Original Papers
655
Authors
Ji-Sun Shin 1, 5, 6, Suran Ryu 1, 2, Young-Wuk Cho 2, 5, 6, Hyun Ji Kim 3, Dae Sik Jang 4, Kyung-Tae Lee 1, 4
Affiliations
The affiliations are listed at the end of the article
Key words " Schisandrae Fructus l " Schisandra chinensis l " Schisandraceae l " sesquiterpene l " nitric oxide l " prostaglandin E l 2 " mPGES‑1 l
Abstract
Abbreviations
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Much is known about the bioactive properties of lignans from the fruits of Schisandra chinensis. However, very little work has been done to determine the properties of sesquiterpenes in the fruits of S. chinensis. The aim of the present study was to investigate the anti-inflammatory potential of new sesquiterpenes (β-chamigrenal, β-chamigrenic acid, α-ylangenol, and α-ylangenyl acetate) isolated from the fruits of S. chinensis and to explore their effect on macrophages stimulated with lipopolysaccharide. Of these four sesquiterpenes, β-chamigrenal most significantly suppressed lipopolysaccharide-induced nitric oxide and prostaglandin E2 production in RAW 264.7 macrophages (47.21 ± 4.54 % and 51.61 ± 3.95 % at 50 µM, respectively). Molecularly, the inhibitory activity of β-chamigrenal on nitric oxide production was mediated by suppressing inducible nitric oxide synthase activity but not its expression. In the prostaglandin E2 synthesis pathway, β-chamigrenal prevented the upregulation of inducible microsomal prostaglandin E synthase-1 expression after stimulation with lipopolysaccharide. Conversely, β-chamigrenal had no effect on the expression and enzyme activity of cyclooxygenase-2. In addition, the expression of early growth response factor-1, a key transcription factor of microsomal prostaglandin E synthase-1 expression, was inhibited by β-chamigrenal. These results may suggest a possible anti-inflammatory activity of β-chamigrenal which has to be proven in in vivo experiments.
AA: COX: cPGES: dNTP: DTT: Egr: EIA: eNOS: IL: iNOS: L-NIL: LPS: MAPEG:
received revised accepted
Dec. 2, 2013 April 9, 2014 May 5, 2014
Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1368544 Published online May 28, 2014 Planta Med 2014; 80: 655–661 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0032‑0943 Correspondence Dae Sik Jang, PhD Department of Life and Nanopharmaceutical Science College of Pharmacy Kyung Hee University Dongdaemun-Ku, Hoegi-Dong 130–701 Seoul Republic of Korea Phone: + 82 29 61 07 19 Fax: + 82 29 66 38 85 [email protected] Correspondence Kyung-Tae Lee, PhD Department of Pharmaceutical Biochemistry College of Pharmacy Kyung Hee University Dongdaemun-Ku, Hoegi-Dong 130–701 Seoul Republic of Korea Phone: + 82 29 66 38 85 Fax: + 82 29 62 08 60 [email protected]
arachidonic acid cyclooxygenase cytosolic prostaglandin E synthase deoxyribonucleotide triphosphate dithiothreitol early growth response factor enzyme immunoassay endothelial nitric oxide synthase interleukin inducible nitric oxide synthase L-N6-(1-iminoethyl)lysine lipopolysaccharide membrane-associated proteins involved in the eicosanoid and glutathion metabolism mPGES: microsomal prostaglandin E synthase MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide NO: nitric oxide NOS: nitric oxide synthase nNOS: neuronal nitric oxide synthase NSAID: nonsteroidal anti-inflammatory drug PG: prostaglandin prostaglandin E2 PGE2: PGES: prostaglandin E synthase PGHS: prostaglandin H endoperoxide synthase PMSF: phenylmethylsulfonylfluoride qRT‑PCR: quantitative real-time reversetranscription polymerase chain reaction TNF: tumor necrosis factor Supporting information available online at http://www.thieme-connect.de/products
Shin J-S et al. Inhibitory Effects of …
Planta Med 2014; 80: 655–661
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Inhibitory Effects of β-Chamigrenal, Isolated from the Fruits of Schisandra chinensis, on LipopolysaccharideInduced Nitric Oxide and Prostaglandin E2 Production in RAW 264.7 Macrophages
Original Papers
Introduction !
Although the inflammatory response is a defense mechanism against infection or injury, sustained inflammation is a pathological condition. An inflammatory response is characterized by the abundant production of proinflammatory mediators such as NO, PGE2, reactive species, and cytokines. NO is produced by macrophages, and acts as a cytotoxic agent during immune and inflammatory responses during the oxidation of L-arginine to L-citrulline [1]. In mammalian cells, NO is synthesized by the three isoforms of NOS: nNOS, eNOS, and iNOS [2]. In particular, iNOS is expressed in response to interferon-γ, LPS, and various proinflammatory cytokines [3]. The NO produced by iNOS has been suggested to have beneficial antiviral and microbiocidal effects [4], but the overproduction of NO can be harmful to the host and, in fact, is involved in the pathogenesis of various inflammatory diseases [5]. Other important mediators overexpressed during inflammation are the PGs. PGE2 is an eicosanoid product synthesized from AA in two steps. The first step is catalyzed by PGHS, also called COX, which converts AA to PGH2. Two different COX enzymes have been reported: COX-1, a constitutive enzyme in many tissues and COX-2, which is induced by cytokines, growth factors, or bacterial products, such as LPS, in different pathologic processes involving inflammation such as infectious diseases, cancer, arthritis, and atherosclerosis [6, 7]. The second step is the conversion of PGH2 into PGE2 by a reaction of isomerization catalyzed by PGES enzymes. Three different PGES have been described: cPGES and two membrane-bound mPGES, mPGES-1 and -2 [8, 9]. Among them, the mPGES-1 enzyme is a membrane-associated perinuclear protein belonging to the MAPEG family. This enzyme, which seems to be preferentially functionally coupled with COX2, is markedly induced by inflammatory stimuli and downregulated by anti-inflammatory drugs such as glucocorticoids [10]. Egr-1 is a nuclear transcription factor and belongs to a group of early response genes that are induced rapidly and transiently by different stimuli such as LPS, cytokines, growth factors, and hypoxia [11]. This factor has been implicated in cell growth and differentiation, and in the development of chronic inflammatory diseases such as atherosclerosis and arthritis [11, 12]. Recent studies demonstrated that Egr-1 is a key transcription factor in regulating the inducible expression of mPGES-1 through the binding to the DNA sequence GCG(G/T)GGCG on the mPGES-1 promoter [13, 14]. The fruits of Schisandra chinensis Baillon (Schisandraceae), also known as Schisandrae Fructus, have traditionally been used in Korea, Japan, and China for the treatment of coughs, spontaneous sweating, dysentery, and insomnia [15]. A previous phytochemical investigation on the fruits of S. chinensis has resulted in the isolation of lignans [16, 17] and nortriterpenoids [18]. Diverse pharmacological effects such as antihepatotoxic, anti-inflammatory, antioxidant and antitumoral activities of the fruits of S. chinensis and dibenzo[a,c]cyclooctene lignans have been reported in numerous studies [19, 20]. However, there are few reports on other pharmacologically active compounds from the fruits of S. chinensis. Therefore, as a part of our ongoing screening program to evaluate the anti-inflammatory potentials of natural compounds, we isolated four sesquiterpenes from the fruits of S. chinensis and investigated their effects on the LPS-induced production of NO and PGE2 in macrophages. In this study, we investigated the underlying molecular mechanisms of β-chamigrenal
Shin J-S et al. Inhibitory Effects of …
Planta Med 2014; 80: 655–661
Fig. 1 Inhibitory effects of sesquiterpene compounds isolated from the fruits of S. chinensis on lypopolysaccharide-induced production of nitric oxide and prostaglandin E2. A Chemical structures of β-chamigrenal, β‑chamigrenic acid, α-ylangenol, and α-ylangenyl acetate isolated from the fruits of S. chinensis. B Following pretreatment with sesquiterpenes (50 µM) for 1 h, cells were treated with LPS (1 µg/mL) for 24 h. Levels of NO and PGE2 in culture media were quantified using the Griess reaction assay and EIAs, respectively. Controls were not treated with LPS and sesquiterpenes. L-NIL (10 µM) and NS-398 (3 µM) were used as positive controls for NO and PGE2 production. Data are presented as the means ± SD of three independent experiments. # P < 0.05 vs. the control group; *** p < 0.001 vs. LPS-stimulated cells.
showing the most potent inhibitory activities on the LPS-induced production of NO and PGE2.
