A Diarylheptanoid from Lesser Galangal (Alpinia officinarum) Inhibits Proinflammatory Mediators via Inhibition of Mitogen- Activated Protein Kinase, p44/42, and Transcription Factor Nuclear Factor- B

The diarylheptanoid 7-(4 -hydroxy-3 -methoxyphenyl)-1-phenylhept-4-en-3-one (HMP) is a naturally occurring phytochemical found in lesser galangal (Alpinia officinarum). In the present study, we have demonstrated the anti-inflammatory properties of this compound on mouse macrophage cell line (RAW 264.7) and human peripheral blood mononuclear cells (PBMCs) in vitro. Treatment of RAW 264.7 cells with HMP (6.25–25 M) significantly inhibited lipopolysaccharide (LPS)-stimulated nitric oxide (NO) production. This compound also inhibited the release of LPS-induced proinflammatory cytokines interleukin-1 (IL-1 ) and tumor necrosis factor(TNF) from human PBMCs in vitro. In addition, Western blotting and reverse transcription-polymerase chain reaction analysis demonstrated that HMP decreased LPS-induced inducible nitric-oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein and mRNA expression in RAW 264.7 cells. Furthermore, HMP treatment also reduced nuclear factorB (NFB) DNA binding induced by LPS in RAW 264.7 cells. To elucidate the molecular mechanism for inhibition of proinflammatory mediators by HMP (25 M), we have studied the effect of HMP on LPS-induced p38 and p44/42 mitogen-activated protein kinase (MAPK). We observed that the phosphorylation of p44/42 MAPK in LPS-stimulated RAW 264.7 cells was markedly inhibited by HMP, whereas activation of p38 MAPK was not affected. These results suggested that HMP from lesser galangal suppressed the LPS-induced production of NO, IL-1 , and TNFand expression of iNOS and COX-2 gene expression by inhibiting NFB activation and phosphorylation of p44/42 MAPK. The use of herbal therapy or alternative medicine is becoming an increasingly attractive approach for the treatment of various inflammatory disorders. The majority of naturally occurring phenolics found in plants possess tremendous antioxidative and anti-inflammatory activities (Surh et al., 2001). Anti-inflammatory properties of various phytochemicals are mediated through the inhibition of nitric oxide (NO), prostaglandins, leukotriene synthesis, and production of cytokines such as interleukin (IL)-1 , IL-6, IL-12, interferon(IFN), and tumor necrosis factor(TNF). Antioxidants such as ( )-epigallocatechin-3-gallate (Lin and Lin, 1997), resveratrol (Tsai et al., 1999) and naturally occurring flavonoids, including apigenin and kaempferol (Liang et al., 1999) have been reported to suppress NO production through inhibition of nuclear factorB (NFB). Previous studies have shown that ginger (Zingiber officinale) and its constituents, which are used for the treatment of cancer, are potent inhibitors of immune cell activation and cytokine secretion (Ageel et al., 1989). Pharmacologically, ginger possess very complex mixture of compounds such as gingerols, -carotene, capsaicin, caffeic acid, and curcumin. Various formulations of ginger have been shown to act as a dual inhibitor of both COX and lipooxygenase (Mustafa et al., 1993), to inhibit leukotriene synthesis (Kiuchi et al., 1992), and to reduce carrageenan-induced rat-paw edema (Mascolo et al., 1989; Jana et This work was supported by New Jersey Agricultural Experiment Station’s Hatch Project (Cook College, Rutgers, The State University of New Jersey). Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. DOI: 10.1124/jpet.103.049171. ABBREVIATIONS: NO, nitric oxide; IL, interleukin; IFN, interferon, TNF, tumor necrosis factor; NFB, nuclear factorB; COX-2, cyclooxygenase-2; NOS, nitric-oxide synthase; iNOS, inducible nitric-oxide synthase; HMP, 7-(4 -hydroxy-3 -methoxyphenyl)-1-phenylhept-4-en-3-one; LPS, lipopolysaccharide; PBMC, peripheral blood mononuclear cell; MAPK, mitogen-activated protein kinase; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; MTT, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide; ELISA, enzyme-linked immunosorbent assay; RT-PCR, reverse transcription-polymerase chain reaction; PBS, phosphate-buffered saline; PMSF, phenylmethylsulfonyl fluoride; PCR, polymerase chain reaction; EMSA, electrophoretic mobility shift assay; DTT, dithiothreitol; bp, base pair(s); I B, inhibitory B; SB203580, (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole); PD98059, (2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one). 