6-Dehydrogingerdione Restrains Lipopolysaccharide-Induced

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6‑Dehydrogingerdione Restrains Lipopolysaccharide-Induced Inflammatory Responses in RAW 264.7 Macrophages Shih-Han Huang,† Chien-Hsing Lee,§ Hui-Min Wang,‡ Yu-Wei Chang,†,⊗ Chun-Yu Lin,†,#,○ Chung-Yi Chen,*,○ and Yen-Hsu Chen*,†,#,⊥ †

Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan Department of Nursing, Min-Hwei Junior College of Health Care Management, Tainan, Taiwan ‡ Department of Fragrance and Cosmetic Science, Kaohsiung Medical University, Kaohsiung, Taiwan ⊗ Department of Pathology and Laboratory, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan # Division of Infectious Diseases, Department of Internal Medicine, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan ○ School of Medical and Health Sciences, Fooyin University, Kaohsiung, Taiwan ⊥ School of Medical, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan §

ABSTRACT: 6-Dehydrogingerdione (6-DG), one important component of ginger, has been reported to possess some medical effects, such as antitumor and antiatherosclerosis. Herein, the anti-inflammatory effects of 6-DG against lipopolysaccharides (LPS) induced pro-inflammation mediators in RAW 264.7 cells were investigated. Results show that 6-DG significantly attenuated inducible nitric oxide synthase (iNOS, NOS2), cyclooxygenase-2 (COX-2), interleukin-1β (IL-1β), interleukin 6 (IL6), and tumor necrosis factor-α (TNF-α) in the LPS-mediated murine macrophages (RAW 264.7 cells). 6-DG inhibited LPSinduced phosphorylation of both p38 and nuclear factor of κ light polypeptide gene enhancer in B-cells inhibitor-α (IκBα), which further prevented p-p65 nuclear factor-κB (NF-κB-p65) translocation to the nucleus. Moreover, 6-DG increased the ratio of phosphorylated signal transducers and activators of transcription 1 (p-STAT1)/p-STAT3 and down-regulated the gene expression of IL-1β, IL-6, and IL-10. KEYWORDS: 6-dehydrogingerdione, lipopolysaccharides, inflammation, signal transducers and activators of transcription 1 and 3 (STAT-1 and -3)

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INTRODUCTION Sepsis, a complex syndrome mainly induced by lipopolysaccharides (LPS), often occurs during a serious infection and is a major cause of death in critical patients and patients from longterm care units.1 Enhanced productions of several inflammatory cytokines are involved in the pathogenesis of sepsis.2 The plasma levels of several cytokines (interleukin (IL)-1β, IL-6, IL10, and tumor necrosis factor-α (TNF-α)) are significantly higher in septic patients who do not survive.3 During sepsis, nitric oxide (NO) is produced in large amounts by inducible nitric oxide synthase (iNOS, NOS2),4 and prostaglandin E2 (PGE2) is mostly produced by COX-2.5 The overproduction of these inflammatory mediators (including NO, PGE2, and cytokines) would lead to poor prognosis. Previous studies have demonstrated that the blockade of iNOS6 and COX-27 can potentially reverse the worse condition, including hyperdynamic circulatory state during sepsis, and lead to better prognosis. Macrophages play key roles in directing the host immune response to infection. LPS is a cell wall polymer present in Gram-negative bacteria and has been reported as a major causative agent of sepsis.8 LPS stimulation of monocytes activates several intracellular signaling transduction pathways, which includes the I kappa B kinase (IKK)-NF-κB pathway and the mitogen-activated protein kinase (MAPK) pathway [c-Jun © 2014 American Chemical Society