Results and Discussion !
The pathology of inflammation is initiated by complex processes triggered by microbial pathogens or their antigens [21]. The prototypic endotoxin LPS can potently activate macrophages and induce various proinflammatory mediators [22]. In light of this, reducing activation signals in activated macrophages has been suggested as a therapeutic strategy in various inflammatory diseases [23]. In the present study, repeated column chromatography of the hexane extract of the fruits of S. chinensis resulted in the isolation of four sesquiterpenes, specifically β-chamigrenal, β-cha" Fig. 1 A). migrenic acid, α-ylangenol, and α-ylangenyl acetate (l Initially, the isolated four sesquiterpenes were screened for their inhibitory effects on the LPS-induced production of NO and PGE2 " Fig. 1 B). β-chamigrenal (51.61 ± in RAW 264.7 macrophages (l 3.95 % at 50 µM) and β-chamigrenic acid (17.87 ± 2.55 % at 50 µM) more potently inhibited LPS-induced PGE2 production in comparison with α-ylangenol and α-ylangenyl acetate. Moreover,
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Fig. 2 Effects of β-chamigrenal on lypopolysaccharide-induced nitric oxide production, inducible nitric oxide synthase expression, and inducible nitric oxide synthase enzyme activity in RAW 264.7 macrophages. A Following pretreatment with β-chamigrenal (15, 30, or 60 µM) for 1 h, cells were treated with LPS (1 µg/mL) for 24 h. Levels of NO in culture media were quantified using the Griess reaction assay. Controls were not treated with LPS and β-chamigrenal. B Lysates were prepared from the cells pretreated with/without β-chamigrenal (15, 30, or 60 µM) for 1 h and then with LPS (1 µg/mL) for 24 h. Total cellular proteins were resolved by SDS-PAGE, transferred to PVDF membranes, and detected with a specific iNOS antibody. β-actin was used as an internal control. C Total RNA was prepared for the qRT‑PCR analysis of iNOS from the cells stimulated with LPS (1 µg/mL), with/without β-chamigrenal (15, 30, or 60 µM) for 4 h. The mRNA levels of iNOS were determined using gene-specific primers, as described in Materials and Methods. D Following pretreatment with LPS (1 µg/mL) for 12 h and washing with PBS, cells were treated with β-chamigrenal (15, 30, or 60 µM) for 12 h. Levels of NO in culture media were quantified using the Griess reaction assay. Data are presented as the means ± SD of three independent experiments. # P < 0.05 vs. the control group; *** p < 0.001 vs. the LPS-stimulated group.
acetated compounds β-chamigrenic acid (23.78 ± 1.03 % at 50 µM) and α-ylangenyl acetate showed much lower inhibitory effects than β-chamigrenal (47.21 ± 4.54 % at 50 µM) and α-ylangenol (47.53 ± 2.12 % at 50 µM) on LPS-induced NO production. Among these compounds, β-chamigrenal reduced the production of NO and PGE2 inflammatory mediators to the greatest extent in LPSstimulated RAW 264.7 macrophages. In addition, these inhibitory effects of these four sesquiterpenes were not caused by nonspecific cytotoxicity, because these compounds had no effect on cell viability as determined by the MTT assay at 50 µM (Fig. 1S, Supporting Information). Therefore, we examined the underlying molecular mechanisms for the inhibitory activities of β-chamigrenal on the LPS-induced NO and PGE2 production in macro" Fig. 2 A, β-chamigrenal inhibited LPS-inphages. As shown in l duced NO production, dose-dependently, in RAW 264.7 macrophages (from 13.62 ± 0.23 to 12.79 ± 0.21, 10.76 ± 0.23, and 7.19 ± 0.61 µM at 15, 30, and 60 µM, respectively). L-NIL (20 µM) was used as a positive control for inhibition of NO. The inhibitory effects on NO production are principally signaled through multiple steps: scavenging of NO, deprivation of arginine, suppression of iNOS enzyme expression, and downregulation of iNOS enzyme activity. Imidazolineoxyl-N-oxide, lactacystin, and imidazole are well-known representatives of each step [24–26]. We first determined the effects of β-chamigrenal on the protein and mRNA expression of iNOS using Western blotting and qRT‑PCR, respec" Fig. 2 B and C, β-chamigrenal up to 60 µM tively. As shown in l did not inhibit LPS-induced iNOS protein and mRNA expression. Because no transcriptional events were involved in the inhibitory effects of β-chamigrenal, we identified the possibility that the reduction in NO accumulation caused by β-chamigrenal could be due to the modulation of iNOS enzyme activity. It was found that LPS-induced iNOS activity was inhibited by β-chamigrenal " Fig. 2 D, from 100.00 ± 3.40 to 68.27 ± 2.13, 52.51 ± 2.25, and (l
48.91 ± 3.52 % at 15, 30, and 60 µM, respectively). A few studies have described a possible regulation of iNOS enzyme activity by the post-translational processing of the protein, including active dimer formation and localization, phosphorylation [27], and by regulation of arginine availability [28]. Unfortunately, we did not establish the inhibitory mechanisms of iNOS enzyme activity by β-chamigrenal yet. However, our results demonstrate that the inhibitory effect of β-chamigrenal on LPS-induced NO production may occur through the inhibition of iNOS enzyme activity rather than through iNOS expression. PGE2, the best known of the prostaglandins, is involved in various inflammatory disorders, including arthritis, rheumatism, cardiovascular thrombosis, and Alzheimerʼs disease [29]. PGE2 is synthesized from arachidonic acid by COX-2 and mPGES-1 in response to inflammatory stimuli [30]. To assess the effects of βchamigrenal on LPS-induced PGE2 production in RAW 264.7 macrophage cells, the amount of PGE2 was measured via EIA. Stimulation of cells with LPS (1 µg/mL) resulted in a significant increase in PGE2 production compared with that in unstimulated " Fig. 3 A). Furthermore, pretreatment with β-chacontrol cells (l migrenal inhibited LPS-induced PGE2 production (from 31.65 ± 4.39 to 28.43 ± 2.42, 23.21 ± 1.55, and 16.71 ± 0.59 ng/mL at 15, 30, and 60 µM, respectively). As a control for the inhibition of PGE2 production, we used NS-398 (3 µM), a positive control for the COX-2 inhibitor. We also determined the effects of β-chamigrenal on the protein and mRNA levels of COX-2, which is an inducible enzyme responsible for converting PGH2 from AA, an intermediate step in a process of producing PGE2 from AA. As " Fig. 3 B and C, COX-2 was markedly upregulated by shown in l LPS at the protein and mRNA levels, whereas β-chamigrenal had no inhibitory effect on these upregulations in RAW 264.7 macrophages. These findings show that β-chamigrenal did not downregulate LPS-induced COX-2 expression. Furthermore, we deter-
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Original Papers
Original Papers
Fig. 3 Effects of β-chamigrenal on lypopolysaccharide-induced prostaglandin E2 production, and cyclooxygenase-2 and microsomal prostaglandin E synthase-1 expression in RAW 264.7 macrophages. A Following pretreatment with β-chamigrenal (15, 30, or 60 µM) for 1 h, cells were treated with LPS (1 µg/mL) for 24 h. The levels of PGE2 in culture media were quantified using EIA. Controls were not treated with LPS and β-chamigrenal. B Lysates were prepared from the pretreated cells with/without βchamigrenal (15, 30, or 60 µM) for 1 h and then with LPS (1 µg/mL) for 24 h. Total cellular proteins were resolved by SDS-PAGE, transferred to PVDF membranes, and detected with a specific COX-2 or mPGES-1 antibody. β-actin was used as an internal control. C, D Total RNA was prepared for the qRT‑PCR analysis of COX-2 and mPGES-1 from the cells stimulated with LPS (1 µg/mL), with/without βchamigrenal (15, 30, or 60 µM) for 4 h. The mRNA levels of COX-2 and mPGES-1 were determined using gene-specific primers, as described in Materials and Methods.
