Docosahexaenoic acid enhances cyclooxygenase-2 induction by facilitating p44/42, but not p38, mitogen-activated protein kinase activation in rat vascular smooth muscle cells

The effect of docosahexaenoic acid (DHA) on cyclooxygenase expression induced by interleukin (IL)-1beta and phorbol 12-myristate 13-acetate (PMA) in rat vascular smooth muscle cells (VSMCs) was investigated in order to clarify the cellular mechanism of cardiovascular protective effects. DHA and eicosapentaenoic acid slightly enhanced IL-1beta-induced cyclooxygenase (COX)-2, but not COX-1, expression, whereas arachidonic acid had no effect. DHA also slightly enhanced PMA-induced COX-2 expression. DHA stimulated both rapid and prolonged activation of p44/42, but not p38, mitogen-activated protein kinase (MAPK) induced by IL-1beta and PMA. These results suggest that DHA enhances the COX-2 expression by selectively facilitating p44/42 MAPK activation in VSMCs.     

C-reactive protein directly inhibits nitric oxide production by cytokine-stimulated vascular smooth muscle cells

C-reactive protein (CRP) is an important risk factor for atherosclerosis and coronary artery disease. The authors investigated the effects of CRP on nitric oxide (NO) synthesis in vascular smooth muscle cells (VSMCs). Production of nitrite, a stable metabolite of NO, was measured, as was the expression of inducible NO synthase (iNOS) messenger RNA and protein in cultured rat VSMCs. Incubation of cultures with interleukin (IL)-1beta (10 ng/mL) caused a significant increase in nitrite production. C-reactive protein significantly decreased the IL-1beta-induced nitrite production by VSMCs in a dose-dependent manner (10-100 microg/mL). Incubation with IL-1beta induced expression of iNOS mRNA and protein in VSMCs, whereas CRP showed a suppressive effect on their expression. Addition of IL-1beta activated nuclear factor-kappaB (NFkappaB) in VSMCs, whereas CRP did not affect IL-1beta-induced NFkappaB activation. In conclusion, CRP acts on VSMCs and inhibits NO synthesis, which might contribute to the development of atherosclerosis and vascular events.     

The role of Rho-associated kinase in differential regulation by statins of interleukin-1beta- and lipopolysaccharide-mediated nuclear factor kappaB activation and inducible nitric-oxide synthase gene expression in vascular smooth muscle cells

An optimal level of NO has protective effects in atherosclerosis, whereas large amounts contribute to septic shock. To study how statins, the potent inhibitors of cholesterol synthesis, regulate NO in the vascular wall, we determined their effects on interleukin-1beta (IL-1beta)- and lipopolysaccharide (LPS)-induced NO production in aortic vascular smooth muscle cells (VSMCs). Compared with the large amounts of NO and inducible NO synthase (iNOS) protein expression induced by LPS, the responses of IL-1beta were modest. Various statins were found to inhibit LPS-induced iNOS expression and NO production, although they potentiated IL-1beta responses. In addition, fluvastatin increased IL-1beta-induced p65 nuclear translocation and nuclear factor kappaB (NF-kappaB) activity, although it inhibited those induced by LPS. To address the role of small G proteins in statin's actions, farnesyl transferase inhibitors [alpha-hydroxyfarne-sylphosphonic acid and (2S)-2-[[(2S)-2-[(2S,3S)-2-[(2R)-2-amino-3-mercaptopropyl]amino]-3-methylpentyl]oxy]-1-oxo-3-phenylpropyl]amino]-4-(methylsulfonyl)-butanoic acid 1-methylethyl ester (L-744382)], Rac inhibitor (NSC23766), and Rho-associated kinase (ROCK) inhibitor [N-(4-pyridyl)-4-(1-aminoethyl)cyclohexanecarboxamide dihydrochloride (Y-27632)] were used. We found that Y-27632 potentiated IL-1beta-induced iNOS expression, p65 nuclear translocation, IkappaB kinase (IKK), and NF-kappaB activation, whereas it had minimal effects on LPS-induced responses. In contrast, farnesyl transferase inhibitors blocked iNOS protein expression induced by LPS and IL-1beta, whereas NSC23766 had no effect. Further studies showed that LPS down-regulated Rho and ROCK activity, whereas IL-1beta increased them, suggesting a negative role of Rho and ROCK signaling, which is regulated in contrary manners by IL-1beta and LPS, in IKK/NF-kappaB activation. Through abrogating this negative signaling, statins differentially regulate iNOS expression induced by LPS and IL-1beta in VSMCs. These differential actions of statins on iNOS gene regulation might provide an additional explanation for the pleiotropic beneficial effects of statins.     

