Carvacrol, a component of thyme oil, activates PPARα and γ and suppresses COX-2 expression[S]

      Cyclooxygenase-2 (COX-2), the rate-limiting enzyme in prostaglandin biosynthesis, plays a key role in inflammation and circulatory homeostasis. Peroxisome proliferator-activated receptors (PPARs) are ligand-dependent transcription factors belonging to the nuclear receptor superfamily and are involved in the control of COX-2 expression, and vice versa. Here, we show that COX-2 promoter activity was suppressed by essential oils derived from thyme, clove, rose, eucalyptus, fennel, and bergamot in cell-based transfection assays using bovine arterial endothelial cells. Moreover, from thyme oil, we identified carvacrol as a major component of the suppressor of COX-2 expression and an activator of PPARα and γ. PPARγ-dependent suppression of COX-2 promoter activity was observed in response to carvacrol treatment. In human macrophage-like U937 cells, carvacrol suppressed lipopolysaccharide-induced COX-2 mRNA and protein expression, suggesting that carvacrol regulates COX-2 expression through its agonistic effect on PPARγ. These results may be important in understanding the antiinflammatory and antilifestyle-related disease properties of carvacrol.
      Cyclooxygenase (COX), the rate-limiting enzyme in prostaglandin (PG) biosynthesis, has two isoforms, COX-1 and -2. COX-1 is constitutively expressed in most cells, whereas COX-2 is typically absent. However, COX-2 is induced by inflammatory stimuli such as endotoxins and lipopolysaccharide (LPS), suggesting that COX-2 plays a role in inflammation (
      • Smith W.L.
      Nutritionally essential fatty acids and biologically indispensable cyclooxygenases.
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      ). Recent studies have shown that COX-2 is involved in not only inflammation but also circulatory homeostasis (
      • Dubois R.N.
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      • Simon L.S.
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      ,
      • Morham S.G.
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      • et al.
      Prostaglandin synthase 2 gene disruption causes severe renal pathology in the mouse.
      ,
      • Grosser T.
      • Fries S.
      • FitzGerald G.A.
      Biological basis for the cardiovascular consequences of COX-2 inhibition: therapeutic challenges and opportunities.
      ).
      The peroxisome proliferator-activated receptors (PPARs) are members of a nuclear receptor family of ligand-dependent transcription factors (
      • Mangelsdorf D.J.
      • Thummel C.
      • Beato M.
      • Herrlich P.
      • Schutz G.
      • Umesono K.
      • Blumberg B.
      • Kastner P.
      • Mark M.
      • Chambon P.
      The nuclear receptor superfamily: the second decade.
      ). The PPAR subfamily comprises three isotypes, PPARα, β/δ, and γ, which play various roles in lipid and carbohydrate metabolism, cell proliferation and differentiation, and inflammation; they are considered molecular targets against lifestyle-related diseases (
      • Michalik L.
      • Auwerx J.
      • Berger J.P.
      • Chatterjee V.K.
      • Glass C.K.
      • Gonzalez F.J.
      • Grimaldi P.A.
      • Kadowaki T.
      • Lazar M.A.
      • O’Rahilly S.
      • et al.
      International Union of Pharmacology. LXI. Peroxisome proliferator-activated receptors.
      ,
      • Sonoda J.
      • Pei L.
      • Evans R.M.
      Nuclear receptors: decoding metabolic disease.
      ). The PGD2 metabolite 15-deoxy-Δ12,14 PGJ2 (15d-PGJ2) was identified as a potent natural ligand of PPARγ (
      • Forman B.M.
      • Tontonoz P.
      • Chen J.
      • Brun R.P.
      • Spiegelman B.M.
      • Evans R.M.
      15-Deoxy-delta 12, 14-prostaglandin J2 is a ligand for the adipocyte determination factor PPAR gamma.
      ,
      • Kliewer S.A.
      • Lenhard J.M.
      • Willson T.M.
      • Patel I.
      • Morris D.C.
      • Lehmann J.M.
      A prostaglandin J2 metabolite binds peroxisome proliferator-activated receptor gamma and promotes adipocyte differentiation.
      ). Previously, we reported that 15d-PGJ2 suppressed LPS-induced expression of COX-2 in differentiated, macrophage-like U937 cells, but not in vascular endothelial cells, and that the expression of COX-2 was regulated by a negative feedback loop mediated through PPARγ, especially in macrophages (
      • Inoue H.
      • Tanabe T.
      • Umesono K.
      Feedback control of cyclooxygenase-2 expression through PPARgamma.