Fig. 4 Inhibitory effects of β-chamigrenal on lipopolysaccharide-induced early growth response factor-1 expression in RAW 264.7 macrophages. A Lysates were prepared from the cells pretreated with β-chamigrenal (50 µM) for 1 h prior to pretreatment with LPS (1 µg/mL) for 1 and 2 h. B Lysates were prepared from the cells stimulated with LPS (1 µg/ mL), with/without β-chamigrenal (15, 30, or 60 µM) for 2 h. Total cellular proteins were resolved by SDSPAGE, transferred to PVDF membranes, and detected with a specific Egr-1 antibody. β-actin was used as an internal control.
mined the COX-2 enzyme activity using Dup697 as the respective positive control and found that β-chamigrenal had no effect on the COX-2 enzyme activities (Fig. 2S, Supporting Information). To study the effects of β-chamigrenal on the expression of mPGES-1, which is an inducible enzyme responsible for producing PGE2 from PGH2, cells were pretreated with LPS (1 µg/mL) alone or in combination with β-chamigrenal, and protein and mRNA were extracted thereafter. mPGES-1 protein and mRNA were measured by Western blotting and qRT‑PCR, respectively. We found that LPS-induced mPGES-1 protein and mRNA levels were significantly inhibited by β-chamigrenal in a dose-depen" Fig. 3 B and D). Previous data indicate that NFdent manner (l κB is a major regulatory component of the inflammatory responses mediated by LPS or proinflammatory cytokines [31]. We therefore measured the effects of β-chamigrenal on the transcriptional activity of NF-κB and found that β-chamigrenal did not reduce the NF-κB-dependent luciferase activity induced by LPS (data not shown). In addition to NF-κB, LPS stimulation activates many transcription factors, including the zinc finger transcription factor Egr-1 [32]. Egr-1 has been described as a strong regulator of mPGES-1 transcription after stimulation of different cells with IL-1β or TNFα [33]. Therefore, we investigated whether the inhibition of β-chamigrenal expression is related to reductions in the expression of Egr-1. Western blot analysis showed
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Planta Med 2014; 80: 655–661
that LPS activation of macrophages results in an increase in Egr1 protein and that β-chamigrenal suppressed LPS-induced Egr-1 " Fig. 4 A). We further found that LPS-induced expression at 2 h (l Egr-1 protein levels were inhibited by β-chamigrenal in RAW " Fig. 4 B). In inflammation, mPGES-1 ex264.7 macrophages (l pression was detected to be upregulated in patients with rheumatoid arthritis and osteoarthritis [34]. This suggests a novel therapeutic target for the treatment of inflammatory diseases with improved selectivity against pathological PGE2 and consequential safety when compared to the traditional NSAIDs or selective COX-2 inhibitors [35]. Recently, inhibition of mPGES-1 by epigallocatechin-3-gallate from green tea was suggested to be the predominant mechanism leading to diminished PGE2 production via the modulation of Egr-1 in A549 human pulmonary epithelial cells [33]. In the present study, we found that β-chamigrenal significantly downregulates the mRNA and protein expression of the mPGES-1 enzyme probably via Erg-1 suppression, but has little effect on COX-2 expression in LPS-stimulated macrophages. This suggests mPGES-1 as the primary target of β-chamigrenal, explaining its inhibitory effect on the production of PGE2. β-chamigrenal belongs to the chamigrene subclass of sesquiterpenes, which have been isolated from a variety of sources including terrestrial and marine plants, and have demonstrated a diverse array of biological activity [36]. β-chamigrenal was origi-
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data measurements and by comparison with published values [41–44].
Chemicals and antibodies DMEM, FBS, penicillin, and streptomycin were obtained from Life Technologies, Inc. iNOS, COX-2, mPGES-1, Erg-1, β-actin monoclonal antibodies, and peroxidase-conjugated secondary antibody were purchased from Santa Cruz Biotechnology, Inc. The ELISA kit for PGE2 was obtained from R&D Systems. RNA extraction kits were obtained from Intron Biotechnology. Random oligonucleotide primers and M–MLV reverse transcriptase were purchased from Promega. SYBR green ex Taq was obtained from TaKaRa. iNOS, COX-2, mPGES-1, and β-actin oligonucleotide primers were purchased from Bioneer. MTT, NS-398 (purity ≥ 98 %), PMSF, DTT, L-NIL (purity > 97 %), LPS (Escherichia coli, serotype 0111:B4), and all other chemicals were obtained from Sigma-Aldrich.
Materials and Methods
Cell culture and sample treatment
!
RAW 264.7 murine macrophages were obtained from the Korean Cell Line Bank. Cells were grown at 37 °C in DMEM medium supplemented with 10% FBS, penicillin (100 units/mL), and streptomycin sulfate (100 µg/mL) in a humidified 5 % CO2 atmosphere. Cells were pretreated with β-chamigrenal (15, 30, or 60 µM) for 1 h and then stimulated with LPS for an indicated time.
Plant material The dried fruits of S. chinensis were purchased from Kyung-dong market, Seoul, Republic of Korea, in June 2011 and were identified by Prof. Dae Sik Jang. A voucher specimen (No. 2011-SCCH01) has been deposited in the Laboratory of Natural Product Medicine, College of Pharmacy, Kyung Hee University.
MTT assay Extraction and isolation The milled plant material (3.5 kg) was extracted with 10 L of 80 % aqueous ethanol (EtOH) three times by maceration. The extracts were combined and concentrated in vacuo at 40 °C to give an 80 % EtOH extract (1.52 kg). The 80 % EtOH extract (1.51 kg) was suspended in distilled water (5 L) and then successively extracted with n-hexane (3 × 5 L), ethyl acetate (EtOAc) (3 × 5 L), and butanol (BuOH) (3 × 500 L) to give n-hexane- (125 g), EtOAc – (80 g), BuOH – (430 g), and water-soluble fractions (875 g), respectively. The n-hexane-soluble extract (120 g) was subjected to column chromatography on silica gel (8.6 × 21 cm, 70–230 mesh) eluted with n-hexane: EtOAc [1 : 0, 99 : 1, 97 : 3, 19 : 1, 4 : 1, 7 : 3, 1 : 1, 0 : 1, methanol (MeOH), 4 L each eluent] to afford 7 fractions (Fr.1–Fr.7). Fr.1 [eluted with n-hexane: EtOAc (9 : 1 v/v); 40 g] was chromatographed over silica gel (6 × 48 cm, 230–400 mesh) eluting with n-hexane : EtOAc (99 : 1, 49 : 1, 97 : 3, 19 : 1, MeOH, 4 L each eluent) to give 10 subfractions (Fr.1–1 – Fr.1–10). α-Ylangenyl acetate (1500–1700 mL, 460 mg) and β-chamigrenal (2900–3300 mL, 2.1 g) were obtained from Fr.1–2 [eluted with n-hexane : EtOAc (97 : 3 v/v); 10.8 g] by silica gel column chromatography (6 × 34 cm, 230–400 mesh) eluting with n-hexane : EtOAc (19 : 1 v/v, 5 L). Fr.1–5 [eluted with n-hexane : EtOAc (19 : 1 v/v); 9.3 g] was chromatographed over silica gel (6 × 38 cm, 230–400 mesh) as a stationary phase with n-hexane : EtOAc (9 : 1 v/v, 4.8 L) as the mobile phase to afford α-ylangenol (2900–3300 mL, 2.1 g). Fr.3 [eluted with n-hexane-EtOAc (9 : 1 v/v); 17.1 g] was subjected to column chromatography on silica gel (7 × 46 cm, 70–230 mesh) eluted with n-hexane: EtOAc [7 : 1 v/v (18 L), 4 : 1 v/v (7 L), 7 : 3 v/v (3 L)] to afford 7 fractions (Fr.3–1 – Fr.3–10). β-Chamigrenic acid (475–525 mL, 170 mg) was obtained from Fr.3–7 [eluted with n-hexane : EtOAc (7 : 1 v/ v); 1.46 g] by Sephadex LH-20 column (3.6 × 72 cm) eluting with a CH2Cl2-MeOH mixture (1 : 1 v/v). The purity of these compounds (> 95 %) was determined by HPLC and NMR. The structures of the isolates were identified by physical and spectroscopic
RAW 264.7 cells were plated at 1 × 104 cells/100 µL in 96-well plates and then incubated with various concentrations of the tested compounds for 24 h. After incubation, the cells were treated with an MTT solution for 4 h at 37 °C under 5 % CO2. One hundred microliters of dimethyl sulfoxide were added to extract the MTT formazan, and the absorbance of each well was read by an automatic microplate reader at 540 nm (Molecular Devices).
