Chemical shifts were referenced relative to the residual solvent peak (δH/δC = 7.26/77.2). Electrospray ionization mass spectrometry (ESI-MS) data were obtained using an ESI quadrupole time-of-flight MS/MS system (AB SCIEX Pte. Ltd.). High-performance liquid chromatography (HPLC) separation was performed using a YOUNGLIN-YL9100 system (Young Lin Instrument Co., Ltd.) and a Phenomenex Synergi 4 µm Polar-RP 80A, AXIA packed column (21.2×150 mm, 5 µm particle size, 2 mg of the sample injection, 25°C) with a flow rate of 5 ml/min. All solvents used for HPLC were of analytical grade.

N. jatamansi hexane fraction (GSH-H) was subjected to Si column chromatography (75×330 mm) and eluted with CHCl₃-MeOH (70:1-1:1), which yielded seven sub-fractions (GSH-H1-7). Sub-fraction GSH-H3 was subjected to Si column chromatography (70×300 mm) and eluted with hexane-EtOAc (20:1-1:1), which yielded six sub-fractions (GSH-H3-1-6). Sub-fraction GSH-H3-5 was subjected to Si column chromatography (70×280 mm) and eluted with hexane-EtOAc (10:1-1:1), which yielded 12 sub-fractions (GSH-H3-5-1-12). Sub-fraction GSH-H3-5-9 (370.7 mg) was subjected to Si column chromatography (180×30 mm) and eluted with MeOH-CHCl₃ (30:1-1:1), which yielded nine sub-fractions (GSH-H3-5-9-1-9). Sub-fraction GSH-H3-5-9-2 (22.0 mg) was further purified using semi-preparative reversed-phase HPLC (20×150 mm), eluting in a gradient of 35–100% ACN in H₂O for over 50 min, which yielded GSH-H3-5-9-2-2 (4.0 mg, tR = 30.9 min).

Nardostachin: Pale yellow powder; ¹H-NMR (400 MHz, CDCl₃,): δ 6.30 (1H, brs, H-3), 5.94 (1H, d, J = 4.2, H-1), 4.57 (1H, d, J = 12.2, H-11), 4.37 (1H, d, J = 12.2, H-11), 4.17 (1H, m, H-7), 2.92 (1H, q, H-5), 1.12 (3H, d, J = 6.8, H-10), 0.97 (3H, d, J = 6.59, H-4′), 0.97 (3H, d, J = 6.59, H-5′), 0.97 (3H, d, J = 6.59, H-4″), 0.97 (3H, d, J = 6.59, H-5″); ¹³C-NMR (100 MHz, CDCl₃) δ: 172.1 (C-1″), 172.1 (C-1′), 139.6 (C-3), 114.3 (C-4), 91.6 (C-1),74.7 (C-7), 63.8 (C-11), 44.9 (C-9), 43.6 (C-2′), 43.6 (C-2″), 40.6 (C-8), 39.8 (C-6), 32.1 (C-5), 25.9 (C-3′), 25.8 (C-3″), 22.5 (C-4′), 22.5 (C-5′), 22.5 (C-4″), 22.5(C-5″), 12.8 (C-10); HRESIMS m/z 391.2092 [M + Na⁺] (calculated for C₂₀H₃₂O₆Na 391.2097).

Tissue culture reagents, including RPMI-1640 medium and fetal bovine serum (FBS), were purchased from Gibco; Thermo Fisher Scientific, Inc. All other chemicals were obtained from Sigma-Aldrich; Merck KGaA. Primary antibodies against IкB-α (cat. no. 4812, 1:1,000, rabbit), p-IкB-α (cat. no. 2859, 1:1,000, rabbit), p65 (cat. no. 8242, 1:1,000, rabbit), TLR4 (cat. no. 14358, 1:1,000, rabbit), MyD88 (cat. no. 4283, 1:1,000, rabbit), p-ERK (cat. no. 9101, 1:1,000, rabbit), ERK (cat. no. 9102, 1:1,000, rabbit), p-JNK (cat. no. 9251, 1:1,000, rabbit), JNK (cat. no. 9252, 1:1,000, rabbit), p-p38 (cat. no. 9211, 1:1,000, rabbit) and p38 (cat. no. 9212, 1:1,000, rabbit) were obtained from Cell Signaling Technology, Inc. Antibody against inducible nitric oxide synthase (iNOS; cat. no. 160862, 1:1,000, rabbit) was obtained from Cayman Chemical and against cyclooxygenase-2 (COX-2; cat. no. ab15191, 1:1,000, rabbit) from Abcam. Antibodies against p50 (cat. no. sc-7178; 1:1,000, rabbit), β-actin (cat. no. sc-47778; 1:1,000, rabbit) and PCNA (cat. no. sc-56, 1:1,000, mouse) were purchased from Santa Cruz Biotechnology. Anti-mouse, anti-goat, and anti-rabbit secondary antibodies were purchased from Merck Millipore ELISA kits for PGE₂ were purchased from R&D Systems, Inc (cat. no. KGE004B).

