Neuroscience Letters 462 (2009) 171–175
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Transcriptional regulation of iNOS and COX-2 by a novel compound from Curcuma comosa in lipopolysaccharide-induced microglial activation Anusorn Thampithak a , Yamaratee Jaisin a , Benjawan Meesarapee a , Sukumal Chongthammakun b,e , Pawinee Piyachaturawat c , Piyarat Govitrapong d,e , Porntip Supavilai a , Yupin Sanvarinda a,e,∗ a
Department of Pharmacology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand Department of Anatomy, Faculty of Science, Mahidol University, Bangkok 10400, Thailand Department of Physiology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand d Neuro-Behavioural Biology Center, Institute of Science and Technology for Research and Development, Mahidol University, Salaya, Nakornpathom 73170, Thailand e Center for Neuroscience, Faculty of Science, Mahidol University, Bangkok 10400, Thailand b c
a r t i c l e
i n f o
Article history: Received 14 May 2009 Received in revised form 30 June 2009 Accepted 30 June 2009 Keywords: Curcuma comosa Microglia iNOS NO COX-2 PGE2
a b s t r a c t Overproduction of pro-inflammatory mediators resulting from chronic activation of microglia has been implicated in many neurodegenerative disorders, such as Parkinson’s disease and Alzheimer’s disease. In this study, we investigated the effects of (3R) 1,7-diphenyl-(4E,6E)-4,6-heptadien-3-ol, or compound 049 on the production of pro-inflammatory mediators in lipopolysaccharide (LPS)-treated microglia. Compound 049 is a pure compound fractionated from the hexane extract of Curcuma comosa, an indigenous plant of Thailand traditionally used as an anti-inflammatory agent for the treatment of uterine inflammation. It was found that pretreatment of the highly aggressively proliferating immortalized (HAPI), rat microglial cell line, with compound 049, at the concentrations of 0.1, 0.5 and 1 M significantly decreased LPS-induced NO and PGE2 production in a concentration-dependent manner. Parallel to the decreases in NO and PGE2 production was a reduction in the expression of inducible NO synthase (iNOS) and cyclooxygenase 2 (COX-2) as measured by mRNA and protein levels. These results indicate that compound 049 possesses an anti-inflammatory activity and may have a therapeutic potential for the treatment of neurodegenerative diseases related to microglial activation. © 2009 Elsevier Ireland Ltd. All rights reserved.
Microglia are the immune cells present in the central nervous system (CNS) responsible for homeostasis regulation and defense against injury. Activated microglia can produce various proinflammatory mediators including cytokines, chemokines, nitric oxide (NO), prostaglandin E2 (PGE2 ) and reactive oxygen species (ROS) [7,16]. Prolonged activation of microglia and subsequent overproduction of pro-inflammatory substances have been identified to be associated with numerous neurological disorders, including Alzheimer’s disease (AD), Parkinson’s disease (PD), multiple sclerosis (MS), AIDS dementia complex, and cerebral ischemia [5,6]. Among the potentially lethal substances produced by activated microglia, NO and PGE2 have emerged as important determinants of the inflammation-associated cytotoxicity observed in AD, MS, and cerebral ischemia [17,19,25]. Therefore, suppressing the expression of these pro-inflammatory mediators would be an effective
∗ Corresponding author at: Department of Pharmacology, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand. Tel.: +66 2 201 5510; fax: +66 2 354 7157. E-mail address:
[email protected] (Y. Sanvarinda). 0304-3940/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2009.06.094
therapeutic approach toward mitigating the progression of several neurodegenerative diseases. The rhizome of Curcuma comosa Roxb. (Zingiberaceae): commonly known as Waan Chak Modluk in Thai, has long been used to alleviate the inflammation in postpartum uterine bleeding, perimenopausal bleeding, and uterine inflammation. Recent study demonstrated that hexane extract from the rhizome of C. comosa reduced NO production and suppressed iNOS mRNA and protein expression in lipopolysaccharide (LPS)-stimulated HAPI cells [10]. Furthermore, the diarylheptanoids from hexane and ethanol extract of C. comosa decreased the release of pro-inflammatory cytokines from phorbol-12-myristate-13-acetate (PMA)-stimulated PBMC and U937 cells [21]. There has not been a demonstration, however, of the anti-inflammatory effect of the pure compound from C. comosa in LPS-activated microglia. LPS is the most commonly used compound to immunostimulate microglia and macrophages. It is able to induce microglia and macrophages to produce various types of inflammatory molecules [18]. In this study, we, therefore investigated the effects of compound 049 or (3R) 1,7-diphenyl-(4E,6E)-4,6-heptadien-3-ol (Fig. 1), a pure compound isolated from the hexane extract of C. comosa, on
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Fig. 1. Chemical structure of compound 049 or (3R) 1,7-diphenyl-(4E,6E)-4,6heptadien-3-ol, a pure compound isolated from the hexane extract of C. comosa.