Nitrite assay RAW 264.7 cells were plated at 5 × 105 cells/well in 24-well plates and then incubated with or without LPS (1 mg/mL) in the absence or presence of various concentrations of the tested compounds for 24 h. Nitrite accumulation was measured in culture media using the Griess reaction assay and presumed to reflect NO levels [45].
Prostaglandin E2 assay RAW 264.7 cells were pretreated with the tested compounds for 1 h and then stimulated with LPS (1 µg/mL) for 24 h. Levels of PGE2 in the culture media were quantified using an EIA kit (R&D Systems).
Inducible nitric oxide synthase enzyme activity assay RAW 264.7 cells were plated at 5 × 105 cells/well in 24-well plates and then incubated with LPS (1 mg/mL) for 12 h. The cells were washed twice with PBS and then incubated in the absence and presence of β-chamigrenal for a further 12 h, with no LPS in the medium. The supernatants were collected and the Griess reaction was performed [45].
Cyclooxygenase enzyme activity assay The compound was evaluated for potency and selectivity of inhibition in vitro using the COX inhibitor screening assay (Cayman). Recombinant COX-2 (human) proteins were preincubated with βchamigrenal for 10 min at 37 °C. The reaction was started by the
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Planta Med 2014; 80: 655–661
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nally isolated from the oil of the fruits of S. chinensis [37]. Sesquiterpene aldehydes, such as polygodial, have been shown to inhibit cell infiltration in a number of in vivo models of acute inflammation [38, 39]. There are some reports that α-methyleneγ-lactone groups and/or conjugated aldehyde groups of sesquiterpenes contribute to their anti-inflammatory effect [40]. Therefore, the conjugated aldehyde group of β-chamigrenal seems to be an active site responsible for its possible anti-inflammatory potential. In summary, to the best of our knowledge, this is the first report to suggest that β-chamigrenal, a sesquiterpene isolated from S. chinensis, inhibited the LPS-induced production of NO and PGE2 in RAW 264.7 macrophages. Molecular data revealed that β-chamigrenal is involved in the inhibition of NO and PGE2 production via the reduction of iNOS enzyme activities and the attenuation of expression of mPGES-1 and Erg-1 transcription factor.
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addition of 100 µM arachidonic acid and allowed to proceed for 2 min. The reaction was terminated by the addition of HCl solution containing SnCl2. The COX activity assay directly measures PGF2α produced by the SnCl2 reduction of COX-derived PGH2. The prostanoid product is quantified via EIA. As a control inhibitor for COX-2, Dup-697 (10 µM, purity > 95%, Merk Milipore) was used.
Science, ICT and Future Planning through the National Research Foundation. The NMR experiments were performed by the Korea Basic Science Institute (KBSI).
Conflict of Interest !
The authors have no conflict of interest to declare.
Western blot analysis Cellular proteins were extracted from RAW 264.7 macrophages, as described previously [46], and protein concentrations were determined using Bio-Rad protein assay reagent, according to the manufacturerʼs instructions. Proteins were electroblotted onto PVDF membranes after being separated by 10% SDS-polyacrylamide gel electrophoresis. Membranes were incubated for 1 h in blocking solution (5% skim milk) with the primary antibody overnight at 4 °C, washed three times with Tween 20/Tris-buffer, incubated with a horseradish peroxidase-conjugated secondary antibody (1 : 2000) for 2 h at room temperature, and washed three times with Tween 20/Tris-buffer. Blots were then developed using an enhanced chemiluminescence (Amersham Life Science).
Quantitative real-time reverse-transcriptase polymerase chain reaction Total cellular RNA was isolated using Easy Blue kits (Intron Biotechnology). For each sample, 1 µg of RNA was reverse-transcribed using MuLV reverse transcriptase, 1 mM dNTP, and 0.5 µg/µL oligo (dT12–18). Real-time PCR was performed using a thermal cycler dice real-time PCR system (Takara). The primers used for SYBR Green real-time reverse transcription–PCR were as follows: for iNOS, sense primer, 5′-CATGCTACTGGAGGTGGGTG‑3′, anti-sense primer, 5′-CATTGATCTCCGTGACAGCCC‑3′; for COX-2, sense primer COX-2, 5′-GGAGAGACTATCAAGATAGT‑3′, anti-sense primer COX-2, 5′-ATGGTCAG- TAGACTTTTACA-3′; for mPGES-1, sense primer 5′-AGGATGCGCTGAAACGTGG- A-3′, anti-sense primer, 5′-CGAAGCCGAGGAAGAGGAAA-3′; and for βactin, sense primer, 5′-ATCACTATT-GGCAACGAGCG‑3′, antisense primer, 5′-TCAGCAAT-GCCTGGGTACAT‑3′.
Statistical analysis The data are expressed as the mean ± SD of at least three experiments performed using different cell preparations. Statistically significant values were compared using one-way ANOVA followed by Dunnettʼs post test, and p values less than 0.05 were considered statistically significant.
Supporting information Bar graphs showing the effects of sesquiterpenes isolated from the fruits of S. chinensis on cell viability in RAW 264.7 macrophages and the inhibitory effects of β-chamigrenal on COX-2 enzyme activity in RAW 264.7 macrophages are available as Supporting Information
Acknowledgements !
This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), which is funded by the Ministry of Education, Science and Technology (No. 2011–0023407) and by a grant from the Bio-Synergy Research Project (NRF-2013M3A9C4078145) of the Ministry of
Shin J-S et al. Inhibitory Effects of …
Planta Med 2014; 80: 655–661
Affiliations 1
2
3
4
5
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Department of Pharmaceutical Biochemistry, College of Pharmacy, Kyung Hee University, Seoul, Republic of Korea Department of Biomedical Science, College of Medical Science, Kyung Hee University, Seoul, Republic of Korea Department of Oriental Pharmaceutical Science, College of Pharmacy, Kyung Hee University, Seoul, Republic of Korea Department of Life and Nanopharmaceutical Science, College of Pharmacy, Kyung Hee University, Seoul, Republic of Korea Reactive Oxygen Species Medical Research Center, School of Medicine, Kyung Hee University, Seoul, Republic of Korea Department of Physiology, School of Medicine, Kyung Hee University, Seoul, Republic of Korea
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34 Clark P, Rowland SE, Denis D, Mathieu MC, Stocco R, Poirier H, Burch J, Han Y, Audoly L, Therien AG, Xu D. MF498 [N-{[4-(5,9-Diethoxy-6-oxo6,8-dihydro-7H-pyrrolo[3,4-g]quinolin-7-yl)-3-methylbe nzyl]sulfonyl}-2-(2-methoxyphenyl)acetamide], a selective E prostanoid receptor 4 antagonist, relieves joint inflammation and pain in rodent models of rheumatoid and osteoarthritis. J Pharmacol Exp Ther 2008; 325: 425– 434 35 Korotkova M, Jakobsson PJ. Characterization of microsomal prostaglandin e synthase 1 inhibitors. Basic Clin Pharmacol Toxicol 2014; 114: 64–69 36 White DE, Stewart IC, Seashore-Ludlow BA, Grubbs RH, Stoltz BM. A general enantioselective route to the chamigrene natural product family. Tetrahedron 2010; 66: 4668–4686 37 Ohta Y, Hirose Y. New sesquiterpenoids from Schisandra chinensis. Tetrahedron Lett 1968; 9: 2483–2485 38 da Cunha FM, Frode TS, Mendes GL, Malheiros A, Cechinel Filho V, Yunes RA, Calixto JB. Additional evidence for the anti-inflammatory and antiallergic properties of the sesquiterpene polygodial. Life Sci 2001; 70: 159–169 39 Martin WJ, Herst PM, Chia EW, Harper JL. Sesquiterpene dialdehydes inhibit MSU crystal-induced superoxide production by infiltrating neutrophils in an in vivo model of gouty inflammation. Free Radic Biol Med 2009; 47: 616–621 40 Merfort I. Perspectives on sesquiterpene lactones in inflammation and cancer. Curr Drug Targets 2011; 12: 1560–1573 41 Lee IS, Jung KY, Oh SR, Kim DS, Kim JH, Lee JJ, Lee HK, Lee SH, Kim EH, Cheong C. Platelet-activating factor antagonistic activity and(13)C NMR assignment of pregomisin and chamigrenal from Schisandra chinensis. Arch Pharm Res 1997; 20: 633–636 42 Kuo YH, Lin YT. Two new sesquiterpenes 3β-hydroxycedrol and widdringtonia acid II‑A co-crystal of β-chamigrenic acid and hinokiic acid. J Chin Chem Soc 1980; 27: 15–18 43 Viera PC, Himejima M, Kubo I. Sesquiterpenoids from Brachylaena hutchinsii. J Nat Prod 1991; 54: 416–420 44 Bohlmann F, Ludwig GW, Jakupovic J, King RM, Robinson H. New spirosequiterpene lactones, germacranolides, and eudesmanolides from Wunderlichia mirabilis. Liebigs Ann Chem 1984; 1984: 228–239 45 Ryu S, Shin JS, Cho YW, Kim HK, Paik SH, Lee JH, Chi YH, Kim JH, Lee KT. Fimasartan, anti-hypertension drug, suppressed inducible nitric oxide synthase expressions via nuclear factor-kappa B and activator protein1 inactivation. Biol Pharm Bull 2013; 36: 467–474 46 Lee SJ, Shin JS, Choi HE, Lee KG, Cho YW, An HJ, Jang DS, Jeong JC, Kwon OK, Nam JH, Lee KT. Chloroform fraction of Solanum tuberosum L. cv Jayoung epidermis suppresses LPS-induced inflammatory responses in macrophages and DSS-induced colitis in mice. Food Chem Toxicol 2013; 63: 53–61
Shin J-S et al. Inhibitory Effects of …
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Original Papers
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Authors
Ji-Sun Shin 1, 5, 6, Suran Ryu 1, 2, Young-Wuk Cho 2, 5, 6, Hyun Ji Kim 3, Dae Sik Jang 4, Kyung-Tae Lee 1, 4
Affiliations
The affiliations are listed at the end of the article
Key words " Schisandrae Fructus l " Schisandra chinensis l " Schisandraceae l " sesquiterpene l " nitric oxide l " prostaglandin E l 2 " mPGES‑1 l
Abstract
Abbreviations
!
