Carpesium abrotanoides Extract Inhibits Cyclooxygenase-2 Expression Induced by Toll-like Receptor Agonists Do-Won Jeong1,*, Eun-Kyeong Lee1,*, Chung-Ho Lee1, Se Jin Lim1, Gyo-Jeong Gu2, Ji Hun Paek3, Songmun Kim4, Soon Sung Lim3 & Hyung-Sun Youn1,2 1 Department of Biomedical Laboratory Science, College of Medical Sciences, SoonChunHyang University, Chungnam, Asan 336-745, Korea 2 Departments of Medical Science, College of Medical Sciences, SoonChunHyang University, Chungnam, Asan 336-745, Korea 3 Department of Food Science and Nutrition, Hallym University, Chuncheon, Gangwon 200-702, Korea 4 Department of Biological Environment, Kangwon National University, Chuncheon, Gangwon-do 200-701, Korea *These authors contributed equally to this work. Correspondence and requests for materials should be addressed to H.-S. Youn (
[email protected])
Received 1 May 2013 / Received in revised form 6 June 2013 Accepted 10 June 2013 DOI 10.1007/s13530-013-0161-x ©The Korean Society of Environmental Risk Assessment and Health Science and Springer 2013
Abstract Toll-like receptors (TLRs) are a group of pattern-recognition receptors that play an important role in the induction of innate immune responses, recognizing conserved microbial structural molecules. The microbial pathogens trigger the activation of myeloid differential factor 88 (MyD88)- and toll-interleukin-1 receptor domain-containing adapter inducing interferon-β (TRIF)-dependent pathways, leading to proinflammatory cytokine production. Here, we investigated the effect of the ethanol extracts of Carpesium abrotanoides (CAB), a traditional Korean medicine, on the expression of cyclooxygenase-2 (COX-2) by TLR agonists in murine macrophages. CAB suppressed COX-2 expression induced by lipopolysaccharide (TLR4 agonist), polyriboinosinic polyribocytidylic acid (TLR3 agonist), and macrophage-activating lipopeptide 2-kDa (TLR2 and TLR6 agonist). These results suggest that CAB can regulate the TLR signaling pathways to treat chronic inflammatory diseases. Keywords: Carpesium abrotanoides, Toll-like receptor, Nuclear factor-κB, Cyclooxygenase-2, Inflammation
Introduction Toll-like receptors (TLRs) are an essential arm of the innate immune system that respond to specific pathogen associated molecular patterns (PAMPs) to bacteria, viruses, and fungi1,2. TLRs are type I transmembrane proteins, and consist of an ectodomain, which contains leucine-rich repeats that mediate the recognition of PAMPs, a transmembrane domain, and a cytosolic signaling domain called the Toll-interleukin 1 receptor (TIR) domain. Homotypic binding of TIR domains on the TLRs with signaling adaptors activate the downstream signaling pathways. Those adaptor molecules, five of which have been identified to date, are myeloid differential factor 88 (MyD88), MyD88 adaptor-like, toll-interleukin-1 receptor domain-containing adapter inducing interferon-β (TRIF), TRIF-related adaptor molecule (TRAM), and Sterile-alpha and Armadillo motif-containing protein (SARM)3. These adaptor molecules facilitate the activation of the MyD88- and TRIFdependent pathways leading to the activation of nuclear factor-κB (NF-κB), mitogen-activated protein kinases (MAPKs), and interferon (IFN) regulatory factors (IRFs), as well as the expression of inflammatory gene products including cyclooxygenase-2 (COX-2), cytokines, chemokines, and type I interferon (IFN)2. Carpesium abrotanoides L., which is a biennial herb, has been used as a traditional Korean medicines to treat bruises4. The main bioactive constituents of this medicinal plant are diverse sesquiterpene lactones5. A recent study has shown that the extract of C. abrotanoides inhibited the expression of COX-2 and inducible nitric oxide synthase (iNOS) as induced by LPS6. However, the mechanism of C. abrotanoides’s beneficial effects is largely unknown. Many plant origin drugs are used to treat many diseases traditionally. As part of our search for COX-2 inhibitors from natural products, ethanol extracts of C. abrotanoides (CAB) were identified as a novel, potent inhibitor of COX-2 expression induced by TLRs agonists. In the present study, we found that CAB inhibited the expression of COX-2 in RAW264.7 murine macrophage cells.