the expression of iNOS, COX-2 and their products, NO and PGE2 in LPS-stimulated microglia. The compound was authenticated by Prof. Apichart Suksamrarn (Department of Chemistry, Faculty of Science, Ramkhamhaeng University, Bangkok, Thailand) and was prepared as previously reported [23]. The pure compound was dissolved in dimethyl sulfoxide (DMSO) at 10 mM as a stock solution and stored at 4 ◦ C until use. The final concentration of DMSO applied to the cells was maintained at 0.1% in all experiments. HAPI microglial cells were generously provided by Prof. James R. Connor (Hershey Medical Center, Hershey, PA, USA). The cells were grown in Dubecco’s modified Eagle’s medium (DMEM; GibcoBRL, Grand Island, NY, USA) supplemented with 5% heat-inactivated fetal bovine serum (FBS; PAA Laboratories, Morningside, Queensland, Australia) at 37 ◦ C in a humidified incubator under 5% CO2 and 95% air. In all experiments, the cells were seeded one day before the experiment to allow them to reach 80% confluence, at which time compound 049 was added to the culture 1 h before subsequent treatment of LPS (LPS from Escherichia coli 026:B6, Sigma, St. Louis, MO, USA) for 24 h. LPS was used at a concentration of 0.1 g/ml in all experiments. The cell viability was determined by the quantitative colorimetric with MTT assay (3-(4,5-dimethylthiazole-2-yl)-2,5diphenyltetrazolium bromide Sigma). Cells were plated at a density of 2 × 104 cells per well on 96-well culture plates. After treatment with various concentrations of compound 049 ranging from 0.1 to 50 M for 24 h or treatment with different concentrations of compound 049 for 1 h followed by 24 h of LPS treatment (0.1 g/ml), the medium was removed and the cells were incubated with a solution of 1 mg/ml MTT for 4 h at 37 ◦ C and 5% CO2 . The supernatant was then removed, and the formazan crystals in the cells were solubilized with DMSO. Absorbance was read at 570 nm on a microplate reader (Bio-Tek Instruments, Winooski, VT, USA). NO production was determined by measurement of nitrite, the stable metabolite of NO, in culture medium. Accumulation of nitrite in the medium was assessed by colorimetric assay using Griess reagent (Sigma). Cells were plated in 24-well plates (1 × 105 cells/well) and then treated with various concentrations of compound 049 for 1 h. After LPS stimulation, 100 l of culture medium from each sample was mixed with the same volume of Griess reagent and incubated at room temperature for 15 min. The optical density at 545 nm was measured using a microplate reader (Bio-Tek Instruments). The amount of PGE2 in the cell culture supernatant was determined by enzyme immunoassay (EIA). Cells were cultured in 24-well plates at a density of 1 × 105 cells/well and incubated with compound 049 at the concentrations of 0.1, 0.5, 1 M for 1 h before treating with LPS. The culture supernatants after centrifugation at 10,000 × g for 10 min were collected and stored at −80 ◦ C until the assay was performed. PGE2 in the culture medium was measured with a PGE2 competitive EIA kit (Assay Designs, Ann Arbor, MI, USA) according to the manufacturer’s instruction. Standards from 39 to 2500 pg/ml were used. To evaluate iNOS and COX-2 mRNA expression, HAPI cells were seeded onto 21 cm2 culture dishes at a density of 1 × 106 cells/dish and treated with various concentrations of compound 049 for 1 h. After LPS (0.1 g/ml) stimulation for 6 h, total RNA was extracted using a GenElute Mammalian Total RNA Miniprep Kit (Sigma)