Pentoxifylline potentiates nitric oxide production in interleukin-1beta-stimulated vascular smooth muscle cells through cyclic AMP-dependent protein kinase A pathway

In the present study, we observed that pentoxifylline (PTX) significantly augmented the nitric oxide (NO) production and the iNOS gene expression by interleukin-1beta (IL-1beta)-stimulated vascular smooth muscle cells (VSMCs). The enhancing effects of PTX on the IL-1beta-induced NO production was associated with an increased intracellular cyclic AMP (cAMP) levels, and the synergistic effects of PTX on the IL-1beta-induced NO production was blocked by cAMP-dependent protein kinase A (PKA) inhibitors. PKA inhibitors, KT5720 and H89, markedly decreased the augmented expression of iNOS gene whereas ODQ, a soluble guanylate cyclase inhibitor, did not affect the enhancing effect. In addition, the pretreatment with KT5720 or H89 abolished the increased translocation of the p65 subunit of NF-kappaB into the nucleus by PTX in the IL-1beta-stimulated VSMCs. These results suggest that enhancing effects of PTX on the iNOS gene expression in the IL-1beta-stimulated VSMCs is mediated predominantly through the activation of NF-kappaB via cAMP-dependent PKA pathway.     

Mitogen-activated protein kinase signalling pathways in IL-1 beta-dependent rat airway smooth muscle proliferation

Asthma is associated with abnormal airway smooth muscle (ASM) growth that may contribute to airway narrowing and hyperresponsiveness. We investigated the role of mitogen-activated protein kinase (MAPK) pathway in IL-1beta induced ASM proliferation in the rat. Rat tracheal ASM cells were dissociated and maintained in culture. We examined the effect of selective MAPK inhibitors, SB239063 (a p38 MAPK inhibitor), U0126 (a mitogen-activated and extracellular regulated kinase kinase, MEK-1, inhibitor which inhibits downstream extracellular regulated kinase, ERK, activity), and SP600125 (a c-jun N-terminal kinase, JNK, inhibitor) on IL-1beta-induced proliferation. Proliferation of ASM cells was significantly increased following exposure to IL-1beta in a dose-dependent manner. p38, JNK and ERK MAPKs were activated by IL-1beta in a time-dependent manner, with peak activation time at 30, 60 min and at 6 h, respectively. This activation was inhibited by their respective inhibitors. SP600125 (20 microM) had no effect on IL-1beta-induced ERK and p38 phosphorylation. SB239063, U0126 and SP600125 dose-dependently inhibited IL-1beta-dependent proliferation at doses that inhibit the activities of p38, ERK and JNK MAPKs, respectively. No additive or synergistic effects were observed on proliferative responses with any combination of these compounds. In conclusion, the three major MAPK pathways, ERK as well as the p38 MAPK and JNK pathways, are independent regulators of IL-1beta-dependent proliferation of rat ASM.     

Modulation of sphingosine 1-phosphate,a new lipid mediator,on nitric oxide production by vascular smooth muscle cells

The effects of sphingosine 1-phosphate (S1P) on nitric oxide (NO) production induced by interleukin (IL)-1 beta in cultured rat vascular smooth muscle cells (VSMCs) have been investigated. Rat VSMCs abundantly expressed mRNA of Edg-3 and Edg-5 receptor subtypes in both unstimulated and IL-1 beta-stimulated cells. S1P at higher than 0.1 microM inhibited the NO production induced by IL-1 beta in a concentration-dependent manner, in which S1P at 10 microM caused over 90% inhibition. S1P also inhibited inducible NO synthase (iNOS) protein and mRNA expressions induced by IL-1 beta in a concentration-dependent manner, which effects were smaller than that in the NO production. S1P also significantly inhibited GTP cyclohydrolase I (GTPCH) mRNA expression induced by IL-1 beta. PTX pretreatment partially prevented the inhibitory effects of S1P on the NO production and iNOS induction, while completely prevented the inhibitory effects on iNOS and GTPCH mRNA expressions. In PTX-pretreated VSMC, S1P showed a bell-shaped enhancing and inhibitory effect on iNOS protein expression. These results suggest that S1P modulates IL-1 beta induction of NO production by rat VSMCs through multiple mechanisms, partially via Edg-3 and Edg-5 receptor subtypes coupled to PTX-sensitive G proteins.     