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Figure 1. CAB suppresses NF-κB activation induced by TLR agonists. A) RAW264.7 cells were treated with CAB (20, 50, 100 μg/mL) for 4 h. Twenty microliters of the CellTiter 96 AQueous One Solution Reagent was added directly to culture wells. The plate was incubated at 37� C for 4 h in a humidified, 5% CO2 atmosphere. The absorbance was recorded at 490 nm with a 96-well plate reader. B-D) RAW264.7 cells were transfected with NF-κB luciferase reporter plasmid and pretreated with 50 or 100 μg/mL CAB for 1 h and was then treated with LPS (10 ng/mL) (B), Poly[I:C] (10 μg/mL) (C), or MALP-2 (10 ng/mL) (D) for an additional 8 h. Cell lysates were prepared and luciferase enzyme activities were determined. Values are expressed as the mean± =3). *, Significantly different from LPS alone, p⁄0.01 (**) (B). +, Significantly different from Poly[I:C] alone, p⁄0.01 SEM (n= (++) (C). #, Significantly different from MALP-2 alone, p⁄0.01 (##) (D). Veh, vehicle; CAB, ethanol extracts of Carpesium abrotanoides.
Results and Discussion κB Activation Induced CAB Suppresses NF-κ by TLR Agonists To evaluate the cytotoxic nature of CAB in RAW 264.7 cells, cytotoxicity was determined using the MTS-based viability assay. It was apparent that CAB was not cytotoxic at the concentrations tested (20-100 μg/mL) (Figure 1A). Therefore, cellular toxicity could be excluded as a possible reason for any observed changes. To identify whether CAB could modulate the TLRmediated signaling pathways, NF-κB activation induced in the presence of several TLR agonists was used as a readout for the activation of TLRs. Appropriately, the effect of CAB on NF-κB activation was determined by the NF-κB luciferase reporter assay. CAB inhibited NF-κB activation induced by LPS (TLR4 agonist) (Figure 1B), Poly[I:C] (TLR3 agonist) (Figure 1C), or MALP-2 (TLR2 and TLR6 agonist) (Figure 1D). NFκB, which is the common downstream signaling component for all TLRs, is a central transcription factor for pro-inflammatory gene expression. Increased activity of NF-κB is known to be associated with an enhanced risk of many chronic diseases such as arthritis, atherosclerosis, cardiovascular disease, Alzheimer’s disease and cancer7. NF-κB is activated in response to stimuli from various pathogens, following the rapid activation of target genes involved in inflammation8.
Therefore, the inhibition of NF-κB activation is considered an important strategy for anti-inflammation.