!
Much is known about the bioactive properties of lignans from the fruits of Schisandra chinensis. However, very little work has been done to determine the properties of sesquiterpenes in the fruits of S. chinensis. The aim of the present study was to investigate the anti-inflammatory potential of new sesquiterpenes (β-chamigrenal, β-chamigrenic acid, α-ylangenol, and α-ylangenyl acetate) isolated from the fruits of S. chinensis and to explore their effect on macrophages stimulated with lipopolysaccharide. Of these four sesquiterpenes, β-chamigrenal most significantly suppressed lipopolysaccharide-induced nitric oxide and prostaglandin E2 production in RAW 264.7 macrophages (47.21 ± 4.54 % and 51.61 ± 3.95 % at 50 µM, respectively). Molecularly, the inhibitory activity of β-chamigrenal on nitric oxide production was mediated by suppressing inducible nitric oxide synthase activity but not its expression. In the prostaglandin E2 synthesis pathway, β-chamigrenal prevented the upregulation of inducible microsomal prostaglandin E synthase-1 expression after stimulation with lipopolysaccharide. Conversely, β-chamigrenal had no effect on the expression and enzyme activity of cyclooxygenase-2. In addition, the expression of early growth response factor-1, a key transcription factor of microsomal prostaglandin E synthase-1 expression, was inhibited by β-chamigrenal. These results may suggest a possible anti-inflammatory activity of β-chamigrenal which has to be proven in in vivo experiments.
AA: COX: cPGES: dNTP: DTT: Egr: EIA: eNOS: IL: iNOS: L-NIL: LPS: MAPEG:
received revised accepted
Dec. 2, 2013 April 9, 2014 May 5, 2014
Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1368544 Published online May 28, 2014 Planta Med 2014; 80: 655–661 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0032‑0943 Correspondence Dae Sik Jang, PhD Department of Life and Nanopharmaceutical Science College of Pharmacy Kyung Hee University Dongdaemun-Ku, Hoegi-Dong 130–701 Seoul Republic of Korea Phone: + 82 29 61 07 19 Fax: + 82 29 66 38 85 [email protected] Correspondence Kyung-Tae Lee, PhD Department of Pharmaceutical Biochemistry College of Pharmacy Kyung Hee University Dongdaemun-Ku, Hoegi-Dong 130–701 Seoul Republic of Korea Phone: + 82 29 66 38 85 Fax: + 82 29 62 08 60 [email protected]
arachidonic acid cyclooxygenase cytosolic prostaglandin E synthase deoxyribonucleotide triphosphate dithiothreitol early growth response factor enzyme immunoassay endothelial nitric oxide synthase interleukin inducible nitric oxide synthase L-N6-(1-iminoethyl)lysine lipopolysaccharide membrane-associated proteins involved in the eicosanoid and glutathion metabolism mPGES: microsomal prostaglandin E synthase MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide NO: nitric oxide NOS: nitric oxide synthase nNOS: neuronal nitric oxide synthase NSAID: nonsteroidal anti-inflammatory drug PG: prostaglandin prostaglandin E2 PGE2: PGES: prostaglandin E synthase PGHS: prostaglandin H endoperoxide synthase PMSF: phenylmethylsulfonylfluoride qRT‑PCR: quantitative real-time reversetranscription polymerase chain reaction TNF: tumor necrosis factor Supporting information available online at http://www.thieme-connect.de/products
Shin J-S et al. Inhibitory Effects of …
Planta Med 2014; 80: 655–661
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Inhibitory Effects of β-Chamigrenal, Isolated from the Fruits of Schisandra chinensis, on LipopolysaccharideInduced Nitric Oxide and Prostaglandin E2 Production in RAW 264.7 Macrophages
Original Papers
Introduction !
Although the inflammatory response is a defense mechanism against infection or injury, sustained inflammation is a pathological condition. An inflammatory response is characterized by the abundant production of proinflammatory mediators such as NO, PGE2, reactive species, and cytokines. NO is produced by macrophages, and acts as a cytotoxic agent during immune and inflammatory responses during the oxidation of L-arginine to L-citrulline [1]. In mammalian cells, NO is synthesized by the three isoforms of NOS: nNOS, eNOS, and iNOS [2]. In particular, iNOS is expressed in response to interferon-γ, LPS, and various proinflammatory cytokines [3]. The NO produced by iNOS has been suggested to have beneficial antiviral and microbiocidal effects [4], but the overproduction of NO can be harmful to the host and, in fact, is involved in the pathogenesis of various inflammatory diseases [5]. Other important mediators overexpressed during inflammation are the PGs. PGE2 is an eicosanoid product synthesized from AA in two steps. The first step is catalyzed by PGHS, also called COX, which converts AA to PGH2. Two different COX enzymes have been reported: COX-1, a constitutive enzyme in many tissues and COX-2, which is induced by cytokines, growth factors, or bacterial products, such as LPS, in different pathologic processes involving inflammation such as infectious diseases, cancer, arthritis, and atherosclerosis [6, 7]. The second step is the conversion of PGH2 into PGE2 by a reaction of isomerization catalyzed by PGES enzymes. Three different PGES have been described: cPGES and two membrane-bound mPGES, mPGES-1 and -2 [8, 9]. Among them, the mPGES-1 enzyme is a membrane-associated perinuclear protein belonging to the MAPEG family. This enzyme, which seems to be preferentially functionally coupled with COX2, is markedly induced by inflammatory stimuli and downregulated by anti-inflammatory drugs such as glucocorticoids [10]. Egr-1 is a nuclear transcription factor and belongs to a group of early response genes that are induced rapidly and transiently by different stimuli such as LPS, cytokines, growth factors, and hypoxia [11]. This factor has been implicated in cell growth and differentiation, and in the development of chronic inflammatory diseases such as atherosclerosis and arthritis [11, 12]. Recent studies demonstrated that Egr-1 is a key transcription factor in regulating the inducible expression of mPGES-1 through the binding to the DNA sequence GCG(G/T)GGCG on the mPGES-1 promoter [13, 14]. The fruits of Schisandra chinensis Baillon (Schisandraceae), also known as Schisandrae Fructus, have traditionally been used in Korea, Japan, and China for the treatment of coughs, spontaneous sweating, dysentery, and insomnia [15]. A previous phytochemical investigation on the fruits of S. chinensis has resulted in the isolation of lignans [16, 17] and nortriterpenoids [18]. Diverse pharmacological effects such as antihepatotoxic, anti-inflammatory, antioxidant and antitumoral activities of the fruits of S. chinensis and dibenzo[a,c]cyclooctene lignans have been reported in numerous studies [19, 20]. However, there are few reports on other pharmacologically active compounds from the fruits of S. chinensis. Therefore, as a part of our ongoing screening program to evaluate the anti-inflammatory potentials of natural compounds, we isolated four sesquiterpenes from the fruits of S. chinensis and investigated their effects on the LPS-induced production of NO and PGE2 in macrophages. In this study, we investigated the underlying molecular mechanisms of β-chamigrenal
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Planta Med 2014; 80: 655–661
Fig. 1 Inhibitory effects of sesquiterpene compounds isolated from the fruits of S. chinensis on lypopolysaccharide-induced production of nitric oxide and prostaglandin E2. A Chemical structures of β-chamigrenal, β‑chamigrenic acid, α-ylangenol, and α-ylangenyl acetate isolated from the fruits of S. chinensis. B Following pretreatment with sesquiterpenes (50 µM) for 1 h, cells were treated with LPS (1 µg/mL) for 24 h. Levels of NO and PGE2 in culture media were quantified using the Griess reaction assay and EIAs, respectively. Controls were not treated with LPS and sesquiterpenes. L-NIL (10 µM) and NS-398 (3 µM) were used as positive controls for NO and PGE2 production. Data are presented as the means ± SD of three independent experiments. # P < 0.05 vs. the control group; *** p < 0.001 vs. LPS-stimulated cells.
