Capsaicin exhibits anti-inflammatory property by inhibiting IkB-a degradation in LPS-stimulated peritoneal macrophages Chu-Sook Kim a , Teruo Kawada b , Byung-Sam Kim c , In-Seob Han c , Suck-Young Choe a , Tadao Kurata d , Rina Yu a, * a Department of Food Science and Nutrition, University of Ulsan, Mugeo-dong, Nam-ku, Ulsan 680-749, South Korea b Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan c Department of Biological Science, University of Ulsan, Ulsan 680-749, South Korea d Institute of Human Environmental Life Science, Ochanomizu University, Tokyo 112, Japan Received 27 April 2002; accepted 26 August 2002 Abstract Capsaicin, a major ingredient of hot pepper, is considered to exhibit an anti-inflammatory property. In order to clarify the signalling mechanism underlying the anti-inflammatory action of capsaicin, we investigated the effect of capsaicin on the production of inflammatory molecules in lipopolysaccharide (LPS)-stimulated murine peritoneal macrophages. The level of PGE2 was measured by EIA. The expression levels of COX-2, iNOS, IkB-a, and vanilloid receptor-1 (VR-1) were determined at the protein and mRNA levels. Significant inhibition of the production of LPS-induced PGE2 by capsaicin was observed in a dose-dependent manner. Capsaicin did not affect the COX-2 expression at either the protein or mRNA level, but inhibited the enzyme activity of COX-2 and the expression of the iNOS protein. Capsaicin completely blocked LPS-induced disappearance of IkB-a and therefore inactivated NF-kB. The inhibitory action of capsaicin on PGE2 production was not abolished by capsazepine, a specific antagonist to VR-1. A high expression level of the VR-1 like protein (VRL-1) was observed in peritoneal macrophages, while the expression of VR-1 was not detected. These findings suggest that the anti-inflammatory action of capsaicin may occur through a novel mechanism, not by a VR-1 receptor-mediated one. Both capsaicin and capsazepine may be a promising drug candidates for ameliorating inflammatory diseases and cancer. D 2002 Elsevier Science Inc. All rights reserved. Keywords: Capsaicin; Capsazepine; Macrophage; PGE2; COX-2; iNOS; IkB-a; Inflammation 1. Introduction Prostaglandins (PGs), cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS) are well-known proinflamamtory key mediators in pathogenesis of inflam- matory diseases. The expression of the inducible COX-2, the key enzyme that catalyzes PGs biosynthesis, and iNOS was upregulated in acute/chronic inflammatory diseases, transformed cells, and malignant tissues of lung or color- ectal cancer [1 –5]. One of the most commonly used non- steroidal anti-inflammatory drugs (NSAIDs), aspirin (ace- tylsalicylic acid), exhibits an anti-inflammatory property by inhibiting the COX activity. Significant reduction in the risk of certain cancers was reported when NSAIDs were treated in vitro, in vivo, and in epidemiologic studies [6,7]. The COX activity and subsequent production of PGE2 were closely related to the generation of NO radicals [8,9]. Recent reports regarding the inhibition of the iNOS expression/ function in murine macrophages indicated another possible mechanism of action for an aspirin-like drug [10] and various natural flavonoids [11,12]. Capsaicin, a major ingredient of hot pepper, is known to exhibit an anti-inflammatory property. Capsaicin inhibited the development of carrageenan-induced paw inflammation and adjuvant-induced arthritis in rats, as well as ethanol- induced inflammation [13 – 16]. It was suggested that the release of proinflammatory mediators, eicosanoids and 0898-6568/02/$ - see front matter D 2002 Elsevier Science Inc. All rights reserved. PII: S 0 8 9 8 - 6 5 6 8 ( 0 2 ) 0 0 0 8 6 - 4 Abbreviations: COX-2, cyclooxygenase-2; PGE2, prostaglandin E2; LPS, lipopolysaccaride; iNOS, inducible nitric oxide