BV2 microglial cells were maintained at a density of 5×10⁵ cells/ml in RPMI-1640 medium, supplemented with 10% heat-inactivated FBS, penicillin G (100 U/ml), streptomycin (100 mg/ml) and L-glutamine (2 mM), and were incubated at 37°C in a humidified atmosphere containing 5% CO₂. Primary microglial cells were cultured from the cerebral cortices or substantia nigra of 1-day-old Sprague-Dawley rats weighing 6–8 g which were purchased from the Orient Co., Ltd. Rats were housed in standard cages in a climate-controlled room with an ambient temperature of 23±2°C only for 3 h and then were sacrificed. A total of 15 rats were used in the isolation of primary microglial cells. Brain tissues were triturated into single cells in Dulbecco's modified Eagle's medium containing 10% FBS and cultured in 75-cm² T-flasks for 2 weeks in a humified incubator at 37°C. The microglial cells were detached from the flasks by mild shaking and applied to a strainer to remove astrocytes and cell clumps. Cells were plated onto 6-well plates (5×10⁵ cells/ml) with B27^® supplement (Thermo Fisher Scientific, Inc.) in the absence of insulin for 2–3 days. Cells were washed to remove unattached cells prior to use in subsequent experiments.

Cell viability was evaluated by determining mitochondrial reductase function using an assay based on reducing MTT into formazan crystals. The formation of formazan is proportional to the number of functional mitochondria in living cells. Cells were maintained at 1×10⁵ cells/ml in each well of the 96-well plate to determine cell viability. Following incubation for 6 h, the cells were treated for 24 h with the indicated concentrations of 1.0. 4.0 µM nardostachin with or without LPS (1 µg/ml). Next, 50 µl MTT (2.5 mg/ml) was added to each well in fresh medium at a final concentration of 0.5 mg/ml, and the mixture was further incubated for 3–4 h at 37°C, following which the liquid was removed from the wells. Next, the formazan formed was dissolved in 150 µl dimethyl sulfoxide, and the optical density was measured at 590 nm wavelength. The optical density of the formazan formed in control (untreated) cells was considered to indicate 100% viability.

Total RNA was isolated from the cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocols, and quantified spectrophotometrically (at 260 nm wavelength). Total RNA (1 µg) was reverse transcribed using the High Capacity RNA-to-cDNA kit for 65 min at 100°C (Applied Thermo Fisher Scientific, Inc.). The cDNA was then amplified using the SYBR Premix Ex Taq kit (Takara Bio, Inc.) on a StepOnePlus Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). In brief, each 20 ml reaction volume contained 10 ml SYBR Green PCR Master Mix, 0.8 mM each primer and diethylpyrocarbonate-treated water. Primer sequences were designed using PrimerQuest (Integrated DNA Technologies, Inc.) and were as follows: TNF-α forward, 5′-CCAGACCCTCACACTCACAA-3′ and reverse, 5′-ACAAGGTACAACCCATCGGC-3′; IL-1β forward, 5′-AATTGGTCATAGCCCGCACT-3′ and reverse, 5′-AAGCAATGTGCTGGTGCTTC-3′; IL-6 forward, 5′-ACTTCCAAGTCGGAGGCTT-3′ and reverse, 5′-TGCAAGTGCATCATCGTTGT-3′; and IL-12 forward, 5′-AGTGACATGTGGAATGGCGT-3′ and reverse, 5′-CAGTTCAATGGGCAGGGTCT-3′. The optimal conditions for PCR amplification of the cDNA were established according to the manufacturer's protocols. The data were analyzed using StepOne software (Applied Biosystems; Thermo Fisher Scientific, Inc.), and the cycle numbers at the linear amplification threshold (Ct) for the endogenous control gene (GAPDH) and target gene were recorded. Relative gene expression (target gene expression normalized to endogenous control gene expression) was calculated using the comparative Ct method (2^−ΔΔCt) (22). The analysis was conducted independently three times.