      ). Likewise, the PPARα agonist fenofibrate inhibited interleukin-1-induced COX-2 expression in smooth muscle cells (
      • Staels B.
      • Koenig W.
      • Habib A.
      • Merval R.
      • Lebret M.
      • Torra I.P.
      • Delerive P.
      • Fadel A.
      • Chinetti G.
      • Fruchart J.C.
      • et al.
      Activation of human aortic smooth-muscle cells is inhibited by PPARalpha but not by PPARgamma activators.
      ). These findings indicate that PPARs participate in cell type-specific control of COX-2 expression.
      Resveratrol, a phytoalexin and antioxidant polyphenolic compound found in red wine and various plant products, has long been suspected to have cardioprotective effects and to be a contributor to the so-called “French paradox” (i.e., the relatively low incidence of coronary heart disease in France compared to other developed countries with comparable diets;
      • St. Leger A.S.
      • Cochrane A.L.
      • Moore F.
      Factors associated with cardiac mortality in developed countries with particular reference to the consumption of wine.
      ,
      • Renaud S.
      • De Lorgeril M.
      Wine, alcohol, platelets, and the French paradox for coronary heart disease.
      ,
      • Ferrières J.
      The French paradox: lessons for other countries.
      ). We demonstrated that resveratrol suppressed COX-2 expression in 184B5/HER-transformed mammary epithelial cells (
      • Subbaramaiah K.
      • Chung W.J.
      • Michaluart P.
      • Telang N.
      • Tanabe T.
      • Inoue H.
      • Jang M.
      • Pezzuto J.M.
      • Dannenberg A.J.
      Resveratrol inhibits cyclooxygenase-2 transcription and activity in phorbol ester-treated human mammary epithelial cells.
      ), activated PPARα and γ in cell-based reporter assays using bovine arterial endothelial cells (BAEC), and protected the brain against ischemic stroke in mice through a PPARα-dependent mechanism (
      • Inoue H.
      • Jiang X.
      • Katayama T.
      • Osada S.
      • Umesono K.
      • Namura S.
      Brain protection by resveratrol and fenofibrate against stroke requires peroxisome proliferator-activated receptor alpha in mice.
      ). Similarly, polyphenolic compounds such as apigenin, chrysin (
      • Liang Y.C.
      • Huang Y.T.
      • Tsai S.H.
      • Lin-Shiau S.Y.
      • Chen C.F.
      • Lin J.K.
      Suppression of inducible cyclooxygenase and inducible nitric oxide synthase by apigenin and related flavonoids in mouse macrophages.
      ,
      • Woo K.J.
      • Jeong Y.J.
      • Inoue H.
      • Park J.W.
      • Kwon T.K.
      Chrysin suppresses lipopolysaccharide-induced cyclooxygenase-2 expression through the inhibition of nuclear factor for IL-6 (NF-IL6) DNA-binding activity.
      ), and humulon (
      • Yamamoto K.
      • Wang J.
      • Yamamoto S.
      • Tobe H.
      Suppression of cyclooxygenase-2 gene transcription by humulon of beer hop extract studied with reference to glucocorticoid.
      ) suppress COX-2 expression, and these compounds activate PPARα and/or γ (
      • Liang Y.C.
      • Tsai S.H.
      • Tsai D.C.
      • Lin-Shiau S.Y.
      • Lin J.K.
      Suppression of inducible cyclooxygenase and nitric oxide synthase through activation of peroxisome proliferator-activated receptor-gamma by flavonoids in mouse macrophages.
      ,
      • Yajima H.
      • Ikeshima E.
      • Shiraki M.
      • Kanaya T.
      • Fujiwara D.
      • Odai H.
      • Tsuboyama-Kasaoka N.
      • Ezaki O.
      • Oikawa S.
      • Kondo K.
      Isohumulones, bitter acids derived from hops, activate both peroxisome proliferator-activated receptor alpha and gamma and reduce insulin resistance.
      ).
      The suppression of COX-2 expression and the agonistic activity for PPARs are useful in evaluating the function of food-related components in lifestyle-related diseases. Similar to the case of 15d-PGJ2, it is possible that COX-2 expression is regulated by PPAR agonism exerted by chemical components in food. In this context, we evaluated commercially available lipids derived from various plants and found that six essential oils have COX-2 suppressive activity. Moreover, we identified carvacrol, from the essential oil of thyme, as a major suppressor of COX-2 expression and activator of PPARα. In addition, PPARγ-dependent suppression of COX-2 promoter activity was observed in response to carvacrol, indicating that carvacrol regulates COX-2 expression through its agonistic effect on PPARγ.

