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INFLAMMATION AND IMMUNOPHARMACOLOGY

Inhibition of Nitric Oxide Generation by 23,24-Dihydrocucurbitacin D in Mouse Peritoneal Macrophages

Department of Biochemistry, College of Medicine, Chung-Ang University, Seoul, Korea (C.S.P., H.L., K.J.H., S.H.B., Y.-G.K., H.-Y.Y., K.J.B., N.S.K.); and Laboratory of Biochemistry, KT&G Central Research Institute, Daejeon, Korea (H.O.S., D.W.L.)

Received December 1, 2003; accepted January 28, 2004.

	Abstract

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Nitric oxide (NO) has various physiological functions. However, uncontrolled overproduction of NO can be toxic in many pathologic conditions involving inflammatory tissue damage. In the present study, we examined effects of 23,24-dihydrocucurbitacin D (DHCD) isolated from the root of Bryonia alba L. on macrophage NO generation. DHCD (<80 µM) effectively abolished NO generation from macrophages activated with lipopolysaccharide and interferon-. DHCD decreased the levels of protein and mRNA for inducible NO synthase (iNOS). DHCD potently blocked nuclear factor-B (NF-B) activation, a process necessary for transcriptional activation of iNOS. These results suggested that DHCD inhibited NO generation by blocking NF-B activation and iNOS gene transcription. Because NF-B activation is necessary not only for NO generation but also for many inflammatory processes, DHCD and its derivatives could be developed as anti-inflammatory drugs.

There are several ways to suppress iNOS activity. One is direct inhibition of the enzyme activity mainly by analogs of L-arginine, a substrate of the enzyme (Babu and Griffith, 1998). However, this mode of inhibition may affect both types of NOS, cNOS and iNOS. Treatment with L-arginine analogs exhibited side effects like hypertension in experimental animals (Molnar et al., 1994). Thus, many researchers have now focused on developing NOS type-specific inhibitors (Babu and Griffith, 1998; Tsymbalov et al., 2002). Another way to suppress NO generation from iNOS is to inhibit the expression of the enzyme. Expression of iNOS is regulated primarily at the stage of transcriptional initiation to produce mRNA from the gene (Nathan and Xie, 1994). The promoter region of the iNOS gene has various transcription factor binding sites, such as interferon--response element, activator protein-1 site, tumor necrosis factor-responsive element, and nuclear factor-B (NF-B) binding site (Lowenstein et al., 1993; Xie et al., 1993). Among them, binding of NF-B to the promoter is considered as a critical step for the expression of iNOS (Xie et al., 1994; Kim et al., 1995).

Several substances isolated from natural products suppress iNOS expression, and have been suggested to be possible anti-inflammatory agents (Lee et al., 1998; Yeo et al., 1998; Seo et al., 2000; Han et al., 2002; Lo et al., 2002; Wang et al., 2002). In several areas of the world, plants in the Cucurbitaceae family have been used as a traditional medicinal herb to suppress various symptoms, such as fever, cough, flu, and headache (Sohn et al., 2000). Several compounds with the core structure of a tetracyclic triterpene were isolated from roots of the plant and named cucurbitacin B, C, E, and I. These have shown anticancer and anti-inflammatory activities (Konoshima et al., 1995; Duncan et al., 1996; Peters et al., 1999). However, these cucurbitacins have not been developed as therapeutic agents because of their strong cytotoxicities.

Several years ago, a new cucurbitacin, 23,24-dihycrocucurbitacin D (DHCD; Fig. 1), was isolated from roots of Bryonia alba L. (Cucurbitaceae) collected in Armenia. Unlike other cucurbitacins, DHCD was not toxic to nontumor cells, whereas it was toxic to tumor cells (Baek et al., 1995; Sohn et al., 2000). Therefore, it would be valuable to test the effect of DHCD on iNOS induction and to examine the possibilities for future development of its derivatives or analogs as anti-inflammatory agents. In the present study, we investigate the inhibitory effect of DHCD on the generation of NO and the activation of NF-B in mouse peritoneal macrophages.

View larger version (11K): [in this window] [in a new window]   Fig. 1. Structure of DHCD.