The DNA-binding activity of NF-κB in nuclear extracts was measured using the TransAM kit (cat. no. 40096; Active Motif, Inc.), according to the manufacturer's protocols. BV2 and rat primary microglial cells were pre-treated for 3 h with the indicated concentrations of nardostachin and stimulated for 30 min with LPS (1 µg/ml) at 37°C, and the nuclear fractions were extracted. The nuclear extracts were added to the oligonucleotide-coated plate and reacted with primary and secondary antibodies each for 1 h at room temperature provided in the kit. A developing buffer was then added to each well, and this buffer reacted with the horseradish peroxidase-conjugated secondary antibodies provided in the kit, yielding a blue color. Next, the stop solution was added, and the optical density was measured at 450 nm wavelength. The assay was conducted independently three times.

The cytosolic and nuclear fractions were extracted using the Caiman Nuclear Extraction kit from Cayman Chemical (cat. no. 10009277) and each fraction was lysed according to the manufacturer's protocols.

The nitrite concentration, an indicator of NO production, was measured using the Griess reaction of the culture medium. The detailed procedure for this assay is described in our previous study (23).

The level of PGE₂ present in each sample was determined using a commercially available kit (cat. no. KGE004B) from R&D Systems, Inc. Three independent assays were performed according to the manufacturer's protocols.

Western blot analysis was performed as previously described (23). In brief, BV2 and primary microglial cells were harvested by centrifugation at 200 × g for 3 min at 4°C, washed with phosphate-buffered saline, and lysed using radioimmunoprecipitation assay (RIPA) buffer (Thermo Fisher Scientific, Inc.). The protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, Inc.) was mixed with RIPA buffer at a 3:100 ratio to evaluate phosphorylation.

Protein concentrations were measured using Bradford protein assay (Bio-Rad Laboratories, Inc.) and normalized to ensure equal amounts of protein were loaded. Subsequently, 30 µg protein from each sample was resolved using 7.5 and 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were electrophoretically transferred onto a Hybond enhanced chemiluminescence (ECL) nitrocellulose membrane. The membrane was blocked with 5% skimmed milk at 4°C for 1 h and sequentially incubated with the primary antibodies diluted at 1:1,000 at 4°C for 1.5 h, and horseradish peroxidase-conjugated secondary antibodies diluted at a rate of 1:1,000 at 4°C for 1 h, followed by ECL detection. The intensity of the protein signals was quantified using ImageJ software (ver. 1.47; National Institutes of Health). The data represent the means of three independent experiments.

BV2 and primary rat microglial cells were cultured on Lab-Tek II chamber slides and treated as described in the figure legends. The cells were treated with 4.0 µM nardostachin for 1 h, fixed in formalin, permeabilized with cold acetone, and then probed with a primary antibody against NF-κB and a fluorescein Isothiocyanate (FITC)-labeled secondary antibody (Alexa Fluor 488; Invitrogen; Thermo Fisher Scientific, Inc.). To visualize the nuclei, the cells were treated with DAPI (1 µg/ml) for 30 min, washed with PBS for 5 min, and treated with 50 µl VectaShield (Vector Laboratories, Inc.). The stained cells were visualized and images captured by using a confocal laser microscope (Olympus Corporation).

Data are expressed as the mean ± standard deviation of at least three independent experiments. To compare three or more groups, one-way analysis of variance, followed by Tukey's multiple comparison tests, was used. Statistical analysis was performed using GraphPad Prism software, version 3.03 (GraphPad Software, Inc.) (24).

Nardostachin was isolated from the hexane-soluble fraction of the methanol extracts of N. jatamansi using C₁₈ flash column chromatography and semi-preparative HPLC (Fig. 1A). The structure of nardostachin was elucidated by analysis of MS and NMR data and a comparison of its spectral data with those reported in the literature (25).