      MATERIALS AND METHODS

       Materials

      Bergamot, castor, clove, croton, eucalyptus, fennel, linseed, olive, orange, sesame, soybean, turpentine oils, and chemical components of thyme oils were from Wako Chemicals (Japan). Cottonseed and safflower oils were from MP Biomedicals. Lavender, lemon, and palm oils were from Spectrum Chemicals. Thyme and rose oils were from ICN Biochemicals and Indofine Chemicals, respectively.

       Cell culture

      U937 cells (
      • Inoue H.
      • Nanayama T.
      • Hara S.
      • Yokoyama C.
      • Tanabe T.
      The cyclic AMP response element plays an essential role in the expression of the human prostaglandin-endoperoxide synthase 2 gene in differentiated U937 monocytic cells.
      ) and BAEC (
      • Inoue H.
      • Taba Y.
      • Miwa Y.
      • Yokota C.
      • Miyagi M.
      • Sasaguri T.
      Transcriptional and posttranscriptional regulation of cyclooxygenase-2 expression by fluid shear stress in vascular endothelial cells.
      ) were grown in RPMI 1640 and DMEM, respectively, supplemented with 10% fetal calf serum and 50 μM 2-mercaptoethanol. For differentiation into monocytes/macrophages, U937 cells were treated with 100 nM 12-O-tetradecanoylphorbol-13-acetate (TPA) and allowed to adhere for 48 h, after which they were fed with TPA-free medium and cultured for 24 h before use (
      • Inoue H.
      • Tanabe T.
      Transcriptional role of the nuclear factor kappa B site in the induction by lipopolysaccharide and suppression by dexamethasone of cyclooxygenase-2 in U937 cells.
      ).