	Materials and Methods

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Materials. DHCD was purified and isolated from roots of B. alba L. from Armenia as described (Baek et al., 1995). Roots of B. alba L. were air dried, powdered, extracted with 80% methanol, and evaporated at <40°C. Ethyl acetate-soluble fraction was obtained from the methanol extract and subjected to a silica gel column chromatography to isolate DHCD. The purity (>99%) and structure were determined by thin-layer chromatography and infrared and nuclear magnetic resonance spectroscopies, respectively (Baek et al., 1995; Sohn et al., 2000). Stock solution of DHCD (800 mM) was dissolved in dimethylsulfoxide, kept at -70°C, and diluted with cell culture medium before use. Interferon- (IFN) was from Genzyme (Cambridge, MA). Fetal bovine serum was from Invitrogen (Carlsdbad, CA). Acrylamide, bis-acrylamide, and ammonium sulfate were from Bio-Rad (Hercules, CA). Antibodies against mouse macrophage iNOS were obtained from Upstate Biotechnology (Lake Placid, NY). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise noted.

Cell Culture and Measurement of NO Production. Thiogly-collate-elicited peritoneal macrophages were obtained from ICR mice (Daehan Experimental Animals, Eumsung, Korea) as described (Kim et al., 1995). Experiments using mice were carried out in accordance with the Guide for the Care and Use of Laboratory Animals, National Academy of Science. Macrophages were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine at 37°C in humidified 5% CO₂/95% air. For the induction of iNOS, cells were dispensed on 96-well plates (1 x 10⁶ cells/ml, 0.1 ml/well) and activated with IFN (12.5 U/ml) and lipopolysaccharide (LPS; 12.5 ng/ml). After 12 h, Griess assay was performed to measure concentrations of accumulated nitrite or nitrate plus nitrite as stable oxidation products of NO in the medium (Kwon et al., 1994; Kim et al., 1995). Cell viability was determined by tetrazolium reduction by using MTT bromide assay (Kim et al., 1995).

Western Analysis and Electrophoretic Mobility Shift Assay (EMSA). Lysates of activated peritoneal macrophages were obtained by a brief sonication after suspension in 60 mM Tris containing 1% SDS and protease inhibitors. Protein concentration was measured by BCA reagent (Pierce, Rockford, IL). Lysates with 40 µg of protein were subjected to 10% SDS-PAGE gels and transferred to nitrocellulose membrane (Trans-Blot; Bio-Rad) at 4°C in a Tris-glycine buffer (39 mM Tris, 48 mM glycine, 0.037% SDS, and 20% methanol, pH 7.4). Membranes were then blocked with 5% skim milk in TTBS (25 mM Tris, pH 7.5; 150 mM NaCl; and 0.1% Tween 20), washed three times with TTBS, and incubated with anti-iNOS antibody (1:1000) for 1 h. The membrane was then washed with TTBS and subjected to a procedure for enhanced chemiluminescence (Pierce).

Nuclear extracts from activated macrophages were prepared and subjected to EMSA for detecting NF-B activation as described (Kim et al., 1995) using NF-B Consensus Oligonucleotide and Gel Shift Assay Core System (Promega, Madison, WI).

Northern Analysis and Reverse Transcription (RT)-Polymerase Chain Reaction (PCR). Total RNA was isolated from macrophages activated with LPS (12.5 ng/ml) and IFN (12.5 U/ml) for 8 h by using the RNeasy kit (Qiagen, Hilden, Germany) according to the manufacturer's instruction. Northern blotting was carried out as described (Kim et al., 1995) and analyzed with a BAS Image Analyzer (BAS-2500; Fuji Film, Tokyo, Japan).

For RT-PCR, cDNA was prepared from total RNA (2 µg) using the Omniscript RT kit (Qiagen). PCR amplification was performed in a 50-µl solution containing 1.5 mM MgCl₂, 10 mM Tris-HCl, 50 mM KCl, four dNTPs (0.2 mM each), 2 U of TaqDNA polymerase (BD Biosciences Clontech, Palo Alto, CA), and either 400 ng of iNOS primers or 2 pmol/ml glyceraldehydes-3-phosphage dehydrogenase (G3PDH) primers. The sense and antisense oligonucleotide for iNOS were 5'-CCCTTCCGAAGTTTCTGGCAGCAGC-3' and 5'-GGCTGTCAGAGCCTCGTGGCTTTGG-3', respectively. The sense and antisense primers for G3PDH were 5'-GAAGGGCTAATGACCACAGTCCAT-3' and 5'-TAGCCATATTCGTTGTCGATCCAGG-3', respectively. The amplification cycle was programmed for 45 s at 94°C, 45 s at 60°C, and 2 min at 72°C (GeneAmp 9600; PerkinElmer, Norwalk, CT). The predicted sizes of PCR products are 496 bp for iNOS, and 465 bp for G3PDH. Quantification of PCR products was performed using the BAS Image Gauger Densitometry Software (Fuji Film).