N-terminal kinase (JNK), extracellular signal-regulated kinases (ERK1/2), and p38]. The purpose of these pathways is to activate a variety of transcription factors that include AP-1 (cFos/c-Jun) and NF-κB (p50/p65), which mediate the expression of many genes encoding inflammatory mediators. After stimulation with LPS, NF-κB translocates into the nucleus and binds to κB-binding sites in the promoter regions of many target genes to enhance the expression of these proinflammatory genes,9 such as iNOS,10 COX-2,11 TNF-α,12 IL6,13 and IL-1β.12 Ginger (Zingiber officinale) is widely used in the world as an important spice and traditional herb. It is well-known that ginger extract has some medical properties such as antiinflammation,14 antibacterial,15 antitumor,16 antioxidation,17 and analgesic effects.18 6-Dehydrogingerdione (6-DG) is an active constituent of dietary ginger and recent studies indicate that it possesses some medical properties, such as wound repair19 and antitumor effects.20 The pathogenesis of tumor is associated with inflammation.21 However, the effects of 6-DG on these inflammatory mediators (including NO, PGE2, Received: Revised: Accepted: Published: 9171

April 12, 2014 August 20, 2014 August 27, 2014 August 27, 2014 dx.doi.org/10.1021/jf501665v | J. Agric. Food Chem. 2014, 62, 9171−9179

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to the manufacturer’s instructions (GE Healthcare, 25-0500-71). The level of iNOS, COX-2, or GAPDH mRNA expression was assessed by RT-PCR. Briefly, total RNA (2 mg) was reverse-transcribed into cDNA using random primers (Promega, C1181) and oligo-dT (Promega, C1101) DNA amplification in 20 cycles of iNOS, 94 °C for 30 s, 60.5 °C for 40 s, and 72 °C for 30 s, followed by a 2 min extension at 72 °C. The thermal cycle conditions of COX-2 were 20 cycles of amplification (94 °C for 30 s, 52 °C for 40 s, 72 °C for 30 s, and 72 °C for 2 min). DNA amplification of GAPDH thermal cycle condition was 94 °C for 30 s, 56 °C for 40 s, and 72 °C for 30 s, followed by a 2 min extension at 72 °C. The PCR products were separated by electrophoresis on a 1.5% agarose gels and visualized by SYBR Safe DNA Gel Stain (Invitrogen, S33102). The amplification of GAPDH acted as an internal control for sample loading. PCR was performed using the following primers: iNOS forward primer, 5′-CCCTTCCGAAGTTTCTGGCAGCAGC-3′’ reverse primer, 5′-GGCTGTCAGAGAGCCTCGTGGCTTTGG-3′. COX-2 forward primer, 5′-CATTGATGGTGGCTGTTTTG-3′; reverse primer, 5′-GTTGCTGGGGGAAGAAATGT-3′. GAPDH forward primer, 5′-TCCACCACCCTGTTGCTGTA-3′; reverse primer, 5′-ACCACAGTCCATGCCATCAC-3′. Western Blotting. Western blotting was performed as mentioned previously.23 Briefly, equal amounts of total proteins were resolved by 10% SDS−polyacrylamide gel and transferred