according to the manufacturer’s instruction. One microgram of total RNA was reverse transcribed to cDNA using an Enhanced Avian RT First Strand Synthesis Kit (Sigma) according to the manufacturer’s instruction. The process yielded 20 l of cDNA, of which 2 l was used for each semi-quantitative PCR analysis. The cDNA was then amplified by PCR with specific primers of iNOS, COX-2, and GAPDH as follows: iNOS sense, 5 -GCAGAATGTGACCATCATGG-3 ; iNOS anti-sense, 5 -ACAACCTTGGTGTTGAAGGC-3 ; COX-2 sense, 5 -TGCGATGCTCTTCCGAGCTGTGCT-3 ; COX-2 anti-sense, 5 -TCAGGAAGTTCCTTATTTCCTTTC-3 ; GAPDH sense, 5 -TCCCTCAAGATTGTCAGCAA-3 ; GAPDH anti-sense, 5 -AGATCCACAACGGATACATT-3 . The following PCR conditions were applied to amplify iNOS, COX-2, and GAPDH: 15 min at 95 ◦ C then 25 cycles of 94 ◦ C for 40 s, 55 ◦ C for 40 s, and 72 ◦ C for 60 s: and a final extension at 72 ◦ C for 10 min. PCR products were analyzed on a 2% agarose gel and visualized by SYBR Green (Sigma) staining. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as an internal control for sample loading and mRNA integrity. The band intensity was quantified using Scion Imaging Software (Scion software, Frederick, MD, USA). To determine protein expression of iNOS and COX-2, HAPI cells were grown in 6-well plates at a density of 5 × 105 cells/well and pretreated with different concentrations of compound 049 for 1 h prior to stimulation with LPS for 6 h. The cells were washed with phosphate buffer saline (PBS) three times and lysed with RIPA buffer. Equal amounts of proteins (20 g) were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). Membranes were blocked with 5% nonfat milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) and incubated overnight at 4 ◦ C with polyclonal rabbit anti-mouse iNOS (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) or polyclonal rabbit anti-mouse COX-2 (1:1000; Abcam, Cambridge, MA, USA). The membranes were subsequently incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:10,000; Zymed, San Francisco, CA, USA). Specific protein bands were detected using the enhanced chemiluminescence (ECL) detection system (Pierce, Rockford, IL, USA). Data were expressed as the mean ± SEM of three independent experiments, in which triplicate samples were performed. Statistical significance was assessed by one-way ANOVA followed by a Tukey comparison test using the GraphPad Prism program version 5 (GraphPad software, San Diego, CA, USA). A value of p < 0.05 was considered statistically significant. In order to evaluate the cytotoxic effect of compound 049 on microglia, HAPI cells were incubated with compound 049 at final concentrations of 0.1, 0.5, 1, 10, and 50 M, for 24 h, before the MTT assay was carried out. As illustrated in Fig. 2A, the cell viability was not significantly decreased after the compound 049 treatment except at the highest concentration of 50 M, at which the cell viability was reduced to 30% of the control. Accordingly, the concentration of compound 049 used in other experiments did not exceed 1 M. Treatment of microglia with compound 049 (0.1, 0.5, and 1 M) in the presence or absence of LPS for 24 h did not significantly alter cell viability (Fig. 2B). These results demonstrate that the effects on activated HAPI microglia induced by LPS were not due to cytotoxicity from compound 049. To investigate the effect of compound 049 on iNOS and NO expression, HAPI cells were pretreated with compound 049 (0.1, 0.5, and 1 M) for 1 h before stimulation with LPS. Following 24 h of LPS stimulation, the accumulated NO in the culture media was estimated by a Griess assay. As shown in Fig. 3A, compound
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duction was evident in the compound 049 pretreatment group, in a concentration-dependent manner. PGE2 synthesis decreased to 102.20 ± 2.78, 72.35 ± 5.10, and 44.15 ± 3.57 pg/ml after treatment with compound 049 at 0.1, 0.5, and 1 M, respectively. We
Fig. 2. Effects of compound 049 on the viability of HAPI cells. Cells were treated with various concentrations of compound 049 for 24 h (A), or various concentrations of compound 049 for 1 h followed by incubation with LPS (0.1 g/ml) for 24 h, the first column represented the viability of cells without any treatment (control group), the second column represented the viability of cells treated only with compound 049 at concentration of 1 M for 24 h (B). Cell viability was assessed by the MTT assay. Data were represented as mean ± SEM of three separate experiments. ***p < 0.001 as compared with the control.