	Results

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Inhibitory Effects of DHCD on NO Production from Macrophages. To examine the effect of DHCD on NO production by macrophages, peritoneal macrophages from ICR mice were treated with DHCD (0-80 µM) for 4 h and then activated with LPS and IFN. After 12 h of activation, nitrite concentration in the media was measured as a replication of cumulative NO production. In peritoneal macrophages, NO production gradually decreased with increasing concentrations of DHCD and was almost completely abolished by 80 µM DHCD (Fig. 2). ED₅₀ of the inhibition was 13 µM.

View larger version (18K): [in this window] [in a new window]   Fig. 2. Effects of DHCD on NO production from macrophages. ICR mouse peritoneal macrophages were dispensed in 96-well plates (100,000 cells/100 µl/well) and treated with indicated concentrations of DHCD for 4 h. Then, macrophages were activated with 12.5 ng/ml LPS and 12.5 U/ml IFN. After 12 h, nitrite concentrations in the media () and cell viabilities () were measured as described and expressed as percentage of the control (sample without DHCD). Data are mean ± S.D. of three samples.

When the viability of the peritoneal macrophages was measured by 3(4,5-dimethylthiazol-2-yl),2,5-diphenyltetrazolium bromide assay, it was not decreased by the concentrations of DHCD used (Fig. 2). Cell viability also was tested by several other methods. Crystal violet staining of viable cells and trypan blue dye exclusion by living cells were not decreased by 80 µM DHCD (data not shown). These results suggested that used concentrations of DHCD in the present study were nontoxic to peritoneal macrophages. Dimethylsulfoxide in 0.01%, the highest concentration used to dissolve DHCD, was neither toxic to macrophages nor inhibitory to NO production. When the concentration of nitrate, a further oxidation product of NO, was measured together with nitrite, similar inhibitory effect of DHCD on NO generation from peritoneal macrophages also was obtained (data not shown).

The inhibitory effect of DHCD on NO generation was also tested in vivo. LPS (5 mg/kg body weight) was injected into the peritoneal cavity of mice, and the serum concentration of nitrate plus nitrite was measured after 12 h. The concentration was increased to 505.5 ± 103.6 µM (n = 5) from 39.6 ± 5.6 µM (n = 5) by the LPS treatment. When DHCD (5 mg/kg body weight) was injected two times, 12 h before and 6 h after the LPS injection, NO production was inhibited by 70% (178 ± 68.6 µM, n = 5). These results demonstrate that DHCD effectively blocks NO production in macrophage culture and in vivo.

Inhibition of iNOS Expression by DHCD. To examine the effect of DHCD on the iNOS protein expression in macrophages, cells were pretreated with DHCD for 4 h, activated with LPS and IFN for 12 h, and then iNOS expression was analyzed in macrophage lysates. The expression level of iNOS protein was markedly reduced by DHCD in a dose-dependent manner (Fig. 3). When the protein level was estimated by densitometry, 80 µM DHCD inhibited protein expression by 89%. These results show that DHCD effectively blocked NO production by inhibiting expression iNOS protein in peritoneal macrophages.

View larger version (17K): [in this window] [in a new window]   Fig. 3. Effects of DHCD on iNOS protein expression in peritoneal macrophages. Peritoneal macrophages on 24-well plates (400,000 cells/400 µl/well) were treated with indicated concentrations of DHCD for 4 h, then activated with 12.5 ng/ml LPS and 12.5 U/ml IFN. After 12 h of activation, nitrite concentrations of media were measured, and iNOS protein content in macrophage lysates was analyzed by immunoblottings as described under Materials and Methods.