Protein kinase calpha but not p44/42 mitogen-activated protein kinase, p38, or c-Jun NH(2)-terminal kinase is required for intercellular adhesion molecule-1 expression mediated by interleukin-1beta: involvement of sequential activation of tyrosine kinase, nuclear factor-kappaB-inducing kinase, and IkappaB kinase 2

IL-1beta induced an increase in ICAM-1 expression in human A549 epithelial cells and immunofluorescence staining confirmed this result. Tyrosine kinase inhibitors (genistein or tyrphostin 23) or phosphatidylcholine-specific phospholipase C inhibitor (D609) attenuated IL-1beta-induced ICAM-1 expression. IL-1beta produced an increase in PKC activity and this effect was abolished by D609. PKC inhibitors (staurosporine, Ro 31-8220, calphostin C, or Go 6976) also inhibited IL-1beta-induced response. TPA, a PKC activator, stimulated ICAM-1 expression as well, this effect being inhibited by tyrosine kinase inhibitors. Treatment of cells with IL-1beta resulted in stimulation of p44/42 MAPK, p38, and JNK. However, neither the mitogen activated protein kinase kinase inhibitor PD 98059 nor the p38 inhibitor SB 203580 affected IL-1beta-induced ICAM-1 expression. NF-kappaB DNA-protein binding and ICAM-1 promoter activity were enhanced by IL-1beta and these effects were inhibited by tyrphostin 23, but not by PD 98059 or SB 203580. TPA also stimulated NF-kappaB DNA-protein binding and ICAM-1 promoter activity as well, these effects being inhibited by tyrosine kinase inhibitors. Dominant-negative PKCalpha, NIK, or IKK2, but not IKK1 mutant, inhibited IL-1beta- or TPA-induced ICAM-1 promoter activity. IKK activity was stimulated by either IL-1beta or TPA, and these effects were inhibited by Ro 31-8220 or tyrphostin 23. Taken together, IL-1beta activates phosphatidylcholine-specific phospholipase C and induces activation of PKCalpha and protein tyrosine kinase, resulting in the stimulation of NIK, IKK2, and NF-kappaB in the ICAM-1 promoter, then initiation of ICAM-1 expression. However, activation of p44/42 MAPK, p38, and JNK is not involved.     

Byakangelicol,isolated from Angelica dahurica,inhibits both the activity and induction of cyclooxygenase-2 in human pulmonary epithelial cells

We examined the inhibitory mechanism of byakangelicol, isolated from Angelica dahurica, on interleukin-1beta (IL-1beta)-induced cyclooxygenase-2 (COX-2) expression and prostaglandin E2 (PGE2) release in human pulmonary epithelial cell line (A549). Byakangelicol (10-50 microM) concentration-dependently attenuated IL-1beta-induced COX-2 expression and PGE2 release. The selective COX-2 inhibitor, NS-398 (0.01-1 microM), and byakangelicol (10-50 microM) both concentration-dependently inhibited the activity of the COX-2 enzyme. Byakangelicol, at a concentration up to 200 microM, did not affect the activity and expression of COX-1 enzyme. IL-1beta-induced p44/42 mitogen-activated protein kinase (MAPK) activation was inhibited by the MAPK/extracellular signal-regulated protein kinase (MEK) inhibitor, PD 98059 (30 microM), while byakangelicol (50 microM) had no effect. Treatment of cells with byakangelicol (50 microM) or pyrrolidine dithiocarbamate (PDTC; 50 microM) partially inhibited IL-1beta-induced degradation of IkappaB-alpha in the cytosol, translocation of p65 NF-kappaB from the cytosol to the nucleus and the NF-kappaB-specific DNA-protein complex formation. Taken together, we have demonstrated that byakangelicol inhibits IL-1beta-induced PGE2 release in A549 cells; this inhibition may be mediated by suppression of COX-2 expression and the activity of COX-2 enzyme. The inhibitory mechanism of byakangelicol on IL-1beta-induced COX-2 expression may be, at least in part, through suppression of NF-kappaB activity. Therefore, byakangelicol may have therapeutic potential as an anti-inflammatory drug on airway inflammation.     