showing the most potent inhibitory activities on the LPS-induced production of NO and PGE2.
Results and Discussion !
The pathology of inflammation is initiated by complex processes triggered by microbial pathogens or their antigens [21]. The prototypic endotoxin LPS can potently activate macrophages and induce various proinflammatory mediators [22]. In light of this, reducing activation signals in activated macrophages has been suggested as a therapeutic strategy in various inflammatory diseases [23]. In the present study, repeated column chromatography of the hexane extract of the fruits of S. chinensis resulted in the isolation of four sesquiterpenes, specifically β-chamigrenal, β-cha" Fig. 1 A). migrenic acid, α-ylangenol, and α-ylangenyl acetate (l Initially, the isolated four sesquiterpenes were screened for their inhibitory effects on the LPS-induced production of NO and PGE2 " Fig. 1 B). β-chamigrenal (51.61 ± in RAW 264.7 macrophages (l 3.95 % at 50 µM) and β-chamigrenic acid (17.87 ± 2.55 % at 50 µM) more potently inhibited LPS-induced PGE2 production in comparison with α-ylangenol and α-ylangenyl acetate. Moreover,
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Fig. 2 Effects of β-chamigrenal on lypopolysaccharide-induced nitric oxide production, inducible nitric oxide synthase expression, and inducible nitric oxide synthase enzyme activity in RAW 264.7 macrophages. A Following pretreatment with β-chamigrenal (15, 30, or 60 µM) for 1 h, cells were treated with LPS (1 µg/mL) for 24 h. Levels of NO in culture media were quantified using the Griess reaction assay. Controls were not treated with LPS and β-chamigrenal. B Lysates were prepared from the cells pretreated with/without β-chamigrenal (15, 30, or 60 µM) for 1 h and then with LPS (1 µg/mL) for 24 h. Total cellular proteins were resolved by SDS-PAGE, transferred to PVDF membranes, and detected with a specific iNOS antibody. β-actin was used as an internal control. C Total RNA was prepared for the qRT‑PCR analysis of iNOS from the cells stimulated with LPS (1 µg/mL), with/without β-chamigrenal (15, 30, or 60 µM) for 4 h. The mRNA levels of iNOS were determined using gene-specific primers, as described in Materials and Methods. D Following pretreatment with LPS (1 µg/mL) for 12 h and washing with PBS, cells were treated with β-chamigrenal (15, 30, or 60 µM) for 12 h. Levels of NO in culture media were quantified using the Griess reaction assay. Data are presented as the means ± SD of three independent experiments. # P < 0.05 vs. the control group; *** p < 0.001 vs. the LPS-stimulated group.
acetated compounds β-chamigrenic acid (23.78 ± 1.03 % at 50 µM) and α-ylangenyl acetate showed much lower inhibitory effects than β-chamigrenal (47.21 ± 4.54 % at 50 µM) and α-ylangenol (47.53 ± 2.12 % at 50 µM) on LPS-induced NO production. Among these compounds, β-chamigrenal reduced the production of NO and PGE2 inflammatory mediators to the greatest extent in LPSstimulated RAW 264.7 macrophages. In addition, these inhibitory effects of these four sesquiterpenes were not caused by nonspecific cytotoxicity, because these compounds had no effect on cell viability as determined by the MTT assay at 50 µM (Fig. 1S, Supporting Information). Therefore, we examined the underlying molecular mechanisms for the inhibitory activities of β-chamigrenal on the LPS-induced NO and PGE2 production in macro" Fig. 2 A, β-chamigrenal inhibited LPS-inphages. As shown in l duced NO production, dose-dependently, in RAW 264.7 macrophages (from 13.62 ± 0.23 to 12.79 ± 0.21, 10.76 ± 0.23, and 7.19 ± 0.61 µM at 15, 30, and 60 µM, respectively). L-NIL (20 µM) was used as a positive control for inhibition of NO. The inhibitory effects on NO production are principally signaled through multiple steps: scavenging of NO, deprivation of arginine, suppression of iNOS enzyme expression, and downregulation of iNOS enzyme activity. Imidazolineoxyl-N-oxide, lactacystin, and imidazole are well-known representatives of each step [24–26]. We first determined the effects of β-chamigrenal on the protein and mRNA expression of iNOS using Western blotting and qRT‑PCR, respec" Fig. 2 B and C, β-chamigrenal up to 60 µM tively. As shown in l did not inhibit LPS-induced iNOS protein and mRNA expression. Because no transcriptional events were involved in the inhibitory effects of β-chamigrenal, we identified the possibility that the reduction in NO accumulation caused by β-chamigrenal could be due to the modulation of iNOS enzyme activity. It was found that LPS-induced iNOS activity was inhibited by β-chamigrenal " Fig. 2 D, from 100.00 ± 3.40 to 68.27 ± 2.13, 52.51 ± 2.25, and (l
48.91 ± 3.52 % at 15, 30, and 60 µM, respectively). A few studies have described a possible regulation of iNOS enzyme activity by the post-translational processing of the protein, including active dimer formation and localization, phosphorylation [27], and by regulation of arginine availability [28]. Unfortunately, we did not establish the inhibitory mechanisms of iNOS enzyme activity by β-chamigrenal yet. However, our results demonstrate that the inhibitory effect of β-chamigrenal on LPS-induced NO production may occur through the inhibition of iNOS enzyme activity rather than through iNOS expression. PGE2, the best known of the prostaglandins, is involved in various inflammatory disorders, including arthritis, rheumatism, cardiovascular thrombosis, and Alzheimerʼs disease [29]. PGE2 is synthesized from arachidonic acid by COX-2 and mPGES-1 in response to inflammatory stimuli [30]. To assess the effects of βchamigrenal on LPS-induced PGE2 production in RAW 264.7 macrophage cells, the amount of PGE2 was measured via EIA. Stimulation of cells with LPS (1 µg/mL) resulted in a significant increase in PGE2 production compared with that in unstimulated " Fig. 3 A). Furthermore, pretreatment with β-chacontrol cells (l migrenal inhibited LPS-induced PGE2 production (from 31.65 ± 4.39 to 28.43 ± 2.42, 23.21 ± 1.55, and 16.71 ± 0.59 ng/mL at 15, 30, and 60 µM, respectively). As a control for the inhibition of PGE2 production, we used NS-398 (3 µM), a positive control for the COX-2 inhibitor. We also determined the effects of β-chamigrenal on the protein and mRNA levels of COX-2, which is an inducible enzyme responsible for converting PGH2 from AA, an intermediate step in a process of producing PGE2 from AA. As " Fig. 3 B and C, COX-2 was markedly upregulated by shown in l LPS at the protein and mRNA levels, whereas β-chamigrenal had no inhibitory effect on these upregulations in RAW 264.7 macrophages. These findings show that β-chamigrenal did not downregulate LPS-induced COX-2 expression. Furthermore, we deter-
Shin J-S et al. Inhibitory Effects of …
Planta Med 2014; 80: 655–661
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Original Papers
Original Papers
Fig. 3 Effects of β-chamigrenal on lypopolysaccharide-induced prostaglandin E2 production, and cyclooxygenase-2 and microsomal prostaglandin E synthase-1 expression in RAW 264.7 macrophages. A Following pretreatment with β-chamigrenal (15, 30, or 60 µM) for 1 h, cells were treated with LPS (1 µg/mL) for 24 h. The levels of PGE2 in culture media were quantified using EIA. Controls were not treated with LPS and β-chamigrenal. B Lysates were prepared from the pretreated cells with/without βchamigrenal (15, 30, or 60 µM) for 1 h and then with LPS (1 µg/mL) for 24 h. Total cellular proteins were resolved by SDS-PAGE, transferred to PVDF membranes, and detected with a specific COX-2 or mPGES-1 antibody. β-actin was used as an internal control. C, D Total RNA was prepared for the qRT‑PCR analysis of COX-2 and mPGES-1 from the cells stimulated with LPS (1 µg/mL), with/without βchamigrenal (15, 30, or 60 µM) for 4 h. The mRNA levels of COX-2 and mPGES-1 were determined using gene-specific primers, as described in Materials and Methods.