Because iNOS protein induction is regulated mainly at the transcription level (Nathan and Xie, 1994), the effect of DHCD on iNOS mRNA levels in macrophages was examined. Cells were treated with DHCD and activated with LPS and IFN for 8 h, their total RNA was extracted, and level of iNOS mRNA was estimated by Northern analysis. The NO production at 8 h was decreased by 66%, 76%, 88%, and 91% at 10, 20, 40, and 80 µM DHCD, respectively (Fig. 4A). Although the iNOS mRNA level seemed not to be decreased by 10 µM DHCD, it was markedly decreased at higher concentrations of DHCD (Fig. 4B).

View larger version (30K): [in this window] [in a new window]   Fig. 4. Effect of DHCD on iNOS mRNA level in peritoneal macrophages activated with LPS and IFN. Cells were preincubated with indicated concentrations of DHCD for 4 h and then activated with 12.5 ng/ml LPS and 12.5 U/ml IFN. After 8 h, nitrite concentrations of media were measured (A), and levels of mRNA of iNOS in cells were analyzed by Northern blotting (B).

To confirm the inhibitory effect of DHCD on iNOS mRNA levels, RT-PCR analyses were performed. Total RNA extracted from activated peritoneal macrophages was reacted with reverse transcriptase to obtain cDNA. mRNA level was estimated by PCR as described under Materials and Methods. When number of the cycles of the chain reaction was 25, the amount of PCR product for iNOS gene from activated macrophages was decreased by treatment with 40 or 80 µM DHCD (Fig. 5A). There was no significant change in the level of G3PDH gene by treatment with DHCD. Furthermore, by changing the number of cycles for chain reactions, a decrease of the amount of PCR product for iNOS from the cells treated with DHCD was the most apparent between 21 and 27 cycles (Fig. 5B). These results show that the inhibitory effect of DHCD on the NO production is due to decreased mRNA level and expression of the iNOS protein.

View larger version (30K): [in this window] [in a new window]   Fig. 5. RT-PCR analysis of mRNA expression in peritoneal macrophages. A, cells were preincubated with indicated concentrations of DHCD for 4 h and then activated with 12.5 ng/ml LPS and 12.5 U/ml IFN. After 8 h, total RNA was extracted, reacted with reverse transcriptase, and then its content for iNOS mRNA was analyzed by 25 cycles of PCR. Amount of products from the chain reaction were analyzed by agarose gel electrophoresis. B, cycle numbers of PCR were varied from 10 to 35. Amount of PCR product (, from the sample treated with 80 µM DHCD; , from the sample treated without DHCD) was analyzed with agarose gel electrophoresis and densitometry. Arrow, PCR product of iNOS gene; arrowhead, artifacts.

Inhibitory Effect of DHCD on the Activation of NF-B. Activation of NF-B was reported to be an essential step in the transcriptional induction of iNOS (Xie et al., 1994; Kim et al., 1995). DHCD has a similar backbone with steroid hormones that have been known to inhibit iNOS induction by blocking NF-B activation (Witkowski and Konopa, 1981; Kleinert et al., 1996). Therefore, inhibitory effect of DHCD on the activation of NF-B was examined by EMSA. Mouse peritoneal macrophages were pretreated with DHCD and activated with LPS/IFN for 30 min. Cells were then harvested, and their nuclear fractions were analyzed by gel shift assay. Treatment with LPS/IFN markedly increased the level NF-B in nuclear fractions from macrophages, indicating activation of the transcription factor (Fig. 6). In macrophages, the subunit composition of NF-B activated by LPS/IFN was reported to be mixed dimers of p50/p50, p50/p65, and p50/c-rel (Xie et al., 1994; Ding et al., 1995). The addition of DHCD (40 and 80 µM) to macrophages blocked the LPS/IFN-induced activation of NF-B. These results are consistent with the result that DHCD was effective in inhibiting NO production in peritoneal macrophages and suggested that the inhibitory effect of DHCD is by blocking the activation of NF-B.

View larger version (30K): [in this window] [in a new window]   Fig. 6. EMSA analysis of the effects of DHCD on NF-B activation in LPS/IFN-treated macrophages. Mouse peritoneal macrophages were treated with indicated concentrations of DHCD for 4 h and then activated with 12.5 ng/ml LPS and 12.5 U/ml IFN. After 30 min, the cells were harvested, and their nuclear extractions were analyzed by EMSA for NF-B activation. Arrow indicates the complex of NF-B with radiolabeled oligonucleotides.