A conventional MTT assay was used to analyze BV2 and rat primary microglial cells treated with different concentrations of nardostachin (1.0–4.0 µM) for 24 h in the absence of LPS to determine the cytotoxicity of nardostachin. As shown in Fig. 1B and C, cell viability was not affected by nardostachin compared with that in the control group. Based on the MTT assay results, the concentration range of nardostachin from 1.0 to 4.0 µM was selected for subsequent experiments.

The effects of nardostachin on LPS-induced secretion of NO and PGE₂ was subsequently analyzed using the Griess method and ELISA, respectively, at non-cytotoxic concentrations. As shown in Fig. 2A-D, nardostachin significantly downregulated the excessive secretion of NO and PGE₂ in LPS-stimulated BV2 and rat primary microglial cells at concentrations ranging from 1.0 to 4.0 µM.

Since nardostachin was demonstrated to suppress the overproduction of NO and PGE₂ in LPS-induced BV2 and rat primary microglial cells, its effects on iNOS and COX-2 expression in activated BV2 and rat primary microglial cells were investigated. As shown in Fig. 3, LPS (1 µg/ml) induced a 6–7-fold increase in iNOS and COX-2 expression after 24 h of treatment. However, nardostachin treatment (1.0–4.0 μM) attenuated LPS-mediated iNOS and COX-2 expression in a dose-dependent manner.

Next, the effects of nardostachin on LPS-induced BV2 and rat primary microglial cells were investigated. The mRNA levels of pro-inflammatory cytokines, including TNF-α, IL-6, IL-12 and IL-1β were assayed at non-cytotoxic concentrations. The production of TNF-α, IL-6, IL-12 and IL-1β was increased in the cells treated with LPS alone, whereas the co-treatment of nardostachin with LPS downregulated the overproduction of TNF-α, IL-6, IL-12 and IL-1β mRNA in a dose-dependent manner (Fig. 4).

Since stimulation of the NF-κB pathway is a critical step in the inflammatory reaction, whether nardostachin inhibits LPS-induced NF-κB activation in LPS-stimulated BV2 and rat primary microglial cells was investigated by examining its effects on NF-κB translocation. As shown in Fig. 5A and B, LPS-stimulated phosphorylation of IκB-α was significantly suppressed by nardostachin. Furthermore, IκB-α was degraded after the cells were stimulated with LPS for 1 h. However, IκBα degradation was markedly inhibited by the pretreatment of the cells with nardostachin. It was also demonstrated to block LPS-stimulated nuclear translocation of the NF-κB p50/p65 dimer. As shown in Fig. 5C-F, the protein expression of cytosolic p65/p50 was decreased, and protein levels of nuclear p65/p50 were increased following stimulation with LPS for 1 h. However, treatment with nardostachin suppressed the LPS-enhanced protein levels of nuclear p65/p50 in a dose-dependent manner. The results of the NF-κB binding assay also indicated that treatment with nardostachin markedly inhibited the DNA-binding activity of NF-κB in the nucleus (Fig. 5G and H). Immunofluorescence analysis indicated the significant translocation of NF-κB/p50 into the nucleus upon LPS stimulation. However, nardostachin pretreatment blocked nuclear translocation in the LPS-induced BV2 and rat primary microglial cells (Fig. 5I and J).

NF-κB activation is alternatively regulated by the intracellular signaling proteins, MAPKs, in LPS-induced BV2 and rat primary microglial cells (26). Therefore, it was investigated whether nardostachin regulates the phosphorylation of these signaling proteins, which may be activated by LPS in BV2 and rat primary microglial cells. As shown in Fig. 6, LPS-induced phosphorylation of JNK was significantly suppressed by pretreatment of the cells with nardostachin for 3 h. This effect was dose-dependent, suggesting an additional characteristic of nardostachin in regulating LPS-induced inflammation. However, ERK and p38 phosphorylation were unaffected upon exposure to nardostachin (Fig. 6).

TLR4 is involved in the regulation of LPS-stimulated inflammatory mediators through stimulation of the NF-κB and MAPK pathways. Therefore, the effect of nardostachin on protein expression of TLR4 was investigated to gain further insight into the mechanism underlying its anti-neuroinflammatory activity. As shown in Fig. 7, TLR4 and MyD88 protein expression was increased in BV2 and rat primary microglial cells upon LPS treatment, whereas pretreatment with nardostachin for 3 h attenuated the LPS-induced TLR4 and MyD88 protein expression in a dose-dependent manner.