onto polyvinylidene difluoride membranes. The membrane was blocked for 30 min at room temperature with blocking buffer (20 mM Tris-HCl, pH 7.4, 125 mM NaCl, 0.2% Tween 20, 2% BSA, and 0.1% sodium azide). Antibodies were all purchased from Cell Signaling Technology, except iNOS (610432, BD PharMingen, San Diego, CA, USA), tubulin (sc-5286, Santa Cruz), GAPDH (MAB374, Millipore), and Lamin B (sc-6216, Santa Cruz). The membrane was then incubated with the primary antibody of COX-2, iNOS p-Erk1/2 (9100), Erk (4695), p-p38 (9215S), p-38 (9212), p-JNK (9250), JNK (9258S), p-c-Jun (9164), cFos (2250), p-IκB (9246), p-p65 (3033S), STAT1 (9172), p-STAT1 (9171), STAT3 (4904), p-STAT3 (9134), p-JAK2 (3776), tubulin, GAPDH, or Lamin B overnight. The membrane was incubated with blocking buffer and primary antibody overnight at 4 °C. The membranes were subsequently probed with anti-mouse (sc-2005, Santa Cruz) or anti-rabbit (7074, Cell Signaling) or anti-goat IgG (811620, Zymed) antibody conjugated to horseradish peroxidase and visualized using enhanced chemiluminescence (ECL, Amersham). The densities of the bands were quantitated with the GelPro 32 analysis program. Transient Cell Transfection and Luciferase Assay. Transfection of RAW 264.7 cells was performed with TurboFect Transfection Reagent (Thermo Scientific) according to the manufacturer’s instructions. Briefly, cells were plated onto 60 mm dish at a density of 2 × 106 cells/dish for 18−24 h and then transfected with 5 mg of pNF-kB-luc or p-AP-1-luc plasmid, cells were cultured for 24 h and then reseed cell onto 24-well plate at a density of 2 × 105 cells/plate overnight, then pretreated with 6-DG for 30 min before 100 ng/mL LPS treatment. Cells were harvested after 6 h, and luciferase activity was assessed using the Luciferase reporter assay system (Promega) according to manufacturer’s instructions. Enzyme-Linked Immunosorbent Assay (ELISA). RAW 264.7 cells were plated at a density of 4 × 105 cells per well in 12-well plates. Cells were pretreated with different concentrations of 6-DG, and the supernatants were collected 24 h later after stimulation with LPS. The amounts of various proteins were measured by ELISA kits for IL-10 (eBioscience; 88-104-88), IL-6 (eBioscience; 88-7064-88), IL-6 (eBioscience; 88-7064-88), IL-1β (eBioscience; 88-7013-88), TNF-α (eBioscience; 88-7324-88), and PGE2 (R&D Systems; KGE004), respectively. Each assay was performed in duplicate. Electroporation. Negative control esiRNA and STAT1 esiRNA were transfected into RAW 264.7 cells by using Amaxa Nucleofector (Lonza), following the manufacturer’s protocol (Nucleofector solution V, Nucleofector program D-032), respectively, with 1.5 ng of esiRNA/ 2 × 106 cells. Cells were incubated at 37 °C for 24 h. Cells were then