synthase; NF-kB, nuclear transcription factor. * Corresponding author. Tel.: +82-52-259-2372; fax: +82-52-259- 2888. E-mail address: rinayu@mail.ulsan.ac.kr (R. Yu). www.elsevier.com/locate/cellsig Cellular Signalling 15 (2003) 299 – 306 hydrolytic enzymes, is associated with the anti-inflamma- tory property of capsaicin [13,17]. It is well known that capsaicin-induced intracellular signalling in neuronal cells occurs via vanilloid receptors (VRs) [31]. However, the mechanism of the anti-inflammatory action of capsaicin in non-neuronal cells is not yet fully understood. Recent in vitro studies demonstrated that capsaicin induces apoptosis in certain human cancer cell lines [18 –20]. Inappropriate upregulation of COX-2 played a pivotal role in apoptotic cell death and cancer progression [21]. Considering that most NSAIDs, including aspirin, inhibit the synthesis and release of PGE2 by suppressing COX-2 expression at the mRNA/protein levels or the enzyme activity [22], capsaicin may also exhibit an anti-inflammatory activity by modulat- ing the expression/catalytic activity of proinflammatory mediators such as PGE2, COX-2, or iNOS. The present study demonstrates that capsaicin exhibits an anti-inflam- matory property by the inhibition of PGE2 and NO pro- duction in peritoneal macrophages via a novel mechanism, not via a VR-1 receptor-mediated one. 2. Materials and methods 2.1. Preparation of peritoneal macrophages BALB/c mice were given an i.p. injection with 2 ml of 3% thioglycollate 4 days prior to sacrifice. Peritoneal macrophages were collected by lavaging the peritoneal cavity with 7 ml of HBSS. The cells were collected by centrifugation, washed, and then suspended in EMEM medium. The cells were purified by adherence to tissue culture plates for 2 h. Peritoneal macrophages at 2 10 6 cells/ml were treated with 25 ng/ml LPS, 10– 50 A M of Capsaicin (CAP), or 10 – 20 A M of Capsazepine (CZE), for 24 h in serum-free medium. 2.2. Measurement of PGE2 release by peritoneal macro- phages Macrophages were seeded in 96-well plates (2 10 5 cells/well) and stimulated with LPS (25 ng/ml) with or without capsaicin or capsazepine for 24 h. The level of PGE 2 production from endogenous arachidonic acid was measured in cell culture supernatants of peritoneal macro- phages by EIA (Assay Designs, Ann Arbor, MI, USA). 2.3. Cell viability An MTT (3-[4,5-2-yl]-2,5-diphenyl tetrazolium bromide) assay was used to measure the viability of macrophages [23]. MTT is a pale yellow substrate that produces a dark blue formazan product when incubated with living cells. An MTT ring is cleaved in active mitochondria, and the process occurs only in living cells. After the supernatants were removed for PGE2 determination, cells were incubated at 37 j C with MTT (0.05 mg/ml) for 4 h and the absorbance was then measured at 570 nm using an ELISA reader. 2.4. Measurement of COX-2 enzyme activity Cells were seeded as described above. Measurement of COX-2 enzyme activity was performed as previously described [24]. Briefly, before activation, cells were treated with 30 A M resveratrol for 30 min to inactivate irreversibly COX-1. Thereafter, cells were washed with PBS and incu- bated in fresh medium. Induction of COX-2 was achieved by adding LPS and further incubation for 16 h. Then, the medium was aspirated and cells were washed with PBS again and supplied with fresh medium (foetal bovine serum- free). Capsaicin or capsazepine was preincubated for 30 min before exogenous arachidonic acid (10 A M) was added. After 15 min, supernatants were removed and the PGE 2 level was measured by EIA. 2.5. Nitrite determination The amount of nitrite in cell-free culture supernatants was measured using Griess reagent [25]. Briefly, 100 A l of supernatant was mixed with an equal volume of Griess reagent [1:1 (v/v) of 0.1% N -1-naphthyl-ethylenediamine in distilled water and 1% sulfanilamide in 5% phosphoric acid] on a 96-well flat-bottom plate. The absorbance at 570 nm was measured after 10 min using a micro-ELISA reader. The amount of nitrite was calculated from the NaNO 2 standard curve. 