Whole plants of GT, which were grown and collected in Gyeongsangbuk-do province in Korea in 2011, were purchased from Omniherb Co. (Daegu, Korea) and authenticated by Professor S. Lee at the College of Korean Medicine, Sangji University, Wonju, Korea. A voucher specimen (no. sjomph003) is kept at the College of Korean Medicine, Sangji University. The air-dried whole plant of GT (50 g) was cut and extracted with 50% EtOH (500 ml, twice) at 60°C for 3 h, assisted by ultrasonic waves (40 kHz). The extract was filtered with filter paper (6 μm; Whatman PLC, Kent, UK) and concentrated using a rotary evaporator (Eyela, Tokyo, Japan). Subsequently, the extract was lypophilized using a freeze dryer (Labconco, Kansas City, MO, USA) to yield 14.2 g of powder (28.4%). The powder was dissolved in distilled water for stock solution (1 mg/ml) and diluted with culture medium prior to use in the experiments.

TLR4-specific E. coli LPS was purchased from Alexis Biochemicals (San Diego, CA, USA). Mouse INF-γ was supplied by R&D Systems (Minneapolis, MN, USA). All antibodies used in this study, including antibodies to Nrf2 (SC-13032), the p65 subunit of nuclear factor-κB (NF-κB; SC-8008), lamin B (SC-365962) and hnRNP (SC-10030R) were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). All chemicals and reagents, including 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), sulforaphane (SFN) and N-acetyl cysteine (NAC) were obtained from Sigma-Aldrich (St. Louis, MO, USA) unless indicated otherwise.

HPLC analysis was performed using an Agilent 1200 series system (Agilent Technologies, Santa Clara, CA, USA), which consisted of a solvent delivery unit, an on-line degasser, a column oven, an autosampler and a multi-wavelength detector. For data analysis, LC solution software (version 1.24) was used. The analytical column used was a Phenomenex Gemini NX-C18 column (Phenomenex Inc., Torrance, CA, USA; 4.6×250 mm; pore size, 3.5 μm). The mobile phases were solvent A [0.1% formic acid aqueous (v/v)] and solvent B (acetonitrile). The gradient flow was as follows: (A)/(B) = 95/5 (0 min) → (A)/(B) = 30/70 (60 min). The column temperature was maintained at 35°C. The analysis was carried out at a flow rate of 0.6 ml/min with detection at 275 nm. The column injection volume was 10 μl. A standard solution, containing geraniin (ChemFaces, Wuhan, China) and quercetin (Sigma-Aldrich), was prepared by dissolving in distilled water (10 mg/100 ml). The solution was filtered through a 0.45 μm membrane filter and HPLC was performed.

Male C57BL/6 mice, inbred in a specific pathogen-free facility, were purchased from Samtako Bio Korea, Ltd. (Osan, Korea). Nrf2 knockout mice, which were generated from C57BL/6 mice, were a generous gifted from Dr Jefferson Chan (Pathology and Laboratory Medicine, University of California, Irvine, CA, USA). The animals were housed in certified, standard laboratory cages, and allowed access to food and water ad libitum prior to use in the experiments. All experimental procedures followed the Guidelines for the Care and Use of Laboratory Animals of the National Institutes of Health of Korea, and all the experiments were approved by the Institutional Animal Care and Use Committee of Pusan National University, Pusan, Korea.

RAW264.7 cells (American Type Culture Collection, Rockville, MD, USA) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, Carlsbad, CA, USA) containing L-glutamine (200 mg/l; Invitrogen) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS; Sigma-Aldrich), 100 U/ml penicillin and 100 μg/ml streptomycin (Invitrogen) and maintained in a humidified incubator at 37°C and 5% CO₂ prior to use in the experiments.

BMDM were prepared as previously described (24). Briefly, following the asphyxiation of the mice with CO₂, cellular material from the femurs was aspirated, pressed through a nylon mesh filter (30 μm; Merck Millipore, Billerica, MA, USA), and centrifuged at 400 × g at 4°C for 5 min. Subsequently, the cells were re-suspended in DMEM containing 10% FBS, 100 U/ml penicillin, 0.1 mg/ml streptomycin, 2 mM L-glutamine and 50 ng/ml macrophage-colony stimulating factor (M-CSF; PeproTech, Rocky Hill, NJ, USA). Following incubation at 37°C and 5% CO₂ for 24 h, the cells were washed 3 times with DMEM to remove non-adherent cells and cultured for 1 week, which was subsequently replaced every 2 days. The cells were then detached, washed, counted and replated for the experiments.