049 treatment alone did not alter the NO level compared to the control, while LPS stimulation resulted in a marked induction of NO production (44.57 ± 0.66 M) as compared to untreated control (11.3 ± 0.66 M). Pretreatment of compound 049 significantly diminished the level of LPS-induced NO production in a concentration-dependent manner. At doses of 0.1, 0.5, and 1 M, compound 049 reduced NO production to 39.97 ± 0.91, 22.98 ± 1.92, and 17.34 ± 0.73 M, respectively. However, after treatment with 1 M of compound 049, the level of NO production was still significantly higher than the untreated control. It could be that compound 049 at 1 M concentration is not enough to suppress the NO production to the basal level. Next, to investigate the effect of compound 049 on iNOS expression, semi-quantitative RT-PCR (Fig. 3B) and Western blot analysis (Fig. 3C) were performed to determine iNOS mRNA and protein levels at 6 h after LPS stimulation. LPS treatment caused a marked increase in iNOS expression consistent with the increase in NO production. Interestingly, compound 049 significantly inhibited both mRNA and protein expression of iNOS. GAPDH mRNA and -actin protein levels were not affected by LPS or compound 049 treatments. The results showed that compound 049 inhibited NO production through a reduction in the expression of iNOS mRNA and thus the protein level. To identify whether compound 049 could inhibit LPS-induced PGE2 and COX-2 expression in HAPI microglia, the cells were pretreated with compound 049 (0.1, 0.5, and 1 M) for 1 h before stimulation with LPS. At 24 h after the stimulation, the production of PGE2 in the supernatant was measured by a PGE2 immunoassay. As illustrated in Fig. 4A, the amount of PGE2 in the culture medium increased from 22.60 ± 3.29 to 119.10 ± 9.07 pg/ml after LPS exposure. A marked reduction in the amount of PGE2 pro-
Fig. 3. Effects of compound 049 on NO production and iNOS expression in LPS-activated HAPI microglia. The cells were incubated for 1 h with different concentrations of compound 049 (0.1, 0.5, 1 M) prior to stimulation with LPS (0.1 g/ml) (A). After 24 h, the amounts of NO were determined using Griess reagent. In parallel experiments, after LPS treatment for 6 h, the expressions of iNOS mRNA (B) and protein (C) were measured by semi-quantitative RT-PCR and Western blot analysis. GAPDH mRNA and -actin protein are used as reference to control equal loading. Values are expressed as mean ± SEM from three individual experiments. # p < 0.001, † p < 0.05 as compared with the control; *p < 0.05, **p < 0.01, ***p < 0.001 as compared with LPS treatment alone.
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next investigated the effect of compound 049 on COX-2 expression; semi-quantitative RT-PCR (Fig. 4B) and Western blot analysis (Fig. 4C) were performed to determine COX-2 mRNA and protein at 6 h after LPS stimulation. There was no significant difference
Fig. 4. Effects of compound 049 on PGE2 production and COX-2 expression in LPS-stimulated HAPI microglia. Cells were pretreated for 1 h with different concentrations of compound 049 (0.1, 0.5, 1 M) prior to stimulation with LPS (0.1 g/ml) (A). After 24 h, the amounts of PGE2 were determined using ELISA. In parallel experiments, after LPS treatment for 6 h, the expression of COX-2 mRNA (B) and protein (C) was measured by semi-quantitative RT-PCR and Western blot analysis. GAPDH mRNA and -actin protein were used as references to control equal loading. Values are expressed as mean ± SEM from three independent experiments. # p < 0.001, † p < 0.05 as compared with the control; **p < 0.01, ***p < 0.001 as compared with LPS treatment alone.
in the mRNA and protein levels of COX-2 between the compound 049 treatment alone and the control. The level was significantly increased after LPS treatment, however, compound 049 (0.1, 0.5, and 1 M) dramatically reduced COX-2 mRNA and protein level in the LPS-activated cells. GAPDH mRNA and -actin protein levels were not affected by LPS or compound 049 treatments. These results indicate that compound 049 suppressed LPS-induced PGE2 synthesis through down-regulation of COX-2 mRNA expression, and thus the protein level. Our study demonstrated the anti-inflammatory effects of compound 049 on LPS-activated HAPI microglia. It should be noted that at high concentration (50 M), compound 049 caused substantial cytotoxicity while a 5-fold lower concentration was non-toxic. This aspect has to be considered in the consumption or application of the plant extract for therapeutic purposes. Microglial activation induced by CNS injury or infection is a critical event associated with the neurodegeneration observed in many neuroinflammatory related diseases such as AD, PD, and MS [5,6,13,22]. As demonstrated in this study activated microglia can release a wide spectrum of pro-inflammatory mediators including NO and PGE2 . These two pro-inflammatory mediators have been known to cause the progression and pathology of many neurodegenerative disorders [19,20,25]. Interestingly, compound 049 at nanomolar concentrations markedly inhibited the production of NO and PGE2 in LPS-activated microglia. Moreover, these