       RNA purification and quantitative RT-coupled real-time PCR

      Total RNA from differentiated U937 cells was isolated using the acid guanidinium thiocyanate procedure and analyzed for gene expression by real-time quantitative RT-PCR (Mx3005, Stratagene). After converting total RNA (5 μg) to cDNA using SuperScript III RT (Invitrogen), quantitative PCR was performed using SYBR green real-time PCR Master Mix-Plus (Toyobo, Japan). The following primer pairs were used for amplification of COX-2 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH): 5′-TGCTGTGGAGCTGTATCCTG-3′ (COX-2 forward), 5′-ACAGCCCTTCACGTTATTGC-3′ (COX-2 reverse), 5′-GGTGAAGGTC GGAGTCAACGGA-3′ (GAPDH forward), and 5′-GAGGGATCTCGCTCCTGGAAGA-3′ (GAPDH reverse). The cycling conditions were 3 min at 95°C, followed by 40 cycles of 95°C for 15 s and 67°C for 30 s. Expression levels of COX-2 mRNA were normalized to those of GAPDH mRNA.

       Western blot analysis

      Cell lysates were subjected to SDS-PAGE in 10% gels. The separated proteins were electroblotted onto polyvinylidene difluoride membrane (Millipore), probed with human COX-2 antisera (IBL, Gunma, Japan) or anti-actin antisera (Santa Cruz), and visualized using an ECL Western blot analysis system (GE Healthcare) according to the manufacturer’s protocol. Chemiluminescence was captured using a cooled CCD Light-Capture camera system (ATTO, Japan) and analyzed using CS Analyzer software version 2.0 (ATTO Densitograph Software, Tokyo, Japan).

       Transcription assays

      In the COX-2 promoter assay, BAEC were transfected using Trans IT-LT-1 (Mirus) (
      • Inoue H.
      • Tanabe T.
      • Umesono K.
      Feedback control of cyclooxygenase-2 expression through PPARgamma.
      ). For each well, 0.2 μg of COX-2 reporter vector phPES2 (−327/+59) (
      • Inoue H.
      • Nanayama T.
      • Hara S.
      • Yokoyama C.
      • Tanabe T.
      The cyclic AMP response element plays an essential role in the expression of the human prostaglandin-endoperoxide synthase 2 gene in differentiated U937 monocytic cells.
      ), human PPARγ1 expression vector pCMX-hPPARg1, and 0.02 μg of pSV-β-gal (Promega) were used for transfection. As a control experiment, expression vector pCDNA3.1-GS (Invitrogen) was used instead of pCMX-hPPARg1. Luciferase and β-galactosidase activities were determined, and luciferase activity was normalized to the β-galactosidase standard in BAEC (
      • Inoue H.
      • Tanabe T.
      • Umesono K.
      Feedback control of cyclooxygenase-2 expression through PPARgamma.
      ). For the PPARα activation assay, BAEC were transfected with 0.15 μg of tk-PPREx3-Luc reporter plasmid (
      • Kliewer S.A.
      • Umesono K.
      • Noonan D.J.
      • Heyman R.A.
      • Evans R.M.
      Convergence of 9-cis retinoic acid and peroxisome proliferator signalling pathways through heterodimer formation of their receptors.
      ), 0.15 μg of human PPARα expression vector pGS-hPPARα (Invitrogen, GeneStorm, clone L02932), and 0.02 μg of pSV-β-gal, as described previously (
      • Inoue H.
      • Jiang X.
      • Katayama T.
      • Osada S.
      • Umesono K.
      • Namura S.
      Brain protection by resveratrol and fenofibrate against stroke requires peroxisome proliferator-activated receptor alpha in mice.
      ). For the PPARβ/δ and γ activation assays, their expression vectors, pCMX-hNUCI (PPAR β/δ) and pCMX-hPPARγ, respectively, were used for the transfection instead of the human PPARα expression vector. pCMX-hNUCI was constructed by Dr. Shiho Osada-Ogawa (Kyoto University).

       Analysis of thyme oil by GC

      The thyme oil was analyzed by GC using a Hewlett Packard 5890 series II plus GC system (now Agilent Technologies, Wilmington, DE). A DB-17 GC column (30 m length × 0.32 mm inner diameter, 0.25 μm film thickness; J and W Scientific, Folsom, CA) was used with He as a carrier gas at a flow of 1 ml/min. Thyme oil (1 ml) was dissolved in ethyl alcohol (10 ml), and 1 μl of this solution was injected. The split flow was adjusted at 50 ml/min. The oven temperature was kept at 50°C for 2 min and increased to 280°C at a rate of 5°C/min. The injector temperature was 240°C. The percentage compositions were obtained from electronic integration measurements, using flame ionization detection (FID; 240°C). The relative FID-area percentages of the characterized components are shown in Fig. 5.
      Figure thumbnail gr5
      Fig. 5Major components of thyme oil and their structures. Thyme oil was analyzed by GC and GC-MS. Eleven major compounds, representing 96.80% (FID area percentage for GC) of the oil, were identified (A). The structures of some of the components and thymol are presented (B).

       Analysis of thyme oil by GC-MS

      Thyme oil was also analyzed by high-resolution MS using a JMS BU-20 high-resolution mass spectrometer (JEOL, Tokyo, Japan) attached to a Hewlett Packard 5890 series II plus GC system. The GC conditions were the same as above. Mass spectra were taken at 70 eV under positive electron impact ionization. The mass range was m/z 50–500. The components of thyme oil were identified by mass spectrum library searches conducted using the NIST/EPA/NIH Mass Spectral Search Program, Windows version 1.50, distributed by the Standard Data Reference Program of the US National Institute for Standards and Technology.