Tumour necrosis factor-alpha- and interleukin-1beta-stimulated cell proliferation through activation of mitogen-activated protein kinase in canine tracheal smooth muscle cells

The elevated levels of inflammatory cytokines such as tumour necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) have been found in the fluid of airways in symptomatic asthmatics. These cytokines have been considered as mitogens to stimulate cell proliferation in tracheal smooth muscle cells (TSMCs). We therefore investigated the effects of TNF-alpha and IL-1beta on cell proliferation and activation of p42/p44 mitogen-activated protein kinase (MAPK) in these cells. TNF-alpha and IL-1beta induced [(3)H]-thymidine incorporation in a time- and concentration-dependent manner. The maximal stimulation of [(3)H]-thymidine incorporation induced by TNF-alpha and IL-1beta was seen 12 h after incubation with these cytokines. In response to TNF-alpha and IL-1beta, p42/p44 MAPK was activated with a concentration-dependent manner in TSMCs. Pretreatment of TSMCs with pertussis toxin did not change DNA synthesis and phosphorylation of MAPK induced by TNF-alpha and IL-1beta. These responses were attenuated by a tyrosine kinase inhibitor herbimycin, a phosphatidyl choline (PC)-phospholipase C (PLC) inhibitor D609, a phosphatidyl inositide (PI)-PLC inhibitor U73122, a protein kinase C inhibitor staurosporine, and removal of Ca(2+) by addition of BAPTA/AM plus EGTA. TNF-alpha- and IL-1beta-induced [(3)H]-thymidine incorporation and phosphorylation of p42/p44 MAPK was completely inhibited by PD98059 (an inhibitor of MEK1/2), indicating that activation of MEK1/2 was required for these responses. These results suggest that the mitogenic effects of TNF-alpha and IL-1beta were mediated through the activation of MEK1/2 and p42/p44 MAPK pathway. TNF-alpha- and IL-1beta-mediated responses were modulated by PLC, Ca(2+), PKC, and tyrosine kinase associated with cell proliferation in TSMCs.     

p38 Mitogen-activated protein kinase regulates vasoconstriction in spontaneously hypertensive rats

We investigated whether p42/p44 mitogen-activated protein kinase (MAPK) and/or p38 MAPK participates in the regulation of vascular smooth muscle contraction by endothelin-1 (ET-1) in Wistar-Kyoto rat (WKY) and spontaneously hypertensive rat (SHR). ET-1 (10 nM) induced a sustained contraction in WKY and SHR aortas. PD98059 (100 microM), an inhibitor of p42/p44 MAPK kinase, partially attenuated the ET-1-induced contraction in WKY and SHR. However, SB203580 (10 microM), an inhibitor of p38 MAPK, relaxed the ET-1-induced contraction to the resting levels in SHR, but not in WKY. ET-1 (10 nM) increased phosphorylation of both p42/p44 MAPK and p38 MAPK in WKY and SHR. However, in SHR, p38 MAPK phosphorylation in response to ET-1 stimulation was increased more than in WKY. PD98059 (100 microM) and SB203580 (10 microM) abolished the phosphorylation of p42/p44 MAPK and p38 MAPK in response to ET-1 stimulation in WKY and SHR, respectively. On the other hand, SB203580 (10 microM) did not affect myosin light chain (MLC) phosphorylation in response to ET-1 (10 nM) stimulation in WKY and SHR. From these results, it is concluded that p42/p44 MAPK and/or p38 MAPK partially regulates the ET-1-induced vasoconstriction in WKY. However, p38 MAPK, rather than p42/p44 MAPK, activation plays an important role for the maintenance of ET-1-induced vasoconstriction in SHR through a MLC phosphorylation-independent pathway.     

Mitogenic effect of oxidized low-density lipoprotein on vascular smooth muscle cells mediated by activation of Ras/Raf/MEK/MAPK pathway