cytokines) in macrophages stimulated with LPS have not yet been investigated. The aim of this study was to determine the effects on these mediators and cytokines and possible regulation mechanisms of 6-DG in LPS-treated macrophages.

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MATERIALS AND METHODS

Reagents. LPS (Escherichia coli O26:B6), negative control esiRNA (EHURLUC), and STAT1 esiRNA (EMU071921) were all purchased from Sigma Chemical (St. Louis, MO, USA). All other chemical buffers and reagents were purchased at the highest commercial purity and quality possible. DG Purification Procedures. The rhizomes (25.6 kg) of Z. of f icinale were chipped and air-dried and extracted repeatedly with CHCl3 (50 L × 4) at room temperature. The combined CHCl3 extracts (896.5 g) were then evaporated and further separated into 20 fractions by column chromatography on silica gel (3.8 kg, 70−230 mesh) with n-hexane/chloroform/methanol. Fraction 8 (81.2 g), eluted with chloroform/methanol (60:1), was next subjected to silica gel CC (chloroform/methanol (100:1)) and yielded 6-DG (163 mg). The other fractions were further processed for other components unrelated to this study. The identity and purity of 6-DG were confirmed by NMR and HPLC.20,22,23 Cytotoxicity. The cytotoxic properties were evaluated in an Alamar Blue assay. Briefly, 200 μL of 10% Alamar Blue reagent (Serotec, BUF012B) was added into medium, 2 × 104 cells/200 μL/ well, the cells of which were pretreated with indicated concentrations of 6-DG 30 min before stimulation with LPS (100 ng/mL) for 24 h. Cells were incubated with Alamar Blue reagent (37 °C, 5% CO2) for 4 h, and the colorimetric readings were evaluated with the ELISA plate reader at 570 and 600 nm. Cell Culture. RAW 264.7 cells, derived from murine macrophages, were obtained from the Bioresource Collection and Research Center (BCRC 60001). RAW 264.7 cells were cultured in DMEM (with 10% heat-inactivated fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin, 25 g/mL amphotericin B, and 2 mM L-glutamine, all purchased from Biological Industries (Kibbutz Beit Haemek, Israel)). Various concentrations of 6-DG were dissolved in DMSO. Nitrite Quantification. The nitrite concentration in the supernatant was measured as an indicator of NO production, determined by Griess Reagent System (G2930, Promega Biotech Co., Ltd., Madison, WI, USA) according to the manufacturer protocols. The cells (4 × 105 cells/mL) were pretreated with indicated concentrations of 6-DG for 30 min before stimulation with LPS (100 ng/mL) for 24 h. Extraction of Cytosol, Nuclear, and Total Proteins of RAW 264.7 Cells. RAW 264.7 cells were plated at a density of 5 × 106 cells in each 6 cm dish. Cells were pretreated with 25 μM 6-DG and harvested protein of cytosol and Nuclear at 15, 30, 60, 120 min after stimulation with LPS. Buffer A (10 mM HEPES (pH 7.9), 10 mM KCl, 1.5 mM MgCl2, 10 mM DTT, 4% 25× protease inhibitors (PI), 0.375% NP-40 in ddH2O) and buffer C (20 mM HEPES, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10 mM DTT, 4% 25× PI in ddH2O) were prepared immediately before use. The supernatant was removed and followed by two washings of 1× PBS. Then 300 μL of buffer A was added into each dish and ensuring that buffer A covered cells completely. Ten minutes later, the edges of the dish were knocked to allow cells to shed from the bottom of dish, and then a scraper was used to collect cells. After 10 s of vortex mixing, the cytosol extract was prepared by 1 min centrifugation at 10000g. The resulting pellet contained the nuclear extract. The pellet was washed twice with 1× PBS, then 100 μL of buffer C was added into each sample, and the nuclear envelope was broken by using a stir bar for 20 min. Nuclear extracts were prepared by 10 min centrifugation at 10000g. All above procedures should be performed on ice. In addition, total proteins were extracted from RAW 264.7 cells with RIPA buffer,23 and the protein concentration was measured using the bicinchoninic acid assay (BCA) (Thermo Fisher Scientific, Rockford, IL, USA). Reverse Transcription (RT) PCR. Total RNA was isolated from RAW 264.7 cells using an RNAspin Mini RNA isolation kit according 9172

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pretreated with 25 μM 6-DG 30 min before stimulation with 100 ng/ mL LPS, and the supernatant was harvested after 24 h. Statistical Analysis. All assays were performed in triplicate, and the mean values were calculated. The data were subjected to analysis of variance (ANOVA), and Student’s t tests were used to assess differences between means. Significant differences were concluded at a level of p < 0.05.

in the group with 6-DG post-treatment after LPS given (Figure 2B,C). 6-DG treatment for 48 h alone has no effect on the cell viability of RAW 264.7 cells at different concentrations, as shown in Figure 2D. 6-DG Down-regulates the IKK-NF-κB Pathway To Inhibit the Expression of p-p38. When LPS stimulates macrophages, MAPK pathways are reported to regulate the transcription factor NF-κB and AP-1 (c-Jun/c-Fos).9 Western blotting data demonstrated that pretreatment with 6-DG (25 μM) down-regulates the phosphorylation of both p38 and IκB induced by LPS for 30 min and inhibits the expression of p-p65 induced by LPS for 15 min, but will up-regulate the phosphorylation of JNK induced by LPS for 10 min. There was no effect on p-Erk, p-c-Jun, or c-Fos expression induced by LPS (Figure 3). 6-DG significantly suppressed the NF-κB signaling pathway (Figure 3C) but had little effect on the AP-1 signaling pathway (Figure 3D). According to a past study, p38 MAPK regulates the LPS-induced NF-κB activation pathway through two different mechanisms that contribute to the phosphorylation of IκB-α or p65 NF-κB.24 It may be concluded that 6-DG might down-regulate both IκB and p65 phosphorylation by attenuating the phosphorylation of p38 (Figure 3A,B). 6-DG Inhibits LPS-Induced Nitrite and PGE2 Production via the Suppression of iNOS and COX-2 Expression. COX-2 and iNOS play the key roles in the production of PGE2 and NO during inflammation, and COX-2 and iNOS inhibitors block their synthesis.6,13 Our PCR data revealed that