      RESULTS

       Suppression of LPS-induced COX-2 promoter activity in the presence of PPARgamma by essential oils

      The human COX-2 promoter region (−327/+59) contains nuclear factor-κB, nuclear factor for interleukin 6 expression, and cAMP response element sites (
      • Inoue H.
      • Yokoyama C.
      • Hara S.
      • Tone Y.
      • Tanabe T.
      Transcriptional regulation of human prostaglandin-endoperoxide synthase-2 gene by lipopolysaccharide and phorbol ester in vascular endothelial cells. Involvement of both nuclear factor for interleukin-6 expression site and cAMP response element.
      ). Previously, transient transfection assays using the COX-2 reporter vector phPES2 (−327/+59) and expression vector pCMX-hPPARg1 showed that BAEC acquired suppressive regulation of the COX-2 gene by 15d-PGJ2 (
      • Inoue H.
      • Tanabe T.
      • Umesono K.
      Feedback control of cyclooxygenase-2 expression through PPARgamma.
      ), indicating the involvement of PPARγ in the regulation of COX-2 expression by 15d-PGJ2. With this assay, we evaluated commercially available oils derived from 21 kinds of plants (supplementary Table I), each at 0.01% concentration, and found that LPS-induced COX-2 promoter activity was suppressed by thyme (65%), clove (40%), rose (30%), eucalyptus (25%), fennel (22%), and bergamot oils (21%), in descending order of activity (Fig. 1). Notably, these oils are essential oils, whereas no suppression of COX-2 promoter activity was found with castor, corn, cottonseed, fusel, lavender, lemon, linseed, olive, orange, palm, safflower, sesame, soybean, or turpentine oils (supplementary Table I). Thyme oil was the strongest suppressor among these essential oils and showed dose-dependent suppression of LPS-induced COX-2 promoter activity (Fig. 2A). Previously, we reported that 100 nM TPA induced COX-2 promoter activity in the reporter assay (
      • Inoue H.
      • Yokoyama C.
      • Hara S.
      • Tone Y.
      • Tanabe T.
      Transcriptional regulation of human prostaglandin-endoperoxide synthase-2 gene by lipopolysaccharide and phorbol ester in vascular endothelial cells. Involvement of both nuclear factor for interleukin-6 expression site and cAMP response element.
      ). As shown in Fig. 1, croton oil containing TPA induced COX-2 promoter activity (264%), confirming that this assay is useful for screening natural chemicals from various plants. TPA is known to be a potent protein kinase C (PKC) activator (
      • Castagna M.
      • Takai Y.
      • Kaibuchi K.
      • Sano K.
      • Kikkawa U.
      • Nishizuka Y.
      Direct activation of calcium-activated, phospholipid-dependent protein kinase by tumor-promoting phorbol esters.
      ). Previously, we also reported that combined treatment with TPA and LPS synergistically induced COX-2 promoter activity (
      • Inoue H.
      • Yokoyama C.
      • Hara S.
      • Tone Y.
      • Tanabe T.
      Transcriptional regulation of human prostaglandin-endoperoxide synthase-2 gene by lipopolysaccharide and phorbol ester in vascular endothelial cells. Involvement of both nuclear factor for interleukin-6 expression site and cAMP response element.
      ), indicating that activation of PKC is involved in inducing COX-2 promoter activity in a manner independent of LPS treatment. Chelerythrine, a selective inhibitor of PKC (
      • Herbert J.M.
      • Augereau J.M.
      • Gleye J.
      • Maffrand J.P.
      Chelerythrine is a potent and specific inhibitor of protein kinase C.
      ), did not suppress the LPS-induced promoter activity of COX-2 (Fig. 2B), suggesting that the suppression by essential oils such as thyme oil is not via inhibition of PKC.
      Figure thumbnail gr1
      Fig. 1Effects on COX-2 promoter activity by essential oils in BAEC. BAEC were transiently transfected with phPES2 (−327/+59) together with pCMX-hPPARg1 and pSV-β-gal as an internal control for the transfection. Following transfection, the cells were incubated for 5 h with no stimulant (control) or with each oil (0.01%) and LPS (1 μg/ml). The cells were harvested, lysed, and assayed for both luciferase and β-galactosidase activities. The results are represented as relative luciferase activities, which were normalized against the β-galactosidase standard and presented as the mean ± SD. *, P < 0.05, **, P < 0.01, compared with cells treated with 1 μg/ml LPS, by an unpaired t-test (n = 3).
      Figure thumbnail gr2
      Fig. 2Dose-dependent suppression of COX-2 promoter activity by thyme oil and the effects of chelerythrine on promoter activity. BAEC were transiently transfected with phPES2 (−327/+59), together with pCMX-hPPARg1, and pSV-β-gal as an internal control for the transfection. Following transfection, the cells were incubated for 5 h with thyme oil (0.002, 0.004, and 0.008%) and LPS (1 μg/ml) (A). In a second experiment, following transfection, the cells were incubated for 5 h with no stimulant (control) or were treated with LPS (1 μg/ml) in the presence or absence with chelerythrine (B). In both cases, the cells were harvested, lysed, and assayed for both luciferase and β-galactosidase activities. The results are represented as relative luciferase activities, which were normalized against the β-galactosidase standard and presented as the mean ± SD. *, P < 0.05, **, P < 0.01, compared with cells treated with 1 μg/ml LPS, by an unpaired t-test (n = 3).