The viability of the RAW264.7 cells was assessed using an MTT-based colorimetric assay. In brief, MTT solution (2.0 mg/ml) was added to each well of the cells cultured in a 96-well plate. At 4 h after incubation at 37°C in a CO₂ cell culture incubator, the supernatants were removed and the formazan crystals formed in viable cells were measured at 540 nm using a microplate reader (VICTOR3; Perkin Elmer, Waltham, MA, USA). The percentage of living cells was calculated against the untreated cells.

Total RNA was isolated from the tissues and cells using a Qiagen RNeasy mini kit (Qiagen, Hilden, Germany). Three micrograms of total RNA were then reverse transcribed using M-MLV reverse transcriptase (Promega, Madison, WI, USA) and single-stranded cDNA was amplified by PCR with a set of specific primers as follows: inducible nitric oxide synthase (iNOS) forward, 5′-CTGCAGCACTTGGATCAGGAACC-3′ and reverse, 5′-GGGAGTAGCCTGTGTGCACCTGGAA-3′; tumor necrosis factor-α (TNF-α) forward, 5′-CTACTCCTCAGAGCCCCCAG-3′ and reverse, 5′-AGGCAACCTGACCACTCTCC-3′; interleukin-1β (IL-1β) forward, 5′-GTGTCTTTCCCGTGGACCTT-3′ and reverse, 5′-TCGTTGCTTGGTTCTCCTTG-3′; NQO1 forward, 5′-GCAGTGCTTTCCATCACCAC-3′ and reverse, 5′-TGGAGTGTGCCCAATGCTAT-3′; HO-1 forward, 5′-TGAAGGAGGCCACCAAGGAGG-3′ and reverse, 5′-AGAGGTCACCCAGGTAGCGGG-3′; GCLC forward, 5′-CACTGCCAGAACACAGACCC-3′ and reverse, 5′-ATGGTCTGGCTGAGAAGCCT-3′; and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) forward, 5′-GGAGCCAAAAGGGTCATCAT-3′ and reverse, 5′-GTGATGGCATGGACTGTGGT-3′. For PCR amplification, Taq PCRx DNA polymerase (Invitrogen) was used. The conditions used for the reaction were as follows: an initial denaturation at 95°C for 5 min followed by 22 cycles for GAPDH; 25 cycles for IL-1β, TNF-α, iNOS, NQO1 and HO-1; and 30 cycles for GCLC of denaturation for 40 sec at 95°C, annealing for 40 sec at 57°C, and extension for 50 sec at 72°C with a final extension for 7 min at 72°C. The amplified DNA were then separated on 1.2% agarose gels in 1X TBE buffer at 100 V for 30 min, stained with ethidium bromide (Sigma-Aldrich) and visualized under UV light.

To assay NF-κB and Nrf2 transcriptional activity, we used previously constructed reporter cell lines stably harboring an NF-κB/luciferase reporter and NQO1/luciferase reporter (25). Luciferase activity was examined using a luciferase assay kit (Promega) and normalized by the amount of total proteins from the total cell extract.

Nuclear proteins of 5×10⁶ cells were isolated using an NE-PER nuclear extraction kit (Thermo Fisher Scientific, Waltham, MA, USA) and the amount of protein was measured using the Bradford method (Bio-Rad Protein assay; Bio-Rad Laboratories, Hercules, CA, USA). Equal amounts of proteins were electrophoresized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes (Bio-Rad Laboratories). The blots were blocked for 1 h with 5% non-fat dry milk prior to incubation with antibodies against Nrf2, NF-κB (p65), lamin B and hnRNP at 4°C overnight. Following incubation with secondary antibodies conjugated to HRP at room temperature for 1 h, the bands of interest were revealed by chemiluminescence (SuperSignalWest Femto; Thermo Fisher Scientific).

Intracellular ROS generation was determined by 5-(and-6)-carboxy-2′,7′-dichlorodihydro-fluorescein diacetate (carboxy-H2DCFDA; Molecular Probes, Eugene, OR, USA). Briefly, the cells were treated with 100 μM carboxy-H2DCFDA in culture medium and incubated at 37°C for 30 min. The cells were then washed with phosphate-buffered saline (PBS) and their fluorescence was measured using a BD FACSCanto II system (BD Biosciences, San Jose, CA, USA) at an excitation wavelength of 488 nm and an emission wavelength of 525 nm. The measured values were calculated as a percentage of the control.

