and 66.0% (p ≤ 0.001) of IL-6 mRNA expression after treatment of differentiated mouse hippocampal HT-22 neurons with 0.5 mM glutamate or NMDA, compared to control (medium alone; Figure 7). Pre-treatment with 5 μg/ml STW3-VI significantly inhibited the pro-inflammatory effects of both, glutamate (p ≤ 0.001) or NMDA (p ≤ 0.05), even completely abolishing it for glutamate (Figure 7). These results also suggest a direct anti-inflammatory effect of STW3-VI on neurons.

CK, HA-A are employed by Steigerwald Arzneimittelwerk GmbH. All the other authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.