0022-3565/03/3053-925–931$7.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 305, No. 3 Copyright © 2003 by The American Society for Pharmacology and Experimental Therapeutics 49171/1065830 JPET 305:925–931, 2003 Printed in U.S.A. 925 at A PE T Jornals on O cber 2, 2017 jpet.asjournals.org D ow nladed from al., 1999). Another closely related plant, commonly known as greater galanga (Alpinia galanga), has also traditionally been used for rheumatic diseases in South East Asian medicine. The German Commission E Monographs lists the use of lesser galangal, which is closely related to greater galanga, for dyspepsia and loss of appetite. The United States Food and Drug Administration lists ginger and lesser galangal as “generally regarded as safe” (21 CFR Section 182.10, 182.20). Various preparations from lesser galangal have been used as traditional medicine in China due to its significant therapeutic properties for spleen and stomach. Most important compounds identified from lesser galangal are flavonoids and diarylheptanoids. The various gingerols and diarylheptanoids, naturally occurring in lesser galangal, have been shown as potent inhibitor of prostaglandin synthase enzymatic activity (Kiuchi et al., 1992). The critical role of NO in various pathological conditions has led to the discovery of new therapeutic agents from varied sources. NO is a short-lived free radical produced from L-arginine in a reaction catalyzed by NO synthase (NOS.) It mediates diverse functions by acting on most cells of the body through the interaction with different molecular targets, which can either be activated or inhibited (Xie and Fidler, 1998). At least three types of NOS isoforms have been reported (Nathan and Xie, 1994a): endothelial NOS, neuronal NOS, and inducible NOS (iNOS). The endothelial NOS and neuronal NOS are constitutively expressed and are Ca / calmodulin-dependent, whereas expression of the high-output isoform iNOS is induced by LPS and various cytokines such as IFN, IFN, IFN, IL-1 , IL-1 , and TNF(Nathan and Xie, 1994b). Low concentrations of NO produced by iNOS possess beneficial roles in antimicrobial activity of macrophages against pathogens (Cook and Cattell, 1996). At the same time, excessive production of NO and its derivatives, such as peroxynitrite and nitrogen dioxide, have been suggested to be mutagenic in vivo and to provoke the pathogenesis of septic shock and diverse autoimmune disorders (Kilbourn et al., 1990; Wink et al., 1991; Nguyen et al., 1992; Miller et al., 1993). Furthermore, NO and its oxidized forms have also been shown to be carcinogenic (Halliwell, 1994). Therefore, agents that can suppress high NO production by inhibiting iNOS expression or its activity can be used as potential therapeutic tools for management of NO-related disorders. In the current study, we have evaluated a diarylheptanoid, HMP [7-(4 -hydroxy-3 -methoxyphenyl)-1-phenylhept-4-en3-one] (chemical structure shown in Fig. 1), isolated from Alpinia officinarum (Kiuchi et al., 1992; Liu et al., 2003) for its anti-inflammatory properties, specifically by using in vitro model systems of inflammation. We demonstrate that HMP suppresses the LPS-induced proinflammatory cytokines (IL-1 and TNF) production from human PBMCs and NO production from mouse macrophage cells (RAW 264.7). HMP also inhibits LPS induced iNOS and COX-2 mRNA and protein expression. Furthermore, we show that HMP reduces the activation of mitogen-activated protein kinase (MAPK) p44/42 and NFB DNA binding activity induced by LPS. Materials and Methods Reagents and Cells. Mouse macrophage cell line RAW 264.7 was obtained from American Type Culture Collection (Manassas, VA) and grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 15% fetal bovine serum (FBS), penicillin (100 U/ml), and streptomycin (100 g/ml). DMEM, RPMI 1640 medium, LPS, Tri-reagent, Ficoll-hypaque, Griess reagent, monoclonal anti-actin antibody, and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO). IL-1 and TNFELISA kits were purchased from R & D Systems (Minneapolis, MN). The relative RT-PCR kit for mouse iNOS and COX-2 were obtained from