2.6. Western blot analysis Macrophages were collected and the protein content was determined. Equal amounts of protein (15 A g/lane) were loaded and electrophoresed on a 10% SDS-polyacrylamide gel. After the fractionated protein was blotted onto a nitro- cellulose membrane, the membrane was incubated overnight in blocking buffer (5% nonfat dry milk, 10 mM Tris, pH 7.5, 100 mM NaCl, 0.1% Tween 20) and then treated with a mouse monoclonal COX-2 antibody (Transduction Lab., Lexington, USA) for 1 h. After washing, the membrane was incubated with a horseradish peroxidase-conjugated anti-mouse IgG antibody. To detect iNOS or IkB-a, the membrane was treated with a monoclonal iNOS antibody (Transduction Lab., Lexington, USA) or monoclonal IkB-a antibody (BD PharMingen). The immunoreactive protein was detected with a chemiluminescent system (ECL kit, Amersham). After exposure to an X-ray film, the band density was calculated from the optical density with an image analyser. 2.7. RT-PCR analysis Total RNA was extracted from 2 10 7 cells (peritoneal macrophage) using an RNeasy kit (Qiagen, USA). An C.-S. Kim et al. / Cellular Signalling 15 (2003) 299–306 300 aliquot of 0.5 A g of RNA was used for reverse transcription and was amplified using a polymerase chain reaction (PCR) technique in a single reaction, using the Access RT-PCR system according to the manufacturer’s instructions in a TaKaRa Thermal cycler (TaKaRa, Biomedicals). The following sets of primers were used in PCR ampli- fication: COX-2 (Gene Bank accession number M94967), forward 5 V -CAGTTTTTCAAGACAGATCATAAGCG-3 V , reverse 5 V -TGCTCCTGCTTGAGTATGTCG-3 V ; iNOS (M92649), forward 5 V -AAGCACATGCAGAATGAGTA- CCG-3 V , reverse 5 V -GTGGGACAGCTTCTGGTCGAT-3 V ; b-actin (X03672), forward 5 V -ATGAAGATCCTGA- CCGAGCGT-3 V , reverse 5 V -AACGCAGCTCAGTAACA- GTCCG-3 V ; VR-1 (AF029310), forward 5 V -GTGAG- ACCCCTAACCGTCATGA-3 V , reverse 5 V -GCCTTCC- ACAGGCCGATAGTA-3 V ; VRL-1 (BC005415), forward 5 V -CAAGTACCTCACTGACTCGGCATAC-3 V , reverse 5 V - TTCTCTACCA GCAGTTCACGCA-3 V . The conditions of PCR for the target genes were as follows: COX-2 gene: 30 cycles at 85 j C for 1 min, 42 j C for 1 min, and 72 j C for 1 min; iNOS: 40 cycle at 95 j C for 30 s, 58 j C for 30 s, and 72 j C for 1 min. The termination cycle included a pro- longed extension at 72 j C for 5 min; VRL-1: preincubation at 94 j C for 5 min, followed by 30 cycles at 94 j C for 0.5 min, 55 j C for 1 min, and 72 j C for 1 min; VR-1: one cycle at 94 j C for 5 min, 94 j C for 1 min, 50 j C for 1 min, 72 j C for 1 min, followed by 34 cycles t at 94 j C for 1 min, 55 j C for 1 min, and 72 j C for 1 min. For semiquantitative analysis, the linearity of amplifica- tion of COX-2, iNOS, VR-1, VRL-1, and b-actin cDNAs was established in preliminary experiments. Amplification products obtained by PCR were electrophoretically separa- ted on a 2% agarose gel. SYBR Green-stained bands corresponding to the target genes and b-actin were photo- graphed with a DS-34 Polaroid camera. The intensity of the bands was densitometrically measured with the NIH Image analyser. All COX-2, iNOS, VR-1, and VRL-1 signals were normalized to the mRNA levels of the housekeeping gene b- actin and expressed as a ratio. 2.8. Statistical analysis All experiments were repeated at least twice. Results are expressed as means F S.E.M. Statistical analysis was per- formed using ANOVA and Dunnett’s multiple comparison test. Differences were considered to be significant when P < 0.05. 