CAB Suppresses COX-2 Expression Induced by TLR Agonists We further investigated whether CAB could inhibit COX-2 expression induced by TLR agonists. Since COX-2 is greatly expressed in most normal mammalian tissues in response to physical, chemical, and biological stimuli, COX-2 is used as a molecular target to control the symptoms of inflammation and pain9. CAB suppressed the COX-2 expression induced by LPS (Figure 2A), Poly[I:C] (Figure 2B), or MALP-2 (Figure 2C), as determined by the COX-2-luciferase reporter assay. CAB also suppressed the COX-2 expression induced by LPS (Figure 3A), Poly[I:C] (Figure 3B), or MALP-2 (Figure 3C) as determined by the COX-2 Western blotting assay. Our results suggest that CAB inhibited NF-κB activation induced by several TLR agonists, resulting in the inhibition of the expression of target genes such as COX-2. COX is an enzyme that is responsible for the formation of prostanoids from arachidonic acid, and is the target of non-steroidal anti-inflammatory drugs (NSAIDs), which play a therapeutic role in the treatment of pain, fever, and inflammation10. COX consists of at least 2 isoforms, COX-1 and COX-211. COX-1 is widely distributed, being constitutively expressed in nearly all cells and tissues at relatively stable levels,
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Figure 2. CAB suppresses COX-2 expression induced by TLR agonists. A-C) RAW264.7 cells were transfected with COX-2 luciferase reporter plasmid and were pretreated with 50 or 100 μg/mL CAB for 1 h and were then treated with LPS (10 ng/mL) (A), Poly[I:C] (10 μg/mL) (B), or MALP-2 (10 ng/mL) (C) for an additional 8 h. Cell lysates were prepared and luciferase =3). *, Significantly different from LPS alone, enzyme activities were determined. Values are expressed as the mean±SEM (n= p⁄0.01 (**) (A). +, Significantly different from Poly[I:C] alone, p⁄0.01 (++) (B). #, Significantly different from MALP-2 alone, p⁄0.01 (##). (C). Veh, vehicle; CAB, ethanol extracts of Carpesium abrotanoides.
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Figure 3. CAB suppresses COX-2 protein induced by TLR agonists. A-C) RAW264.7 cells were pretreated with 50 or 100 μg/mL CAB for 1 h and were then stimulated with LPS (10 ng/mL) (A), Poly[I:C] (10 μg/mL) (B), or MALP-2 (10 ng/mL) (C) for 8 h. Cell lysates were immunoblot analyzed for COX-2 and β-actin protein. Veh, vehicle; CAB, ethanol extracts of Carpesium abrotanoides.
and is believed to have a number of housekeeping functions, such as the production of prostaglandin (PG) precursors for thromboxane in platelets. In contrast to COX-1, COX-2 is almost undetectable in most tissues, and is induced by various inflammatory and proliferative stimuli, such as ultraviolet light and LPS9. COX2 plays an important role in prostaglandin (PG) formation during pathophysiologic states, such as inflammation and tumorigenesis. Therefore, the inhibition of COX-2 expression is a very important therapeutic target to control inflammation and pain12. The activation of TLRs can trigger the MyD88- and TRIF-dependent pathways13. MyD88 is the immediate adaptor molecule to all TLRs, except for TLR3. MyD88 recruits IL-1 receptor-associated kinases (IRAK4 and IRAK1) and then phosphorylates all these kinases. The phosphorylated IRAK-1 associates with tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6), leading to the activation of the canonical inhibitor-κB
kinase (IKK) complex, followed by the activation of NF-κB14. TRIF is another adaptor molecule for TLR3 and TLR4. TRIF activates the downstream kinases, TRAF family member associated NF-κB activator-binding kinase1 (TBK1) and inducible IKK (IKKi)/IKKepsilon (IKKε), leading to IRF3 activation15 and the receptor interacting protein-1 (RIP1), leading to delayed NF-κB activation16. The activated IRF3 induces IFN-β and IFN inducible genes, such as interferon inducible protein-10 (IP-10), and regulates normal T-cell expression and secretion (RANTES)17-19. In this study, we investigated whether CAB could regulate the expression of COX-2 induced by several TLR agonists. CAB suppressed the expression of COX-2 induced by MALP-2 (TLR2 and TLR6 agonist), Poly [I:C] (TLR3 agonist), or LPS (TLR4 agonist). Whereas TLR2 and TLR6 induced COX-2 expression mediated through the MyD88-dependent
COX-2 Inhibition by C. abrotanoides Extract
pathway, TLR3 induces COX-2 expression mediated through the TRIF-dependent pathway20. However, TLR4 can induce COX-2 expression mediated through both the MyD88- and TRIF-dependent pathways. Taken together, the results suggest that CAB can regulate both MyD88- and TRIF-dependent pathways of TLRs, leading to decreased inflammatory gene expression. Further understanding of the interaction of TLRs and its signaling components may provide important advances in the study of chronic inflammatory diseases. Therefore, our finding suggests that CAB can be developed as a new immune drug to treat chronic inflammatory diseases. In the future, the mechanisms of inhibition of CAB mediated through TLRs signaling pathways will be identified.