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RESULTS 6-DG Has an Unapparent Toxicity and Suppresses iNOS in RAW 264.7 Stimulated with LPS. To investigate the cell toxicity of 6-DG (Figure 1), four indicated

Figure 1. Chemical structure of 6-DG.

concentrations (5, 10, 25, and 50 μM) were selected to treat macrophages stimulated with LPS, as shown in Figure 2A. The cell toxicity of 6-DG was determined by Alamar Blue assay and showed there was no significant difference between these four concentrations compared to the vehicle. Results revealed that pretreatment with 25 and 50 μM 6-DG was more efficient in the attenuation of LPS-induced iNOS protein expression than post-treated 6-DG. However, no suppression effect was found

Figure 2. Effect of 6-DG treatment in RAW 264.7 stimulated with LPS. (A) Effects of 6-DG in cell viability. Cells were pretreated with 6-DG for 30 min in various concentrations (5, 10, 25, and 50 μM) before being stimulated with LPS (100 ng/mL) for 24 h and then measured by Alamar Blue assay. Data are expressed as the mean ± SD of three independent experiments. (B) Different treatment conditions: pretreatment where 6-DG was administered 30 min before LPS and post-treatment where 6-DG was administered 30 min after LPS treatment. (C) Corresponding quantification data of iNOS protein levels. (D) 6-DG treatment for 48 h alone to examine the cell viability in RAW 264.7. Results show the mean ± SD of three experiments. (∗) p < 0.05 and (∗∗) p < 0.001 versus LPS treatment, by Student’s t test, n = 3−5. The relative intensity was normalized to tubulin and relative to the control. 9173

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Figure 3. Effects of 6-DG on LPS-induced phosphorylation and the MAPK and Ikk-NFκB pathway. (A) Western blot analysis of the phosphorylation of Erk, p38, and JNK expression induced by LPS. Cells were pretreated with 25 μM 6-DG for 30 min, and the total protein was harvested at four different time points (10, 15, 30, and 60 min) after stimulation with LPS (100 ng/mL). (B) Western blot analysis of the phosphorylation of p65 NF-κB, and IκBα induced by LPS. Cells were pretreated with 25 μM 6-DG for 30 min, and cytosol and nuclear extract were harvested at four different time points (15, 30, 60, and 120 min) after stimulation with LPS (100 ng/mL). Expression levels of p-c-Jun and c-Fos were determined by Western blot analysis. Cells were pretreated with 25 μM 6-DG for 30 min, and total protein was harvested at four different time points (10, 15, 30, and 60 min) after stimulation with LPS (100 ng/mL). RAW 264.7 macrophages were transfected with the luciferase reporter pAP1-luc (C) and pNF-κB-luc (D). Twenty-four hours after transfection, cells were pretreated with 6-DG for 30 min and then exposed to LPS (100 ng/ mL) at the indicated concentrations. After 6 h, the luciferase activity was determined. (∗) p < 0.05 versus LPS treatment group. Bars represent the mean ± SD of three independent experiments. All intensity of results was normalized to internal control and relative to the control. These experiments were repeated three times with similar results.

pretreatment with 5, 10, 25, and 50 μM 6-DG could inhibit the RNA expression of both COX-2 and iNOS induced by LPS (Figure 4A). Western blotting results indicated that pretreatment with 10, 25, and 50 μM 6-DG can inhibit the protein production of both COX-2 and iNOS induced by LPS (Figure 4B). Furthermore, pretreatment with 5, 10, 25, and 50 μM 6DG significantly down-regulated the production of both NO and PGE2 induced by LPS (Figure 4C,D). 6-DG Suppresses LPS-Induced TNFα, IL-6, IL-1β, and IL-10 Production. In patients with sepsis, plasma concentrations of TNF-α, IL-1β, IL-6, and IL-10 were significantly higher in nonsurvivors than in survivors.3 According to our ELISAs, pretreatment with 25 and 50 μM 6-DG was found to significantly inhibit the production of TNF-α, IL-6, and IL-10 in RAW 264.7 macrophages through LPS simulation (Figure 5A,C,D), and pretreatment with 5, 10, 25, and 50 μM 6-DG