Fig. 4 Inhibitory effects of β-chamigrenal on lipopolysaccharide-induced early growth response factor-1 expression in RAW 264.7 macrophages. A Lysates were prepared from the cells pretreated with β-chamigrenal (50 µM) for 1 h prior to pretreatment with LPS (1 µg/mL) for 1 and 2 h. B Lysates were prepared from the cells stimulated with LPS (1 µg/ mL), with/without β-chamigrenal (15, 30, or 60 µM) for 2 h. Total cellular proteins were resolved by SDSPAGE, transferred to PVDF membranes, and detected with a specific Egr-1 antibody. β-actin was used as an internal control.
mined the COX-2 enzyme activity using Dup697 as the respective positive control and found that β-chamigrenal had no effect on the COX-2 enzyme activities (Fig. 2S, Supporting Information). To study the effects of β-chamigrenal on the expression of mPGES-1, which is an inducible enzyme responsible for producing PGE2 from PGH2, cells were pretreated with LPS (1 µg/mL) alone or in combination with β-chamigrenal, and protein and mRNA were extracted thereafter. mPGES-1 protein and mRNA were measured by Western blotting and qRT‑PCR, respectively. We found that LPS-induced mPGES-1 protein and mRNA levels were significantly inhibited by β-chamigrenal in a dose-depen" Fig. 3 B and D). Previous data indicate that NFdent manner (l κB is a major regulatory component of the inflammatory responses mediated by LPS or proinflammatory cytokines [31]. We therefore measured the effects of β-chamigrenal on the transcriptional activity of NF-κB and found that β-chamigrenal did not reduce the NF-κB-dependent luciferase activity induced by LPS (data not shown). In addition to NF-κB, LPS stimulation activates many transcription factors, including the zinc finger transcription factor Egr-1 [32]. Egr-1 has been described as a strong regulator of mPGES-1 transcription after stimulation of different cells with IL-1β or TNFα [33]. Therefore, we investigated whether the inhibition of β-chamigrenal expression is related to reductions in the expression of Egr-1. Western blot analysis showed
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that LPS activation of macrophages results in an increase in Egr1 protein and that β-chamigrenal suppressed LPS-induced Egr-1 " Fig. 4 A). We further found that LPS-induced expression at 2 h (l Egr-1 protein levels were inhibited by β-chamigrenal in RAW " Fig. 4 B). In inflammation, mPGES-1 ex264.7 macrophages (l pression was detected to be upregulated in patients with rheumatoid arthritis and osteoarthritis [34]. This suggests a novel therapeutic target for the treatment of inflammatory diseases with improved selectivity against pathological PGE2 and consequential safety when compared to the traditional NSAIDs or selective COX-2 inhibitors [35]. Recently, inhibition of mPGES-1 by epigallocatechin-3-gallate from green tea was suggested to be the predominant mechanism leading to diminished PGE2 production via the modulation of Egr-1 in A549 human pulmonary epithelial cells [33]. In the present study, we found that β-chamigrenal significantly downregulates the mRNA and protein expression of the mPGES-1 enzyme probably via Erg-1 suppression, but has little effect on COX-2 expression in LPS-stimulated macrophages. This suggests mPGES-1 as the primary target of β-chamigrenal, explaining its inhibitory effect on the production of PGE2. β-chamigrenal belongs to the chamigrene subclass of sesquiterpenes, which have been isolated from a variety of sources including terrestrial and marine plants, and have demonstrated a diverse array of biological activity [36]. β-chamigrenal was origi-
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658
Original Papers
data measurements and by comparison with published values [41–44].
Chemicals and antibodies DMEM, FBS, penicillin, and streptomycin were obtained from Life Technologies, Inc. iNOS, COX-2, mPGES-1, Erg-1, β-actin monoclonal antibodies, and peroxidase-conjugated secondary antibody were purchased from Santa Cruz Biotechnology, Inc. The ELISA kit for PGE2 was obtained from R&D Systems. RNA extraction kits were obtained from Intron Biotechnology. Random oligonucleotide primers and M–MLV reverse transcriptase were purchased from Promega. SYBR green ex Taq was obtained from TaKaRa. iNOS, COX-2, mPGES-1, and β-actin oligonucleotide primers were purchased from Bioneer. MTT, NS-398 (purity ≥ 98 %), PMSF, DTT, L-NIL (purity > 97 %), LPS (Escherichia coli, serotype 0111:B4), and all other chemicals were obtained from Sigma-Aldrich.
Materials and Methods
Cell culture and sample treatment
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RAW 264.7 murine macrophages were obtained from the Korean Cell Line Bank. Cells were grown at 37 °C in DMEM medium supplemented with 10% FBS, penicillin (100 units/mL), and streptomycin sulfate (100 µg/mL) in a humidified 5 % CO2 atmosphere. Cells were pretreated with β-chamigrenal (15, 30, or 60 µM) for 1 h and then stimulated with LPS for an indicated time.
Plant material The dried fruits of S. chinensis were purchased from Kyung-dong market, Seoul, Republic of Korea, in June 2011 and were identified by Prof. Dae Sik Jang. A voucher specimen (No. 2011-SCCH01) has been deposited in the Laboratory of Natural Product Medicine, College of Pharmacy, Kyung Hee University.