Ambion (Austin, TX), mouse monoclonal anti-iNOS and COX-2 were purchased from BD Biosciences PharMingen (San Diego, CA), and anti-mouse and anti-rabbit IgG conjugated with horseradish peroxidase were purchased from DAKO (Carpinteria, CA). Monoclonal antibody against phospho-p44/42 was obtained from Cell Signaling Technology, Inc. (Beverly, MA). Polyclonal antibodies against phospho-p38, total p38, and total p44/42 were also obtained from Cell Signaling Technology Inc. Isolation and Identification of HMP. We have isolated a diarylheptanoid from the rhizomes of lesser galangal by bioassay-directed fractionation. Normal phase column chromatography followed by semipreparative reversed-phase high-performance liquid chromatography was used to isolate this diarylheptanoid, which was identified to be HMP (Liu et al., 2003). This compound was confirmed to be 99% pure by high-performance liquid chromatography and NMR studies. MTT Assay for Cell Viability. MTT is a pale yellow substrate that is reduced by living cells to yield a dark blue formazan product. This process requires active mitochondria, and even freshly dead cells do not reduce significant amounts of MTT. Mouse macrophage cell line RAW 264.7 were cultured in 96-well flat-bottom plate at concentration of 0.25 million/ml and after 12 h of preconditioning, cells were treated with various concentrations of HMP for 48 h. Thereafter, culture medium was aspirated and 100 l of MTT dye (1 mg/ml in PBS) was added to the cultures and further incubated for 4 h at 37°C. The formazan crystals made due to dye reduction by viable cells were dissolved using acidified isopropanol (0.1 N HCl). Index of cell viability was calculated by measuring the optical density of color produced by MTT dye reduction at 570 nm. Nitric Oxide Measurement. The RAW 264.7 cells were cultured in DMEM supplemented with 15% FBS, 50 units/ml penicillin, and 50 g/ml streptomycin. The cell suspension of 0.5 million cells/well was cultured for 12 h. Cells were then treated with either LPS (0.5 g/ml) alone or LPS with various concentrations of HMP (6.25–25 M) for 24 h. The cell supernatants were collected at the end of culture for nitrite assay, which were used as a measure of NO production (Eigler et al., 1995). Equal volume of Griess reagent (Sigma-Aldrich) was mixed with each group of cell supernatant (100 l), and the absorbance was measured at 570 nm. The concentration of nitrite (micromolar) was calculated from standard curve drawn with known concentration of sodium nitrite dissolved in DMEM. The results are presented as mean S.D. of four replicates of one representative experiment and this experiment was repeated five times with similar results. TNFand IL-1 ELISA. The PBMCs were separated from peripheral blood of normal healthy human volunteers. Cell suspension of 0.5 10 cells/ml in RPMI 1640 medium supplemented with 10% Fig. 1. The structure of diarylheptanoid HMP, isolated from A. officinarum. 926 Yadav et al. at A PE T Jornals on O cber 2, 2017 jpet.asjournals.org D ow nladed from FBS was prepared, and 200 l/well of this cell suspension was cultured in 96-well flat-bottom plate. PBMC suspension was treated with LPS (10 ng/ml) either alone or in combination of different concentration of HMP (6.25–25 M). Cell supernatants from each group were harvested after 18 h and stored at 70°C until tested. The quantity of IL-1 and TNFpresent in supernatants was estimated by ELISA (R & D Systems) following manufacturer’s instructions. The concentrations of IL-1 and TNFin samples were calculated from standard curve drawn with known concentration of recombinant ILand TNF. The results are presented as mean (picograms per milliliter) S.D. of three replicates of one representative experiment and this experiment was repeated three times with similar results. Preparation of Total Protein Lysate for Western. The RAW 264.7 cells were cultured with LPS alone or with various concentrations of HMP (12.5 and 25 M) for indicated time points. At the end of incubation, cells were rapidly washed with ice-cold PBS and solubilized in cold lysis buffer containing 10 mM Tris-base, 5 mM EDTA, 50 mM NaCl, 1% Triton X-100, 5 mM phenylmethylsulfonyl fluoride (PMSF), 2 mM sodium orthovanadate, 10 g/ml leupeptin, 25 g/ml aprotinin, 1 mM sodium pyrophosphate, and 