could inhibit the secretion of IL-1β induced by LPS (Figure 5B). 6-DG Inhibited STAT3-Mediated Genes through Elevating the p-STAT1/p-STAT3 Ratio. A past study indicated that LPS-stimulated macrophages could activate JAK2, STAT1, and STAT3.25 Many studies revealed that the JAK−STAT pathway is one of the main pathways that regulated cytokine gene expression. Western blotting data showed that pretreatment with 25 μM 6-DG can decrease the phosphorylation of both JAK2 and STAT3 (ser) after stimulation with 100 ng/mL LPS as shown in (Figure 6A). Reversely, pretreatment with 25 μM 6-DG enhanced the expression of p-STAT1 (Figure 6A). There were no changes of these three total protein levels. STAT1 and STAT3 mutually inhibited one another, and another study indicated that STAT1 levels and activity increased in the absence of STAT3.26−28 Our results revealed 9174

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Figure 4. Effects of 6-DG on LPS-induced iNOS, COX-2, NO, and PGE2 expression and production in RAW 264.7 cells. (A) RAW 264.7 cells were treated with the indicated concentration of 6-DG for 30 min followed by LPS (100 ng/mL) for 3 h. RT-PCR analysis of the expression of iNOS and COX-2 was performed. Intensities of the bands for iNOS and COX-2 were normalized to GAPDH and relative to the control. (B) RAW 264.7 cells were treated with the indicated concentration of 6-DG for 30 min followed by LPS (100 ng/mL) for 6 h. Western blot analysis of the expressions of iNOS and COX-2 was performed. The intensities of the bands for iNOS and COX-2 were normalized to tubulin and relative to the control. (C) RAW 264.7 cells were pretreated with the indicated concentration of 6-DG for 30 min followed by LPS (100 ng/mL) for 24 h. Griess reagent assay analysis of the production of nitrite was performed. (D) RAW 264.7 cells were treated with the indicated concentration of 6-DG for 30 min followed by LPS (100 ng/mL). PGE2 assay analysis of the production of PGE2 was performed. Results show the mean ± SD of three experiments. (∗) p < 0.05 and (∗∗)p < 0.001 versus LPS treatment, by Student’s t test, n = 3−6. These experiments were repeated three times with similar results.

Figure 5. Effects of 6-DG on LPS-induced IL-1β, IL-6, IL-10, and TNF-α productions. Cells were pretreated with 6-DG for 30 min in various concentrations (5, 10, 25, and 50 μM) before stimulation with LPS (100 ng/mL) for 24 h. The concentrations of IL-1β, IL-6, IL-10, and TNF-α were measured by using ELISA. Results show the mean ± SD of three experiments. (∗) p < 0.05 and (∗∗) p < 0.001 versus LPS treatment, by Student’s t test, n = 3−5.

similarly mutual phenomenon p-STAT1 upregulation and pSTAT3 downregulation in Figure 6A. We postulate that 6-DG

inhibits the gene mediated by STAT3 possibly through elevating the p-STAT1/p-STAT3 ratio. Our STAT1 knock9175

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Figure 6. Effects of 6-DG on LPS-induced p-JAK2, p-STAT1, and p-STAT3 expression and effects of STAT1 knockdown on 6-DG inhibition of IL1β, IL-6, and IL-10 productions induced by LPS. (A) Western blot analysis of phosphorylated and total amount of key proteins in the JAK/STAT pathway induced by LPS. Intensities of the bands for p-JAK2, p-STAT3, p-STAT1 were normalized to tubulin and relative to the control. There were no changes of these three total protein levels. (B−D) Cells were transfected with negative control or STAT1 esiRNA for 24 hand then pretreated with 6-DG for 30 min in various concentrations (5, 10, 25, and 50 μM) before stimulation with LPS (100 ng/mL) for 24 h. The concentrations of IL-1β, IL-6, and IL-10 were meausred by using ELISA. Results show the mean ± SD of three experiments. (∗) p < 0.05 versus LPS treatment, by Student’s t test, n = 3−5.