       Activation of PPARs by essential oils

      A natural ligand for PPARγ, 15d-PGJ2, suppressed the LPS-induced COX-2 mRNA expression in macrophage-like differentiated U937 cells (
      • Inoue H.
      • Tanabe T.
      • Umesono K.
      Feedback control of cyclooxygenase-2 expression through PPARgamma.
      ). Resveratrol suppressed COX-2 mRNA expression (
      • Subbaramaiah K.
      • Chung W.J.
      • Michaluart P.
      • Telang N.
      • Tanabe T.
      • Inoue H.
      • Jang M.
      • Pezzuto J.M.
      • Dannenberg A.J.
      Resveratrol inhibits cyclooxygenase-2 transcription and activity in phorbol ester-treated human mammary epithelial cells.
      ) and also activated PPARα and γ in our cell-based assay using BAEC (
      • Inoue H.
      • Jiang X.
      • Katayama T.
      • Osada S.
      • Umesono K.
      • Namura S.
      Brain protection by resveratrol and fenofibrate against stroke requires peroxisome proliferator-activated receptor alpha in mice.
      ). Thus, using this assay system, we evaluated the activation of PPARs by the essential oils that suppressed LPS-induced COX-2 promoter activity (Fig. 1). Figure 3A shows that thyme, rose, clove, and bergamot oils had PPARα agonistic activity and that thyme oil also had PPARγ agonistic activity. In the positive control experiment, selective activators of PPARs, such as Wy-14643 (α), GW501516 (β/δ), and pioglitazone (γ), had PPARα, β/δ, and γ agonistic activities, respectively (Fig. 3B). Remarkably, thyme oil activated PPARα and γ with about the same dose dependency (Fig. 4), within the range for suppression of the LPS-induced COX-2 promoter activity was observed (Fig. 2A). As a control experiment, pioglitazone suppressed LPS-induced COX-2 promoter activity in BAEC in the presence of the vector encoding PPARγ (supplementary ). In contrast, we previously found that LPS rapidly downregulates PPARγ expression in U937 cells (
      • Inoue H.
      • Tanabe T.
      • Umesono K.
      Feedback control of cyclooxygenase-2 expression through PPARgamma.
      ). By this mechanism, the suppressive effect of the PPARγ agonist 15d-PGJ2 on COX-2 expression is less potent than that of dexamethasone, an agonist of the glucocorticoid receptor, which is upregulated by LPS (
      • Inoue H.
      • Tanabe T.
      • Umesono K.
      Feedback control of cyclooxygenase-2 expression through PPARgamma.
      ). We also examined the effects of LPS on the expression of PPARs and COX-2 in U937 cells and found that PPARγ mRNA alone was rapidly suppressed in response to LPS (supplementary ).
      Figure thumbnail gr3
      Fig. 3Effect on activation of PPARs by essential oils. BAEC were transiently transfected with PPRE-luc together with GS-hPPARα (shaded column), pCMX-NUC1 (open column), or pCMX-hPPARg1 (solid column) and pSV-β-gal as an internal control for the transfection. Following transfection, the cells were incubated for 24 h with each essential oil (0.01%) (A). In the positive control experiment, the indicated concentration of selective PPAR activators such as Wy-14643 (α), GW501516 (β/δ), and pioglitazone (γ) were applied to the BAEC transiently transfected with PPRE-luc together with GS-hPPARα, pCMX-NUC1, or pCMX-hPPARg1, respectively (B). In both experiments, the cells were harvested, lysed, and assayed for both luciferase and β-galactosidase activities. The results are represented as relative luciferase activities, which were normalized against the β-galactosidase standard and presented as the mean ± SD. *, P < 0.05, **, P < 0.01, compared with cells treated with ethanol (control), by an unpaired t-test (n = 3).
      Figure thumbnail gr4
      Fig. 4Dose-dependent activation of PPARα and PPARγ by thyme oil. BAEC were transiently transfected with PPRE-luc together with GS-hPPARα (A) or pCMX-hPPARg1 (B) and pSV-β-gal as an internal control for the transfection. Following transfection, the cells were incubated for 24 h with thyme oil (0.002, 0.004, and 0.008%). The cells were harvested, lysed, and assayed for both luciferase and β-galactosidase activities. The results are represented as relative luciferase activities, which were normalized against the β-galactosidase standard and presented as the mean ± SD. *, P < 0.05, **, P < 0.01, compared with cells treated with ethanol (control), by an unpaired t-test (n = 3).

       Carvacrol, the major component involved in the activation of PPARalpha and gamma, suppresses LPS-induced COX-2 mRNA and protein expression