3. Results 3.1. Effect of capsaicin on LPS-induced PGE2 release by peritoneal macrophages Capsaicin (10 – 50 A M) inhibited PGE2 release by LPS- stimulated peritoneal macrophages (25 ng/ml) in a dose- dependent manner (Fig. 1). Under the experimental con- ditions described above, cell viability was determined to be >95% by the MTT assay (data not shown). Since the effects of capsaicin are mediated by VR-1 [26], we examined whether capsazepine, a competitive antagonist to the VR-1 receptor, abolishes the inhibitory effect of capsaicin on PGE2 release by peritoneal macrophages. Interestingly enough, the inhibitory effect of capsaicin was not blocked by capsazepine, while capsazepine sub- stantially inhibited LPS-induced PGE2 release (Fig. 1). The inhibitory effect of capsazepine on PGE2 release was rather additive with capsaicin. 3.2. Effect of capsaicin on the levels of COX-2 expression at the protein and mRNA levels in peritoneal macrophages In order to clarify whether the inhibitory effect of capsaicin on PGE2 release could result from a decreased COX-2 protein level, we further examined the effect of capsaicin on LPS-induced COX-2 protein levels by Western blot analysis. Interestingly, capsaicin did not alter LPS- induced COX-2 protein levels in peritoneal macrophages, while a marked decrease in COX-2 protein expression level was induced by capsazepine (Fig. 2A). We also determined mRNA steady-state levels of COX-2 in peritoneal macrophages by RT-PCR. Capsaicin did not affect COX-2 mRNA expression (Fig. 2B), while capsaze- pine downregulated mRNA expression of COX-2 in a dose- dependent manner. Fig. 1. Effect of capsaicin on LPS-induced PGE2 release by murine peritoneal macrophages. Macrophages were stimulated with LPS and incubated for 24 h with capsaicin and/or capsazepine. The PGE2 level in the culture medium was measured by EIA. Values are means F S.E.M. Values are the mean F S.E.M. from four independent experiments. * P < 0.01, significantly different from LPS-stimulated control. C.-S. Kim et al. / Cellular Signalling 15 (2003) 299–306 301 3.3. Effect of capsaicin on COX-2 enzyme activity in LPS- stimulated peritoneal macrophages Since capsaicin did not reduce the expression level of the LPS-induced COX-2 protein or mRNA, we investi- gated whether the inhibitory effect of capsaicin results from a direct inhibition of COX-2 enzyme activity. COX-2 activity was measured after adding exogenous arachidonic acid to the cell culture [24]. Significant inhibition of the conversion of exogenous arachidonic acid to PGE2 was observed (Fig. 2C). 3.4. Effect of capsaicin on LPS-induced NO release and iNOS protein/mRNA expression in peritoneal macro- phages It has been reported that PGE2 production by COX-2 increased NO production [27]. We investigated whether capsaicin inhibits the release of PGE2 by inhibiting iNOS expression and/or NO release. Capsaicin markedly down- regulated NO release (Fig. 3A), and significant reduction in iNOS expression level was also induced by capsazepine (Fig. 3B). When mRNA steady-state levels of iNOS in Fig. 2. Effect of capsaicin on COX-2 expression level and catalytic activity in peritoneal macrophages. Macrophages were stimulated with LPS and incubated for 24 h with capsaicin and/or capsazepine. (A) COX-2 protein expression level was analysed by Western blot analysis. (B) The COX-2 mRNA expression level was determined by RT-PCR. (C) COX-2 enzyme activity was measured after adding exogenous arachidonic acid to the cell culture medium as described in Materials and methods. Values are means F S.E.M. from four independent experiments. # P < 0.05, * P < 0.01, significantly different from LPS stimulated control. C.