Carvacrol, a component of thyme oil, activates PPARalpha and gamma and suppresses COX-2 expression

Abstract

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 PPARalpha and gamma. PPARgamma-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 PPARgamma. These results may be important in understanding the antiinflammatory and antilifestyle-related disease properties of carvacrol.

Fig. 1.

Effects 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).

Fig. 2.

Dose-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).

Fig. 3.

Effect 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).

Fig. 4.

Dose-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).

Fig. 5.

Major 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).

Fig. 6.

Effects 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).

Fig. 7.

PPARγ-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).

Fig. 8.

Activation 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).

Fig. 9.

Suppression 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).

Fig. 10.

Suppression 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).