overexpression of the pro-inflammatory mediators of the inflammatory process.31 In the present study, we showed that the pretreatment of LPS-stimulated RAW 264.7 macrophages with 6-DG (5−50 μM) inhibited COX-2 and iNOS expression, thereby suppressing COX-2-derived PGE2 and iNOS-derived NO production (Figure 4). PGE2 has been proved able to upregulate the levels of both IL-10 and IL-6 produced by activated murine macrophages.32 Moreover, 6-DG suppressed LPSinduced TNF-α and IL-1β production in RAW 264.7 macrophages. These results suggest that 6-DG exerts its antiinflammatory effects via the suppression of inflammatory enzymes and mediators such as iNOS, COX-2, NO, PGE2, TNF-α, and IL-1β (Figures 4 and 5). Recent studies showed that emphasis on attenuating inflammatory response may exacerbate the progressive development of immunosuppression.3,33,34 MAPKs regulate inflammatory and immune responses,35 and the MAPK signaling pathways are known to be involved in expressing iNOS and COX-2, as well as producing pro-inflammatory cytokines in LPS-induced macrophages.36 The inhibition of p38 MAPK pathways is sufficient to block the induction of proinflammatory mediators by LPS.37 One of the past studies suggested that p38 MAPK mediates the LPS-induced NF-κB activation pathway through two different mechanisms that contributed to the phosphorylation of p65 NF-κB.24 NF-κB activation regulates many gene encoding inflammatory

down experiments confirmed this assumption, whereas the knockdown of STAT1 significantly reversed the 6-DG inhibition of LPS-induced cytokines (IL-6, IL-1β, and IL-10) (Figure 6B−D).

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DISCUSSION

Our study demonstrated 6-DG, one novel component of ginger, restrained several key inflammatory responses in RAW 264.7 macrophages induced by LPS and further investigated their potential molecular regulation mechanisms. 6-DG has the potent ability to inhibit the production of several major LPSinduced inflammatory mediators and cytokines, including NO, PGE2, IL-1β, IL-6, IL-10, and TNF-α induced by LPS via the regulation of several inflammation-related signal transduction pathways, including IKK-NF-κB, p38, and JAK2/STAT1/ STAT3. The inflammation response is a complex reaction of the immune system regulated by many inflammatory mediators, such as NO, prostaglandins, and cytokines. An overproduction of iNOS-derived NO and COX-2-derived PGE2 can have cytotoxic effects in pathological processes, especially in inflammatory and autoimmune disorders.29 These mediators play a regulatory role in a variety of physiological and pathological processes following an immune response and inflammation.30 In addition, macrophages play a central role to mediate several immunopathological conditions and inducing 9176

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Figure 7. Schematic representation of suppression of LPS-induced production of NO, PGE2, IL-1β, IL-6, and TNF-α on 6-DG may be through the inhibition of p38 and IκBα phosphorylation to prevent p-p65 NFκB from entering the nucleus. Besides, 6-DG decreased LPS-induced IL-1β, IL-6, and IL-10 may be through elevating the p-STAT1/p-STAT3 ratio and down-regulating those genes (IL-1β, IL-6, and IL-10) mediated by STAT3.