Materials and Methods Plant Materials The aerial part of C. abrotanoides L. was collected from Cheonsun Bee Bong Mountain in Gangwon Province, Korea, in August 2011. The botanical identity was confirmed by Emeritus Professor Heung Jun Chi, Department of Pharmacy, Seoul National University, Seoul, South Korea. The voucher specimen (No. RIC0569) was deposited and maintained at the center for efficacy assessment and development of functional foods and drugs (Regional Innovation Center), Hallym University, Chuncheon, Korea. Preparation of the Crude Extract of C. Abrotanoides L. The aerial part of C. abrotanoides (500 g) was mixed with 3 L of 70% ethanol water solution in a 5 L round bottom flask fitted with a cooling condenser, which was used to perform the extraction. The extraction temperature was controlled at 80� C with a water bath to allow ethanol to boil continuously. Extraction was carried out for 4 h. The extracts were combined and concentrated under reduced pressure with a Model EYELA N-1000 rotary evaporator (Tokyo Rikakikai, Tokyo, Japan) and were freeze-dried (yield: 65.8 g, 13.2%). The dried extract was dissolved in dimethyl sulfoxide. Reagents Lipopolysaccharide (LPS) was obtained from List Biological Laboratories (San Jose, CA, USA). Macrophage-activating lipopeptide 2-kDa (MALP-2) was purchased from Alexis Biochemical (San Diego, CA, USA). Polyriboinosinic polyribocytidylic acid (Poly [I:C]) was purchased from Amersham Biosciences (Piscataway, NJ, USA). All other reagents were pur-
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chased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise stated.
Cell Culture Cells of the RAW264.7 murine monocytic cell line (ATCC TIB-71; American Type Culture Collection, Manassas, VA, USA) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% (v/v) fetal bovine serum (FBS), 100 units/mL penicillin, and 100 μg/mL streptomycin. Cells were maintained at 37� C in a 5% CO2/air environment. Cell Viability Test Cell viability was assessed using a 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) based colorimetric assay. Viability tests were performed by adding a small amount of the CellTiter 96 AQueous One Solution Reagent (Promega, Madison, WI, USA) directly to culture wells, incubating for 4 h, and then recording the absorbance at 490 nm with a 96-well plate reader. Transfection and Reporter Gene Luciferase Assay The assays were performed as previously described21,22. Cells were co-transfected with a luciferase plasmid and a plasmid containing heat shock protein, (HSP)70β-galactosidase, as an internal control using SuperFect transfection reagent (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. Luciferase enzyme activities were determined using a commercial Luciferase Assay System (Promega) according to the manufacturer’s instructions. Luciferase activity was normalized by β-galactosidase activity. Western Blotting Western blotting was performed as previously described23. Equal amounts of cell extracts were subjected to 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the separated proteins were electrotransferred to a polyvinylidene difluoride membrane. The membrane was blocked to prevent the nonspecific binding of antibody in phosphate-buffered saline containing 0.1% Tween-20 and 3% nonfat dry milk. Immunoblotting was performed with the indicated antibodies and secondary antibodies conjugated to horseradish peroxidase (Amersham Biosciences, Arlington Heights, IL, USA). The reactive bands were visualized with enhanced chemiluminescence Western blot detection reagents (Intron, Seongnam, Gyeonggi-do, South Korea). Statistical Analysis Data were obtained from triplicate experiments. Val-
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ues are expressed as the mean±standard error of the mean (SEM). Differences in the data were evaluated using Student’s t test. Results were considered statistically significant when p values were p⁄0.05.
Acknowledgements This work was supported by the Soonchunhyang University Research Fund.
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