effects were carried out by a concentration-dependent suppression of iNOS and COX-2 mRNA and protein levels. These results suggest that the compound possesses a potent anti-inflammatory property and should be further studied to investigate other aspects of this mechanism as well as its toxicity. Accumulating evidence has demonstrated that medicinal herbs or anti-inflammatory agents that inhibit microglial activation or the production of pro-inflammatory mediators have the ability to attenuate neuronal degeneration [12,15,26]. Recent studies showed the anti-inflammatory effects of C. comosa hexane extract and its two diarylheptanoids, 5-hydroxy-7-(4-hydroxyphenyl)-1phenyl-(1E)-1-heptene and 7-(3,4-dihydroxyphenyl)-5-hydroxy1-phenyl-(1E)-1-heptene [10,21]. Our results support these studies. We have demonstrated that compound 049 significantly inhibits LPS-stimulated NO/PGE2 production and iNOS/COX-2 mRNA and protein expression. These results suggest that compound 049 exerts anti-inflammatory action via inhibition of iNOS and COX-2. However, this inhibition may result from the action of compound 049 at the upstream signaling pathways or directly inhibit the activity of iNOS/COX-2 causing a reduction in the levels of NO/PGE2 or enhances the degradation of iNOS/COX-2 mRNA. However, further studies are necessary to fully understand these mechanisms. There is a considerable amount of evidence suggesting that overproduction of NO by activated microglia is involved in neurodegeneration and its progression, whereas inhibition of NO production contributes to significant neuroprotection [1,3,8,11,14]. NO is a diffusible free radical synthesized from l-arginine by iNOS. NO passively diffuses through the membrane and reacts rapidly with other species containing unpaired electrons such as superoxide anion to generate peroxynitrite which can cause DNA damage, and finally lead to cell death [16]. Several lines of evidence have demonstrated that PGE2 plays an important role in the initiation and progression of many neurodegenerative disorders [2,4,9,24]. PGE2 is converted from arachidonic acid by COX-2. Once synthesized, PGE2 is translocated to the extracellular space and exerts its actions by binding with its receptors. PGE2 can be converted non-enzymatically into PGA2 , PGB2 and other metabolites. Overproduction of PGE2 may contribute to cell damage by perturbing neuronal cell membranes, affecting the activity of ion channels, and altering mitochondrial respiratory activities [16]. Therefore, suppressing the high level of NO/PGE2 production by inhibiting
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iNOS/COX-2 expression or its activity may be potentially beneficial in the treatment of NO/PGE2 -related diseases. To the best of our knowledge, this is the first study to show that compound 049 or (3R) 1,7-diphenyl-(4E,6E)-4,6-heptadien-3ol, a pure compound isolated from the hexane extract of C. comosa, exerts a strong anti-inflammatory property through suppression of the release of pro-inflammatory mediators in LPS-stimulated microglia. Compound 049 significantly inhibited the production of NO and PGE2 in a concentration-dependent manner. This antiinflammatory activity occurs via the down-regulation of iNOS and COX-2 mRNA transcription level and protein translational level. These data suggest that compound 049 is a novel anti-inflammatory agent for the treatment of many neurodegenerative disorders in which microglial activation is the critical step in their pathogenesis. Moreover, compound 049 has the unique property of mediating the action on both iNOS and COX-2, which are known to be the major targets of most anti-inflammatory agents, suggesting that compound 049 may have an application as a novel neuroprotective agent. Acknowledgements The authors wish to thank Professor Apichart Suksamrarn for providing the pure compound of plant extract. This study was partially supported by the Commission on Higher Education, Ministry of Education, Thailand and a TRF-Senior Research Scholar Fellowship. References [1] G.C. Brown, Mechanisms of inflammatory neurodegeneration: iNOS and NADPH oxidase, Biochem. Soc. Trans. 35 (2007) 1119–1121. [2] E. Candelario-Jalil, A. González-Falcón, M. García-Cabrera, O.S. León, B.L. Fiebich, Wide therapeutic time window for nimesulide neuroprotection in a model of transient focal cerebral ischemia in the rat, Brain Res. 1007 (2004) 98–108. [3] P.E. Chabrier, C. Demerle-Pallardy, M. Auguet, Nitric oxide synthases: targets for therapeutic strategies in neurological diseases, Cell. Mol. Life Sci. 55 (1999) 1029–1035. [4] C. Consilvio, A.M. Vincent, E.L. Feldman, Neuroinflammation, COX-2, and ALS—a dual role? Exp. Neurol. 187 (2004) 1–10. [5] P.D. Drew, P.D. Storer, J. Xu, J.A. Chavis, Hormone regulation of microglial cell activation: relevance to multiple sclerosis, Brain Res. Rev. 48 (2005) 322–327. [6] F. González-Scarano, G. Baltuch, Microglia as mediators of inflammatory and degenerative diseases, Annu. Rev. Neurosci. 22 (1999) 219–240. [7] U.K. Hanisch, Microglia as a source and target of cytokines, Glia 40 (2002) 140–155.
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