      We screened for active chemicals in thyme oil that were involved in the suppression of LPS-induced COX-2 promoter activity and activation of PPARα and γ. Thyme oil was analyzed by GC and GC-MS. Eleven major compounds, representing 96.80% (FID-area percentage for GC) of the oil, were identified; the main constituents of the oil were carvacrol (58.29%), p-cymene (24.15%), linalyl acetate (4.07%), γ-terpinene (2.39%), β-caryophylene (2.26%), β-myrcene (2.15%), limonene (1.11%), 2-carene (1.05%), and borneol (0.60%; Fig. 5). Next, commercially available compounds were examined for their effects on LPS-induced COX-2 promoter activity at a concentration of 1 mM; carvacrol and borneol suppressed promoter activity by 80% and 55%, respectively (Fig. 6). As shown in Fig. 2A, COX-2 promoter activity was suppressed to 48% by 0.008% thyme oil, which contained about 300 μM carvacrol and 3 μM borneol (Fig. 5). Figure 7 shows the dose-dependent suppression of COX-2 promoter activity by carvacrol (open columns: 40% and 60% suppression by 200 and 400 μM carvacrol, respectively), indicating that carvacrol is the major component of thyme oil involved in the suppression of the LPS-induced COX-2 promoter activity. On the other hand, COX-2 promoter activity was only marginally suppressed in the absence of PPARγ expression vector (closed column); however, this suppression was statistically significant in the presence of the PPARγ expression vector (open column) in BAEC (Fig. 7), indicating that carvacrol-mediated suppression was partly PPARγ dependent. In the control experiments, carvacrol alone had no effect on COX-2 promoter activity in the transfection assay using BAEC and no suppressive effect on COX-2 mRNA in U937 cells (supplementary Table II). Next, we evaluated carvacrol-mediated PPAR activation. Carvacrol activated PPARα (Fig. 8A) and γ (Fig. 8B) at the same concentrations at which the suppression of COX-2 promoter activity was observed (Fig. 7). These carvacrol concentrations are similar to those at which PPARα and γ activities were observed in response to thyme oil (Fig. 4). Moreover, 400 μM carvacrol induced the mRNA expression of the PPARα-dependent carnitine palmitoyltransferase I gene in U937 cells (supplementary ). These results indicate that carvacrol is both a suppressor of COX-2 promoter activity and an activator of PPARα and γ.
      Figure thumbnail gr6
      Fig. 6Effects on COX-2 promoter activity by components of thyme oil and thymol. BAEC were transiently transfected with phPES2 (−327/+59) together with pCMX-hPPARg1 and pSV-β-gal as an internal control for the transfection. Following transfection, the cells were incubated for 5 h with no stimulant (control), with LPS alone (1 μg/ml), or with LPS (1 μg/ml) and each component (1 mM). The cells were harvested, lysed, and assayed for both luciferase and β-galactosidase activities. The results are represented as relative luciferase activities, which were normalized against the β-galactosidase standard. *, P < 0.05, **, P < 0.01, compared with cells treated with 1 μg/ml LPS alone, by an unpaired t-test (n = 3).
      Figure thumbnail gr7
      Fig. 7PPARγ-dependent suppression of COX-2 promoter activity in response to carvacrol. BAEC were transiently transfected with phPES2 (−327/+59), along with either the human PPARγ expression vector, pCMX-hPPARg1 (open column), or the expression vector pCDNA3.1-GS alone (closed column) and pSV-β-gal as an internal control for the transfection. Following transfection, the cells were incubated for 5 h with no stimulant (control) or with carvacrol (100, 200, or 400 μM) and LPS (1 μg/ml). The cells were harvested, lysed, and assayed for both luciferase and β-galactosidase activities. The results are represented as relative luciferase activities, which were normalized against the β-galactosidase standard. *, P < 0.05, compared with open and closed columns, by an unpaired t-test (n = 3).
      Figure thumbnail gr8
      Fig. 8Activation of PPARα and PPARγ in response to carvacrol and thymol. BAEC were transiently transfected with PPRE-luc together with GS-hPPARα (A, C, E), or pCMX-hPPARg1 (B, D, F), and pSV-β-gal as an internal control for the transfection. Following transfection, the cells were incubated for 24 h with carvacrol, p-cymene, or thymol (100, 200, or 400 μM). The cells were harvested, lysed, and assayed for both luciferase and β-galactosidase activities. The results are represented as relative luciferase activities, which were normalized to the β-galactosidase standard. *, P < 0.05, **, P < 0.01, compared with cells treated with ethanol (control), by an unpaired t-test (n = 3).
      In BAEC, dehydroxyl-carvacrol, p-cymene, did not suppress LPS-induced COX-2 promoter activity, whereas thymol, a structural isomer of carvacrol, did (Fig. 6). Similarly, thymol but not p-cymene, activated PPARα and γ (Fig. 8C–F). In addition, 400 μM carvacrol and thymol, but not p-cymene, weakly activated PPARβ/δ (supplementary ). These results suggest that the hydroxyl group of carvacrol is essential for both the suppression of COX-2 promoter activity and the activation of PPARα and γ.
      Next, we examined whether carvacrol suppressed COX-2 mRNA and protein expression in differentiated macrophage-like U937 cells. LPS-induced COX-2 mRNA and protein expression was suppressed by carvacrol to about 14% (1,000 μM in Fig. 9) and 40% (400 μM in Fig. 10), respectively. Carvacrol-mediated suppression of LPS-induced COX-2 protein expression (Fig. 10) is consistent with the results of the COX-2 reporter assays (Fig. 7). The LPS-induced expression of COX-2 mRNA was suppressed to about 14% by treatment with 1000 μM carvacrol but was not suppressed in response to 400 μM carvacrol (Fig. 9). This discrepancy may be due, at least in part, to the dual regulation of COX-2 mRNA at the transcriptional and posttranscriptional levels (
      • Herbert J.M.
      • Augereau J.M.
      • Gleye J.
      • Maffrand J.P.
      Chelerythrine is a potent and specific inhibitor of protein kinase C.
      ,
      • Nishimori T.
      • Inoue H.
      • Hirata Y.
      Involvement of the 3’-untranslated region of cyclooxygenase-2 gene in its post-transcriptional regulation through the glucocorticoid receptor.
      ). The 3′-untranslated region of COX-2 mRNA has 17 copies of the Shaw-Kamen sequence (AUUUA), which is involved in the regulated stabilization of COX-2 mRNA by LPS (
      • Nishimori T.
      • Inoue H.
      • Hirata Y.
      Involvement of the 3’-untranslated region of cyclooxygenase-2 gene in its post-transcriptional regulation through the glucocorticoid receptor.
      ). Regarding this point, we found no evidence that carvacrol is involved in the control of COX-2 mRNA stability, based on reporter assays using the 3′-untranslated region of COX-2 (supplementary ). From our data, carvacrol suppressed not only promoter activity in BAEC transfected with hPCMX-hPPARg1 but also LPS-induced COX-2 mRNA and protein expression in U937 cells expressing PPARγ.
      Figure thumbnail gr9
      Fig. 9Suppression of COX-2 mRNA expression by carvacrol in macrophage-like differentiated U937 cells. Macrophage-like differentiated U937 cells were treated for 2 h with 10 μg/ml LPS in the presence or absence of 1 mM carvacrol. Isolated RNA was used for quantitative RT-PCR analysis. The amount of COX-2 mRNA was normalized to that of GAPDH mRNA, and the normalized amount of COX-2 mRNA induced by LPS was taken as 100%. The results represent the means ± SD of three separate dishes. **, P < 0.01, compared with cells treated with 10 μg/ml LPS, by an unpaired t-test (n = 3).
      Figure thumbnail gr10
      Fig. 10Suppression of COX-2 protein expression in response to carvacrol in macrophage-like differentiated U937 cells. Macrophage-like differentiated U937 cells were treated for 5 h with 10 μg/ml LPS in the presence or absence of the indicated concentrations of carvacrol. Cellular lysate proteins (5 μg/lane) were separated by 10% SDS-PAGE and transferred onto a polyvinylidene difluoride membrane. Immunoblots were probed with antibodies specific for COX-2 and actin. The expression level of COX-2 was normalized against that of actin, and the normalized amount of COX-2 protein induced by LPS was taken as 1.0. The results represent the mean ± SD of three separate dishes. *, P < 0.05, **, P < 0.01, compared with cells treated with 10 μg/ml LPS, by an unpaired t-test (n = 3).