20% glycerol. After incubation for 30 min on ice, lysates were centrifuged (12,500 rpm, 15 min.) and supernatants were collected and protein concentration in samples was estimated by Bio-Rad protein assay reagent (Bio-Rad, Hercules, CA) following manufacturer’s instructions. Western Blotting. Equal amount of protein (40 g) from each sample was resolved on SDS-polyacrylamide electrophoresis gel (8 and 10% separating gels for iNOS and p42/44, respectively). After electrophoresis the proteins were transferred to Hybond enhanced chemiluminescence nitrocellulose membrane (Amersham Biosciences, Inc., Piscataway, NJ). Membrane was then blocked in blocking buffer containing 20 mM Sodium phosphate buffer, pH 7.6, 150 mM NaCl, 0.1% Tween 20, and 5% nonfat dry milk for 1 h at room temperature. Thereafter, membrane was incubated with primary antibody at 4°C overnight and membrane was then washed four times with PBS-Tween 20 and further incubated with secondary antibody for 1 h at room temperature. Specific bands were detected using enhanced chemiluminescence’s detection system (Amersham Biosciences, Inc.), and the membrane was exposed to X-ray film. Densitometry was performed using image analysis software (Scion; National Institutes of Health). Determination of Relative Change in iNOS and COX-2 mRNA Expression. The RAW 264.7 cells were cultured (10/well) in six-well plate for 24 h followed by treatment with LPS either alone or with different concentrations of HMP for 12 h. Total RNA was isolated using Tri-reagent (Sigma-Aldrich), and 5 g of this total RNA was reverse transcribed to make cDNA using random hexamer and superscript reverse transcriptase (Invitrogen, Carlsbad, CA), following manufacturer’s instructions. Linear range of amplification of iNOS and COX-2 cDNA was determined using gene-specific primers from Ambion, following manufacturer’s instructions. Briefly, the optimum amount of 18S primer and competitor for iNOS and COX-2 gene was determined. The PCR for iNOS (2 l of cDNA, 30 cycles) and COX-2 (1 l of cDNA, 25 cycles) was performed in a final volume of 50 l containing dNTPs (each at 2.5 mM), 1 PCR buffer, 5 units of TaqDNA polymerase, 0.4 M gene specific primer, and optimum ratio of 18S primer and competitor (3:7). Finally, PCR products from each sample (10 l) were resolved in 2% agarose gel (Fisher Scientific Co., Fair Lawn, NJ), stained with ethidium bromide, and image of gel was captured at appropriate exposure time. Densitometric analysis was performed using image analysis software (Scion). Electrophoretic Mobility Shift Assay (EMSA). RAW 264.7 cells were treated with either LPS (0.5 g/ml) alone or with various concentration of HMP for 2 h. Thereafter, nuclear extracts were prepared using a modified method (Lahti et al., 2000). Briefly, cells were washed once with PBS (pH 7.2) and were suspended in hypotonic buffer A [10 mM HEPES (pH 7.6), 10 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.5 mM PMSF] for 10 min on ice, and vortexed for 10 s. Nuclei were pelleted by centrifugation at 12,000g for 5 min. Then the pellets were suspended in buffer B [20 mM HEPES (pH 7.6), 25% glycerol, 0.4 M NaCl, 1 mM EDTA, 1 mM DTT, 0.5 mM PMSF] for 30 min on ice. The supernatants containing nuclear proteins were collected by centrifugation at 12,000g for 20 min and stored at 80°C. For electrophoretic mobility shift assays, 6 g of each nuclear extract was mixed with the P-labeled double-stranded NFB binding consensus oligonucleotides (5 -AGTTGAGGGGACTTTCCCAGGC-3 ) (Promega, Madison, WI) and incubated at room temperature for 20 min. The incubation mixture contains 1 g of poly(dI-dC) in a binding buffer [25 mM HEPES (pH 7.9), 0.5 mM EDTA, 0.5 mM DTT, 1% Nonidet P-40, 5% glycerol, and 50 mM NaCl]. The DNA/protein complex was electrophoresed on 5% nondenaturing polyacrylamide gels in Tris/acetate/EDTA buffer. The specificity of binding was also examined by competition with the unlabeled oligonucleotides. Mobility shift of DNA due to binding of NFB complex was detected by PhosphorImager-445 SI (Amersham Biosciences, Inc.). Statistical Analysis. Data are expressed as mean standard deviation of indicated experiments. Statistical significance between two groups was determined by Student’s t test. The significance level was set at p 0.05.