-S. Kim et al. / Cellular Signalling 15 (2003) 299–306 302 peritoneal macrophages were measured by RT-PCR, cap- saicin and capsazepine markedly downregulated iNOS expression at the mRNA level in a dose-dependent manner (Fig. 4). Fig. 3. Effect of capsaicin on the levels of iNOS protein expression in peritoneal macrophages. Macrophages were stimulated with LPS (25 ng/ ml) and IFNr (5 ng/ml), and incubated for 24 h with capsaicin and/or capsazepine. The amount of nitrite accumulation level (A) was determined by Griess reaction in the culture medium, and iNOS protein expression level (B) was measured by Western blot analysis as described in Materials and methods. Values are means F S.E.M. from three independent experi- ments. * P < 0.01, significantly different from LPS-stimulated control. Fig. 4. Effect of capsaicin on levels of iNOS mRNA expression in peritoneal macrophages. Macrophages were stimulated with LPS (25 ng/ ml) and IFNr (5 ng/ml), and incubated for 24 h with capsaicin and/or capsazepine. Total RNA was extracted from peritoneal macrophages and the iNOS mRNA expression level was determined by RT-PCR analysis. Values are means F S.E.M. from three independent experiments. * P < 0.01, significantly different from LPS-stimulated control. Fig. 5. Effect of capsaicin on levels of IkB-a in peritoneal macrophages. Macrophages were stimulated with LPS and incubated for 24 h with capsaicin or/and capsazepine. Cell extract from peritoneal macrophages was assayed for IkB-a by Western blot analysis. The figure shows results of the representative Western blot analysis from three separate experiments. Values are means F S.E.M. from three independent experiments. * P < 0.01, significantly different from LPS-stimulated control. C.-S. Kim et al. / Cellular Signalling 15 (2003) 299–306 303 3.5. Effect of capsaicin on IkB-a degradation in peritoneal macrophages The iNOS or COX-2 gene is known to be regulated by transcription factors such as nuclear transcription factor kB (NF-kB) [6]. We investigated whether the inhibitory action of capsaicin or capsazepine on iNOS expression can be induced by the inactivation of NF-kB. LPS resulted in complete degradation of IkB-a in peritoneal macrophages. Capsaicin or capsazepine completely blocked LPS-induced disappearance of IkB-a (Fig. 5), indicating that the sub- sequent NF-kB inactivation was induced by both com- pounds. 3.6. Expression of vanilloid receptor-like protein 1, but not VR-1 in peritoneal macrophages Since capsazepine could not abolish the inhibitory action of capsaicin on PGE2 release, we investigated whether VR- 1 mRNA is expressed in peritoneal macrophages by RT- PCR analysis. The specific expression of RT-PCR products of VR-1 in peritoneal macrophages was not detected although the products of VR-1 in rat and mouse brain (positive controls) were clearly expressed (Fig. 6A). Fur- thermore, it is interesting to note that VRL-1 (nonsensitive to capsaicin) mRNA was highly expressed in peritoneal macrophages (Fig. 6B). 4. Discussion The present study demonstrates that capsaicin suppresses PGE2 production by inhibiting the COX-2 enzyme activity and iNOS expression in a dose-dependent manner in LPS- stimulated murine peritoneal macrophages. Capsaicin has been shown to exhibit an analgesic –anti- inflammatory property [13 –15]. NSAIDs such as aspirin inhibited proinflammatory PGE2 biosynthesis as well as NO release [6,7]. Curcumin, a yellow pigment with a structure similar to that of capsaicin, inhibited phorbol ester-mediated induction of COX-2 as well as LPS-stimu- lated iNOS expression [6]. The biological action of these drugs was considered to be chemopreventive and anti- inflammatory. Based on our data, capsaicin markedly inhibits PGE2 release by inhibiting the catalytic activity of the COX-2 enzyme. The inhibitory action of capsaicin on PGE2 release appears to mimic the action of aspirin- like drugs, by inhibiting the COX-2 catalytic activity. It has been shown that the COX activity and subsequent produc- tion of proinflammatory PGE2 are regulated by NO [8,9]. Joe et al. [13,17] have reported