and activity elevated in the absence of STAT3.28 The balance of pSTAT1 and pSTAT3, which was investigated in cancer immunity, played a pivotal role in regulation of cancer immune responses.47,48 The higher pSTAT1/pSTAT3 ratio in tumor cells or in immune cells is related to the longer survival time of cancer patients.47,49−51 However, this balance has not been studied in the regulation of IL-1β, IL-10, or IL-6 induced by LPS. On the basis of our Western blotting results, the expression of p-STAT increased and p-STAT3 was reduced (Figure 6A). We postulated that 6-DG may decrease LPSinduced IL-1β, IL-6, and IL-10 through elevating the pSTAT1/p-STAT3 ratio and down-regulating the genes (IL-1β, IL-6, and IL-10) mediated by STAT3. Furthermore, we have verified this assumption by using STAT1 esiRNA (Figure 6). JNKs have been implicated in apoptotic response of cells exposed to pro-inflammatory cytokines.52,53 We have previously shown that blocking the 6-DG-induced activation of JNK by chemical inhibitor could prevent Bax up-regulation and caspase9 activity.20 In this paper, our data suggested that 6-DGinduced JNK activation may contribute to 6-DG-induced mitochondrial apoptotic pathways. In summary, we demonstrated that 6-DG decreased LPSinduced production of iNOS, COX-2, IL-1β, IL-6, and TNF-α, which may be through the inhibition of p38 and IκBα phosphorylation to prevent p-p65 NFκB from translocating into the nucleus. Furthermore, 6-DG decreased LPS-induced IL-1β, IL-6, and IL-10 perhaps also through elevating the pSTAT1/p-STAT3 ratio and down-regulating the IL-1β, IL-6, and IL-10, mediated by STAT3 (Figure 7). The inhibition of

mediators, and our results suggested that 6-DG may downregulate both IκB and p65 phosphorylation by attenuating the phosphorylation of p38 (Figure 3) expression and further decreases the expression of iNOS, COX-2, IL-1β, IL-6, TNF-α (Figures 4 and 5). Another important pathway in inflammatory mediator production is Janus-kinase/signal transducers and the activators of transcription (Jak/STAT) pathway.38 Prompt activation of JAK2, STAT1, and STAT3 was demonstrated after LPS stimulation. STAT1-mediated production will regulate proinflammatory mediators, such as COX-239 and iNOS.40 Another previous study indicated that STAT3 activation is also critical for IL-1β,41 IL-6,42 and IL-1043 production. A recent study indicated the regulatory mechanism of iNOS gene expression via cooperation of ISGF3 (a complex containing STAT1, STAT2, and IRF9 subunits) and NF-κB.44 The DNA binding for STAT3 in the promoter of acute-phase proteins was found to compete with NF-κB.45 Our data showed that 6-DG could inhibit the expression of p-p65 NF-κB, but increase the expression of STAT1, and we presume that 6-DG decreases the expression of iNOS through the disruption of ISGF3 and NFκB cooperation (Figure 6A). Past research revealed that the overexpression of STAT1 suppresses the COX-2 promoter activity in insulin-secreting RINm5F cells.46 Our data indicated 6-DG increased p-STAT1 (Figure 6A), but suppressed COX-2 (Figure 4), which might link to this regulation mechanism. STAT1 and STAT3 have similar structures and were found to not only act as functional antagonists but also work to mutually inhibit one another.26 A previous study revealed STAT1 levels 9177

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p38 MAPK has been proposed as a mechanism by which the 5hydroxytryptamine receptor type 3 (5-HT3) antagonist tropisetron could manifest its anti-inflammatory effects.53 6DG is a potent antioxidant with 5-HT3 inhibitor.20 Our experimental results indicate that 6-DG showed potential for the development of novel therapeutic targets for patients with sepsis in the future.

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AUTHOR INFORMATION

Corresponding Authors

*(Y.-H.C.) Mail: Division of Infectious Diseases, Department of Internal Medicine, Kaohsiung Medical University Hospital, No. 100 Tzyou 1st Road, Kaohsiung 807, Taiwan. Phone: 8867-3121101-5677. Fax: 886-7-3228547. E-mail: infchen@gmail. com *(C.-Y.C.) Mail: Department of Medical Laboratory Science and Biotechnology, School of Medical and Health Sciences, Fooyin University, 151 Ching-Hsueh Road, Ta-Liao District, Kaohsiung 831, Taiwan. Phone: 886-7-7811151, ext. 6200. Fax: 886-7-7834548. E-mail: [email protected]. Author Contributions

S.-H.H. and C.-H.L. contributed equally to this work. Funding

This work was financially supported by grants from the Taiwan National Science Council (NSC96-2314-B-037-018-MY3) and the Kaohsiung Medical University Hospital (KMUH-1000R12). Notes

The authors declare no competing financial interest.

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REFERENCES

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