MTT assay Extraction and isolation The milled plant material (3.5 kg) was extracted with 10 L of 80 % aqueous ethanol (EtOH) three times by maceration. The extracts were combined and concentrated in vacuo at 40 °C to give an 80 % EtOH extract (1.52 kg). The 80 % EtOH extract (1.51 kg) was suspended in distilled water (5 L) and then successively extracted with n-hexane (3 × 5 L), ethyl acetate (EtOAc) (3 × 5 L), and butanol (BuOH) (3 × 500 L) to give n-hexane- (125 g), EtOAc – (80 g), BuOH – (430 g), and water-soluble fractions (875 g), respectively. The n-hexane-soluble extract (120 g) was subjected to column chromatography on silica gel (8.6 × 21 cm, 70–230 mesh) eluted with n-hexane: EtOAc [1 : 0, 99 : 1, 97 : 3, 19 : 1, 4 : 1, 7 : 3, 1 : 1, 0 : 1, methanol (MeOH), 4 L each eluent] to afford 7 fractions (Fr.1–Fr.7). Fr.1 [eluted with n-hexane: EtOAc (9 : 1 v/v); 40 g] was chromatographed over silica gel (6 × 48 cm, 230–400 mesh) eluting with n-hexane : EtOAc (99 : 1, 49 : 1, 97 : 3, 19 : 1, MeOH, 4 L each eluent) to give 10 subfractions (Fr.1–1 – Fr.1–10). α-Ylangenyl acetate (1500–1700 mL, 460 mg) and β-chamigrenal (2900–3300 mL, 2.1 g) were obtained from Fr.1–2 [eluted with n-hexane : EtOAc (97 : 3 v/v); 10.8 g] by silica gel column chromatography (6 × 34 cm, 230–400 mesh) eluting with n-hexane : EtOAc (19 : 1 v/v, 5 L). Fr.1–5 [eluted with n-hexane : EtOAc (19 : 1 v/v); 9.3 g] was chromatographed over silica gel (6 × 38 cm, 230–400 mesh) as a stationary phase with n-hexane : EtOAc (9 : 1 v/v, 4.8 L) as the mobile phase to afford α-ylangenol (2900–3300 mL, 2.1 g). Fr.3 [eluted with n-hexane-EtOAc (9 : 1 v/v); 17.1 g] was subjected to column chromatography on silica gel (7 × 46 cm, 70–230 mesh) eluted with n-hexane: EtOAc [7 : 1 v/v (18 L), 4 : 1 v/v (7 L), 7 : 3 v/v (3 L)] to afford 7 fractions (Fr.3–1 – Fr.3–10). β-Chamigrenic acid (475–525 mL, 170 mg) was obtained from Fr.3–7 [eluted with n-hexane : EtOAc (7 : 1 v/ v); 1.46 g] by Sephadex LH-20 column (3.6 × 72 cm) eluting with a CH2Cl2-MeOH mixture (1 : 1 v/v). The purity of these compounds (> 95 %) was determined by HPLC and NMR. The structures of the isolates were identified by physical and spectroscopic
RAW 264.7 cells were plated at 1 × 104 cells/100 µL in 96-well plates and then incubated with various concentrations of the tested compounds for 24 h. After incubation, the cells were treated with an MTT solution for 4 h at 37 °C under 5 % CO2. One hundred microliters of dimethyl sulfoxide were added to extract the MTT formazan, and the absorbance of each well was read by an automatic microplate reader at 540 nm (Molecular Devices).
Nitrite assay RAW 264.7 cells were plated at 5 × 105 cells/well in 24-well plates and then incubated with or without LPS (1 mg/mL) in the absence or presence of various concentrations of the tested compounds for 24 h. Nitrite accumulation was measured in culture media using the Griess reaction assay and presumed to reflect NO levels [45].
Prostaglandin E2 assay RAW 264.7 cells were pretreated with the tested compounds for 1 h and then stimulated with LPS (1 µg/mL) for 24 h. Levels of PGE2 in the culture media were quantified using an EIA kit (R&D Systems).
Inducible nitric oxide synthase enzyme activity assay RAW 264.7 cells were plated at 5 × 105 cells/well in 24-well plates and then incubated with LPS (1 mg/mL) for 12 h. The cells were washed twice with PBS and then incubated in the absence and presence of β-chamigrenal for a further 12 h, with no LPS in the medium. The supernatants were collected and the Griess reaction was performed [45].
Cyclooxygenase enzyme activity assay The compound was evaluated for potency and selectivity of inhibition in vitro using the COX inhibitor screening assay (Cayman). Recombinant COX-2 (human) proteins were preincubated with βchamigrenal for 10 min at 37 °C. The reaction was started by the
Shin J-S et al. Inhibitory Effects of …
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nally isolated from the oil of the fruits of S. chinensis [37]. Sesquiterpene aldehydes, such as polygodial, have been shown to inhibit cell infiltration in a number of in vivo models of acute inflammation [38, 39]. There are some reports that α-methyleneγ-lactone groups and/or conjugated aldehyde groups of sesquiterpenes contribute to their anti-inflammatory effect [40]. Therefore, the conjugated aldehyde group of β-chamigrenal seems to be an active site responsible for its possible anti-inflammatory potential. In summary, to the best of our knowledge, this is the first report to suggest that β-chamigrenal, a sesquiterpene isolated from S. chinensis, inhibited the LPS-induced production of NO and PGE2 in RAW 264.7 macrophages. Molecular data revealed that β-chamigrenal is involved in the inhibition of NO and PGE2 production via the reduction of iNOS enzyme activities and the attenuation of expression of mPGES-1 and Erg-1 transcription factor.
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addition of 100 µM arachidonic acid and allowed to proceed for 2 min. The reaction was terminated by the addition of HCl solution containing SnCl2. The COX activity assay directly measures PGF2α produced by the SnCl2 reduction of COX-derived PGH2. The prostanoid product is quantified via EIA. As a control inhibitor for COX-2, Dup-697 (10 µM, purity > 95%, Merk Milipore) was used.
Science, ICT and Future Planning through the National Research Foundation. The NMR experiments were performed by the Korea Basic Science Institute (KBSI).
Conflict of Interest !
The authors have no conflict of interest to declare.
Western blot analysis Cellular proteins were extracted from RAW 264.7 macrophages, as described previously [46], and protein concentrations were determined using Bio-Rad protein assay reagent, according to the manufacturerʼs instructions. Proteins were electroblotted onto PVDF membranes after being separated by 10% SDS-polyacrylamide gel electrophoresis. Membranes were incubated for 1 h in blocking solution (5% skim milk) with the primary antibody overnight at 4 °C, washed three times with Tween 20/Tris-buffer, incubated with a horseradish peroxidase-conjugated secondary antibody (1 : 2000) for 2 h at room temperature, and washed three times with Tween 20/Tris-buffer. Blots were then developed using an enhanced chemiluminescence (Amersham Life Science).
Quantitative real-time reverse-transcriptase polymerase chain reaction Total cellular RNA was isolated using Easy Blue kits (Intron Biotechnology). For each sample, 1 µg of RNA was reverse-transcribed using MuLV reverse transcriptase, 1 mM dNTP, and 0.5 µg/µL oligo (dT12–18). Real-time PCR was performed using a thermal cycler dice real-time PCR system (Takara). The primers used for SYBR Green real-time reverse transcription–PCR were as follows: for iNOS, sense primer, 5′-CATGCTACTGGAGGTGGGTG‑3′, anti-sense primer, 5′-CATTGATCTCCGTGACAGCCC‑3′; for COX-2, sense primer COX-2, 5′-GGAGAGACTATCAAGATAGT‑3′, anti-sense primer COX-2, 5′-ATGGTCAG- TAGACTTTTACA-3′; for mPGES-1, sense primer 5′-AGGATGCGCTGAAACGTGG- A-3′, anti-sense primer, 5′-CGAAGCCGAGGAAGAGGAAA-3′; and for βactin, sense primer, 5′-ATCACTATT-GGCAACGAGCG‑3′, antisense primer, 5′-TCAGCAAT-GCCTGGGTACAT‑3′.
Statistical analysis The data are expressed as the mean ± SD of at least three experiments performed using different cell preparations. Statistically significant values were compared using one-way ANOVA followed by Dunnettʼs post test, and p values less than 0.05 were considered statistically significant.
Supporting information Bar graphs showing the effects of sesquiterpenes isolated from the fruits of S. chinensis on cell viability in RAW 264.7 macrophages and the inhibitory effects of β-chamigrenal on COX-2 enzyme activity in RAW 264.7 macrophages are available as Supporting Information
Acknowledgements !
This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), which is funded by the Ministry of Education, Science and Technology (No. 2011–0023407) and by a grant from the Bio-Synergy Research Project (NRF-2013M3A9C4078145) of the Ministry of
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Affiliations 1
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Department of Pharmaceutical Biochemistry, College of Pharmacy, Kyung Hee University, Seoul, Republic of Korea Department of Biomedical Science, College of Medical Science, Kyung Hee University, Seoul, Republic of Korea Department of Oriental Pharmaceutical Science, College of Pharmacy, Kyung Hee University, Seoul, Republic of Korea Department of Life and Nanopharmaceutical Science, College of Pharmacy, Kyung Hee University, Seoul, Republic of Korea Reactive Oxygen Species Medical Research Center, School of Medicine, Kyung Hee University, Seoul, Republic of Korea Department of Physiology, School of Medicine, Kyung Hee University, Seoul, Republic of Korea
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