The intensity of the bands obtained from RT-PCR and western blot analysis were quantified using ImageJ software (NIH, Bethesda, MD, USA). The values were calculated as fold increases over the control and are expressed as the means ± SD. The differences between 2 groups were determined by a one-way analysis of variance (ANOVA) with a post hoc test. The minimum significance level was set at a p-value of 0.05 for all analyses. All experiments were independently performed at least 3 times.

At first, the extract was analyzed for the constituents of GT by comparing it with the known components, geraniin and quercetin (23,26) (Fig. 1A and B). Subsequently, we determined an optimal dose of GT, at which does not show any significant cytotoxicity, by MTT assay. GT showed no significant cytotoxicity to the RAW264.7 cells at concentrations of up to 100 μg/ml (Fig. 1C). The half maximal inhibitory concentration (IC₅₀) of GT to affect the viability of the RAW264.7 cells was >1,000 μg/ml. Thus, we used GT at concentrations of up to 100 μg/ml for the subsequent experiments.

In order to examine the effects of GT on INF-γ- and LPS-induced inflammation, the cells were pre-treated with GT for 4 h prior to stimulation with INF-γ- and LPS, and the expression levels of NF-κB-dependent genes were analyzed by RT-PCR. The results revealed that GT significantly reduced the expression levels of NF-κB-dependent pro-inflammatory genes, including iNOS, TNF-α and IL-1β (Fig. 2). The IC₅₀ values of GT to affect the expression of inflammatory genes were approximately 60 μg/ml for iNOS, 63 μg/ml for TNF-α and 75 μg/ml for IL-1β.

To elucidate the mechanisms responsible for the anti-inflammatory effects of GT, we first examined the suppressive effects of GT on the INF-γ- and LPS-induced NF-κB activity. Of note, the transcriptional activity of NF-κB, measured by luciferase assay using NF-κB reporter cells, was not reduced by treatment with GT (Fig. 3A). The nuclear translocation of NF-κB was also not altered (Fig. 3B and C). However, GT induced the transcriptional activity of Nrf2 in a dose-dependent manner (Fig. 4A). The nuclear translocation of Nrf2 was also significantly increased by treatment with GT in a dose-dependent manner (Fig. 4B and C).

The time-dependent activation of Nrf2 was determined by detecting the nuclear translocation of Nrf2 by western blot analysis. The results revealed that the nuclear translocation of Nrf2 was significantly increased at 2 to 8 h of treatment with 100 μg/ml of GT (Fig. 5A and B). In order to exclude the possibility that ROS activates Nrf2 to protect the cells from oxidative damage (27), we examined the production of ROS by the GT-treated RAW264.7 cells. GT did not significantly increase ROS production. Taken together, these results indicate that the GT-induced transcriptional activity of Nrf2 is not mediated by ROS (Fig. 5C).

The expression of Nrf2-dependent antioxidant genes may contribute to the suppression of inflammatory responses (28). Thus, we examined the expression levels of Nrf2-dependent genes, including NQO1, HO-1 and GCLC. The results revealed a dose-dependent increase in the expression of Nrf2-dependent genes, which positively correlated with Nrf2 activity (Fig. 6). The expression levels of Nrf2-dependent genes were increased by 3.4-fold for NQO1, 2.8-fold for HO-1 and by 3.1-fold for GCLC when the cells were treated with 100 μg/ml of GT.

Subsequently, we investigated the possibility that Nrf2 activation has a direct association with the anti-inflammatory effects of GT. The macrophages derived from the bone marrow of wild-type C57BL/6 mice showed a similar response to GT treatment. Namely, the expression of NF-κB-dependent pro-inflammatory genes was significantly reduced by treatment with GT in a dose-dependent manner (Fig. 7A and C). However, in the macrophages derived from the bone marrow of Nrf2 knockout mice, the expression of NF-κB-dependent genes was not diminished. On the contrary, the expression of TNF-α was increased in accordance with the concentration of GT (Fig. 7B and D). These results clearly indicate that the anti-inflammatory effects of GT are related to Nrf2 activity.