that capsaicin modulates the release of inflammatory mediators secreted by macro- phages as well as the generation of oxygen free radicals [28]. Our data, showing that capsaicin reduced iNOS protein expression level and inhibited the subsequent NO release from LPS-stimulated peritoneal macrophages, sug- gest that the anti-inflammatory action of capsaicin may be associated with the reduced iNOS expression level. Tran- scription factors such as NF-kB are known to regulate the expression of iNOS as well as COX-2 [6]. Kopp and Ghosh [29] reported that aspirin suppressed NF-kB activa- tion through stabilization of IkB. Considering that capsai- cin also inhibited LPS-induced IkB-a degradation in macrophages, capsaicin might antagonize the transcrip- tional activity of NF-kB, and thereby induced the down- regulation of iNOS. Capsaicin receptors in primary sensory neurons, which are known as vanilloid receptors (VRs), play a fundamental role in the signal transduction of peripheral tissue injury and/or inflammation responses [30]. Capsaicin-induced intracellular signalling [31] or heat-evoked currents [32] via VRs were completely blocked by capsazepine, one of the most competitive VR antagonist against the action of capsaicin [33], in sensory neurons. Interestingly enough, however, our data showed that the inhibitory action of capsaicin on PGE2 release was not blocked by capsaze- pine, while capsazepine exerted rather agonistic on the capsaicin action. Capsazepine, similar to capsaicin, sup- Fig. 6. Steady state levels of VR-1 or VRL-1 mRNA expression in peritoneal macrophages. The VR-1 (A) or VRL-1 (B) mRNA expression level was determined by RT-PCR. Total RNA was extracted from peritoneal macrophages and used for RT-PCR analysis as described in Materials and methods. R, rat; M, mouse. Results are representative of three separate experiments. C.-S. Kim et al. / Cellular Signalling 15 (2003) 299–306 304 pressed COX-2 and iNOS expression at the mRNA/protein level, and the NF-kB signalling pathway, suggesting that capsaicin and capsazepine may share a common antago- nistic/agonistic role in intracellular signalling cascades. Recently, a capsazepine-insensitive VR has also been reported [34]. A VR 5 V -splice variant (VR.5 V sv), which differs from VR-1, is insensitive to capsaicin and heat [35]. The VR.5 V sv and other postulated VR subtypes are expressed differentially in non-neuronal peripheral blood mononuclear cells [35]. Considering that the peritoneal macrophages expressed the VR-1 receptor rarely, but ex- pressed VR-like protein 1 highly, the anti-inflammatory action of capsaicin in murine macrophages may occur in a VR-1 independent manner. The proposed signalling path- way for the anti-inflammatory action of capsaicin or capsazepine is shown in Fig. 7. Further study is necessary to elucidate the alternative molecular mechanism of the action of capsaicin. In conclusion, capsaicin showed a significant inhibitory activity on the production of LPS-induced PGE2 by peri- toneal macrophages. The anti-inflammatory activity of cap- saicin was directly related to the inhibition of the COX-2 enzyme activity and iNOS expression. Capsazepine, a VR-1 antagonist, did not abolish the capsaicin action, but rather inhibited the COX-2 and iNOS expressions. The inhibitory action of both compounds was associated with NF-kB inactivation through the stabilization of the IkB-a protein. Murine peritoneal macrophages did not express VR-1, but highly expressed VRL-1. Our findings suggest that the anti- inflammatory action of capsaicin might be induced by a novel mechanism, not by the VR-1 receptor-mediated one. Both compounds may be useful for ameliorating inflamma- tory diseases, accompanied by PGE2 overproduction, and for developing a chemopreventive agent. 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