      DISCUSSION

      The prevention of lifestyle-related diseases, such as cardiovascular disease, diabetes, and stroke, is of worldwide interest. There have been many studies on the functionality of natural chemicals in food and drink. In this context, resveratrol in red wine is one of the most attractive of the characterized compounds, and experimental information regarding its actions has accumulated (
      • Lastra C.A.
      • Villegas I.
      Resveratrol as an anti-inflammatory and anti-aging agent: mechanisms and clinical implications.
      ,
      • Baur J.A.
      • Sinclair D.A.
      Therapeutic potential of resveratrol: the in vivo evidence.
      ). We have focused on COX-2 (
      • Subbaramaiah K.
      • Chung W.J.
      • Michaluart P.
      • Telang N.
      • Tanabe T.
      • Inoue H.
      • Jang M.
      • Pezzuto J.M.
      • Dannenberg A.J.
      Resveratrol inhibits cyclooxygenase-2 transcription and activity in phorbol ester-treated human mammary epithelial cells.
      ) and PPARs (
      • Inoue H.
      • Jiang X.
      • Katayama T.
      • Osada S.
      • Umesono K.
      • Namura S.
      Brain protection by resveratrol and fenofibrate against stroke requires peroxisome proliferator-activated receptor alpha in mice.
      ) as possible molecular targets of resveratrol in preventing lifestyle-related diseases, while others have studied sirtuins and PGC-1α (
      • Baur J.A.
      • Pearson K.J.
      • Price N.L.
      • Jamieson H.A.
      • Lerin C.
      • Kalra A.
      • Prabhu V.V.
      • Allard J.S.
      • Lopez-Lluch G.
      • Lewis K.
      • et al.
      Resveratrol improves health and survival of mice on a high-calorie diet.
      ,
      • Lagouge M.
      • Argmann C.
      • Gerhart-Hines Z.
      • Meziane H.
      • Lerin C.
      • Daussin F.
      • Messadeq N.
      • Milne J.
      • Lambert P.
      • Elliott P.
      • et al.
      Resveratrol improves mitochondrial function and protects against metabolic disease by activating SIRT1 and PGC-1alpha.
      ). Given that humulon (
      • Yamamoto K.
      • Wang J.
      • Yamamoto S.
      • Tobe H.
      Suppression of cyclooxygenase-2 gene transcription by humulon of beer hop extract studied with reference to glucocorticoid.
      ,
      • Yajima H.
      • Ikeshima E.
      • Shiraki M.
      • Kanaya T.
      • Fujiwara D.
      • Odai H.
      • Tsuboyama-Kasaoka N.
      • Ezaki O.
      • Oikawa S.
      • Kondo K.
      Isohumulones, bitter acids derived from hops, activate both peroxisome proliferator-activated receptor alpha and gamma and reduce insulin resistance.
      ) and apigenin (
      • Liang Y.C.
      • Tsai S.H.
      • Tsai D.C.
      • Lin-Shiau S.Y.
      • Lin J.K.
      Suppression of inducible cyclooxygenase and nitric oxide synthase through activation of peroxisome proliferator-activated receptor-gamma by flavonoids in mouse macrophages.
      ), as well as resveratrol, suppress COX-2 expression and activate PPARs, we screened commercially available oils, including essential oils, for their effects on COX-2 and PPARs and found that carvacrol, a major component of thyme oil, was active. It is noteworthy that carvacrol activates PPARα and γ (Fig. 8A, B) and suppresses LPS-induced COX-2 protein expression (Fig. 10) in the same concentration range. Moreover, PPARγ-dependent suppression of COX-2 promoter activity was observed in response to carvacrol (Fig. 7), which is consistent with our previous finding that 15d-PGJ2, a potent natural ligand of PPARγ, suppressed LPS-induced COX-2 expression in differentiated macrophage-like U937 cells and that the expression of COX-2 was regulated by a negative feedback loop mediated through PPARγ (
      • Inoue H.
      • Tanabe T.
      • Umesono K.
      Feedback control of cyclooxygenase-2 expression through PPARgamma.
      ). Taken together, it is likely that carvacrol regulates COX-2 expression, at least in part, via its agonistic effect on PPARγ.
      Because of the high concentration of carvacrol necessary to suppress COX-2 promoter activity and activate PPARs, we must consider the possible existence of a minor contaminant associated with carvacrol. To address this point, we confirmed our results using another source of carvacrol (100% purity) via both HPLC and NMR analyses; these analyses indicated that a minor contaminant was unlikely. Another point to consider, given the high carvacrol concentration, is the possibility of nonspecific effects on the cell. Concerning the binding pocket of PPARs, the molecular size of carvacrol is smaller than that of other synthetic PPAR agonists, which may be one of the reasons for its relatively weak activation of PPARs. We also observed weak activation of PPARβ/δ with 400 μM carvacrol and thymol, both of which elicited an approximate 2-fold increase in activity (supplementary ). In contrast, carvacrol and thymol elicited 4- and 8-fold increases in PPARα activity, respectively, and 2.5- and 3.5-fold increases in PPARγ activity, suggesting that carvacrol and thymol activate these factors in a somewhat specific manner. Fatty acids are considered to be intrinsic ligands for PPARs at higher concentrations compared with synthetic ligands. In this case, the presence of fatty acid-binding molecules, which include specific binding proteins and nonspecific interacting lipids, both inside and outside of the cells appears to modulate PPAR activation. A similar mechanism may be at work in the activation of PPARs by carvacrol and other chemical components in essential oils. Recently, carvacrol was reported to be an agonist of transient receptor potential (TRP) V3, a thermosensitive ion channel expressed predominantly in the skin and neural tissues (
      • Xu H.
      • Delling M.
      • Jun J.C.
      • Clapham D.E.
      Oregano, thyme and clove-derived flavors and skin sensitizers activate specific TRP channels.
      ,
      • Vogt-Eisele A.K.
      • Weber K.
      • Sherkheli M.A.
      • Vielhaber G.
      • Panten J.
      • Gisselmann G.
      • Hatt H.
      Monoterpenoid agonists of TRPV3.
      ). The EC50 of carvacrol for TRPV3 is 0.49 mM, similar to the effective concentration for COX-2 and PPARs in our study. Moreover, thymol, but not p-cymene, is also an agonist of TRPV3 (EC50 = 0.86 mM), suggesting a structural requirement for a hydroxyl group (Fig. 5), which is also similar to our results on the effects on COX-2 and PPARs (Fig. 6, Fig. 8). Members of TRPV activate upon heating: TRPV1 at 42°C, TRPV2 at 52°C, TRPV3 at 39°C, and TRPV4 at 27−42°C. Further study on the relationships among COX-2, PPARs, and TRPV3 may be helpful.
      We demonstrated that LPS-induced COX-2 promoter activity was suppressed by not only thyme oil but also clove, rose, eucalyptus, fennel, and bergamot oils (Fig. 1). It is noteworthy that statistically significant activations of PPARs were not found with rose or eucalyptus oils, but there was a tendency for PPARβ/δ activation by thyme, clove, rose, eucalyptus, and fennel oils (Fig. 3A). These results may indicate that some natural chemicals in essential oils act as COX-2 suppressors but not PPAR activators, or that some minor oil components act as PPARβ/δ agonists. Further identification of the chemical compounds in these essential oils is under investigation.
      In summary, we identified carvacrol, a chemical component of thyme oil, as a suppressor of COX-2 and activator of PPARα and γ. Although most essential oils such as thyme oil have a wide range of applications in pharmaceutical, medical, and cosmetic fields, their mechanisms of action are not completely understood for many of the described effects, which include antimicrobial, antitumor, antimutagenic, antigenotoxic, analgesic, antiinflammatory, angiogenic, antiparasitic, antiplatelet, antihepatotoxic, and hepatoprotective properties (
      • Baser K.H.
      Biological and pharmacological activities of carvacrol and carvacrol bearing essential oils.
      ). Our results may be important in understanding the antiinflammatory and antilifestyle-related disease properties of carvacrol, although further studies in vivo will be necessary to address the physiological significance of our findings. Our screening system using reporter assays for COX-2 and PPARs may be applicable for studying the functionality of food-related materials.

      Acknowledgments

      The authors thank Dr. Takanobu Matsubara, Haruka Takeuchi, Tomoko Tsukamoto, Kanako Kojima, and Mariko Akita for technical assistance.

      Supplementary Material

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