	Discussion

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In the present study, we examined the effect of DHCD on NO production from mouse peritoneal macrophages. DHCD effectively blocked NO production from peritoneal macrophages with an ED₅₀ of 13 µM. DHCD did not show apparent cytotoxic to peritoneal macrophages. Inhibitory effects of DHCD on the expression of iNOS protein and mRNA were demonstrated by Western analysis, Northern blotting, and RT-PCR. Activation of NF-B, an essential process in iNOS induction, was almost completely blocked. These results suggest that DHCD is effective in primary macrophages for inhibiting NO production, and the effect might occur via inhibiting NF-B activation.

Cucurbitacins isolated from plants in Cucurbitaceae were studied as anticancer agents (Konopa et al., 1974a,b; Cordell and Farnsworth, 1977). Cucurbitacin was demonstrated to inhibit the biosyntheses of DNA, RNA, and proteins in the HeLa S3 cell line (Witkowski et al., 1984) and to block two-stage carcinogenesis in skin (Konoshima et al., 1994). Cucurbitacin E was also demonstrated to disrupt the cytoskeleton in prostate carcinoma cells (Duncan et al., 1996). Recently, it was shown that cucurbitacin I effectively blocked the growth of several tumor cells by disrupting STAT3 signaling pathways (Blaskovich et al., 2003). Despite all these results on cancer cells, cucurbitacins have not been developed as anticancer drugs because many cucurbitacins are toxic not only to cancer cells but also to normal cells (Konopa et al., 1974a,b). However, the cucurbitacin used in the present study, DHCD, did not show apparent toxicities to normal cells (Sohn et al., 2000; this study). Thus, DHCD might be an excellent lead candidate for developing anticancer agents.

On the other hand, because cucurbitacin B was identified as an effective substance in a fruit juice used as an herbal medicine for allergic rhinitis (Yesilada et al., 1988), some researchers have studied anti-inflammatory activities of cucurbitacins. Later, it was suggested that anti-inflammatory actions of cucurbitacins were attributable to inhibition of prostaglandin E2 biosynthesis (Peters et al., 1999). The present study revealed that DHCD inhibited NO generation by peritoneal macrophages. Excessive generation of NO by improper regulation of its production could contribute to inflammation (Nathan, 1992). Thus, the inhibitory effect of DHCD on NO generation could help explain the anti-inflammatory action of cucurbitacins.

DHCD blocked the activation of NF-B in the mouse peritoneal macrophages. Activation of NF-B has been demonstrated as a major control process in the induction iNOS. Thus, the present study showed that the inhibition of iNOS induction by DHCD was caused by blocking of NF-B activation. Furthermore, activation of NF-B is also known to regulate the expression of various cytokines related to inflammation, such as tumor necrosis factor, interleukin-1, and interleukin-2 (Lewis and Manning, 1999; McKay and Cidlowski, 1999). In addition to the in vitro studies, we demonstrated the inhibitory effect of DHCD on LPS-induced NO generation in experimental animals. Therefore, the present study strongly suggests that DHCD is a potent inhibitor for the activation of NF-B and NO production; thus, DHCD could be a useful substance for developing anti-inflammatory drugs.

	Acknowledgements

 
We acknowledge Dr. Carl Nathan for critical review of the manuscript and Dr. Dae-Myung Jue for helpful discussion.

	Footnotes

 
This research was supported by a special grant from Chung-Ang University.

DOI: 10.1124/jpet.103.063693.

ABBREVIATIONS. NO, nitric oxide; NOS, nitric-oxide synthase; iNOS, inducible nitric-oxide synthase; cNOS, constitutive nitric-oxide synthase; NF-B, nuclear factor-B; DHCD, 23,24-dihydrocucurbitacin D; IFN, interferon-; EMSA, electrophoretic mobility shift assay; RT, reverse transcription; PCR, polymerase chain reaction; LPS, lipopolysaccharide; G3PDH, glyceraldehyde-3-phosphage dehydrogenase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium.

Address correspondence to: Dr. Nyoun Soo Kwon, Department of Biochemistry, College of Medicine, Chung-Ang University, 221 Heuksukdong, Dongjakgu, Seoul 156-756, Korea. E-mail: nskwon{at}cau.ac.kr

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