1. It has been demonstrated that oxidized low-density lipoprotein (OX-LDL) is a risk factor in atherosclerosis by stimulating vascular smooth muscle cell (VSMC) proliferation. However, the mechanisms of OX-LDL-induced cell proliferation are not completely understood. Therefore, we investigated the effect of OX-LDL on cell proliferation associated with mitogen-activated protein kinase (MAPK) activation in rat cultured VSMCs. 2. Both native-LDL (N-LDL) and OX-LDL induced a time- and concentration-dependent incorporation of [(3)H]-thymidine in VSMCs. 3. OX-LDL induced time- and concentration-dependent phosphorylation of p42/p44 MAPK. Pretreatment of these cells with pertussis toxin or U73122 attenuated the OX-LDL-induced responses. 4. Pretreatment with PMA for 24 h, preincubation with a PKC inhibitor staurosporine or the tyrosine kinase inhibitors, genistein and herbimycin A for 1 h, substantially reduced [(3)H]-thymidine incorporation and p42/p44 MAPK phosphorylation induced by OX-LDL. 5. Removal of Ca(2+) by BAPTA/AM or depletion of the internal Ca(2+) pool by thapsigargin significantly inhibited OX-LDL-induced [(3)H]-thymidine incorporation and p42/p44 MAPK phosphorylation. 6. OX-LDL-induced [(3)H]-thymidine incorporation and p42/p44 MAPK phosphorylation was inhibited by PD98059 (an inhibitor of MEK1/2) and SB203580 (an inhibitor of p38 MAPK) in a concentration-dependent manner. 7. Overexpression of dominant negative mutants of Ras (H-Ras-15A) and Raf (Raf-N4) significantly suppressed MEK1/2 and p42/p44 MAPK activation induced by OX-LDL and PDGF-BB, indicating that Ras and Raf may be required for activation of these kinases. 8. These results suggest that the mitogenic effect of OX-LDL is mediated through a PTX-sensitive G protein-coupled receptor that involves the activation of the Ras/Raf/MEK/MAPK pathway similar to that of PDGF-BB in rat cultured VSMCs.     

Interleukin-1beta expression in murine J774A.1 macrophages exposed to platinum compounds: the role of p38 and ERK 1/2 mitogen-activated protein kinases.

Although skin and respiratory sensitizing properties of platinum compounds have been proved in humans and mice, little is known about signal transduction pathways leading to cytokine production in the induction phase. It is generally assumed that induction of skin sensitization, but not skin irritation, is associated with a rapid increase in the IL-1beta mRNA expression. In this study, IL-1beta expression and a role of mitogen-activated protein kinases (MAPKs) in this process were investigated in murine macrophages J774A.1 exposed to four platinum compounds. Potassium tetrachloroplatinate (K(2)PtCl(4); TCPP), ammonium tetrachloroplatinate ((NH(4))(2)PtCl(4); TCPA), ammonium hexachloroplatinate ((NH(4))(2)PtCl(6); HCPA) showed a very similar range of cytotoxic concentrations (IC(50) values: 238 microM+/-30; 269 microM+/-39 and 245 microM+/-31, respectively) as assessed in the 24-h MTT reduction test. Cytotoxicity of cis-diammineplatinum dichloride (cisplatin) was considerably higher (IC(50) of 23 microM+/-4). While increased expression of IL-1beta mRNA was observed in the macrophages exposed to each test compound, IL-1beta protein production was detected in cell lysates after treatment with TCPP, TCPA and HCPA for 24h (concentration range of 150-350 microM) as well as for 2h (450-650 microM). The treatment with each compound resulted in the phosphorylation of both p38 MAPK and ERK 1/2 (p44/42). Blocking the activation of p38 MAPK as well as ERK 1/2 with specific inhibitors (SB203580 and U0126, respectively) down-regulated the IL-1beta expression. Interestingly, the skin irritant sodium dodecyl sulfate did not trigger phosphorylation of these kinases, nor induced IL-1beta production. These data suggest that p38 MAPK and ERK 1/2 play an important role in induction of IL-1beta expression in J774A.1 macrophages exposed to test platinum compounds.     

Interleukin-1beta induces MUC2 and MUC5AC synthesis through cyclooxygenase-2 in NCI-H292 cells

Interleukin-1beta (IL-1beta) has been implicated in the pathogenesis of inflammatory diseases of the airway. In this study, we investigated the regulation of MUC2 and MUC5AC expression and of their regulatory mechanisms through cyclooxygenase-2 (COX-2) and prostaglandin E(2) (PGE(2)). Cells activated by IL-1beta showed increased COX-2, MUC2, and MUC5AC expressions at both the mRNA and protein levels. Mucin production was blocked by the selective COX-2 inhibitor NS398, and PGE(2) directly induced MUC2 and MUC5AC expression at both the mRNA and protein levels in a dose-dependent manner. These results suggest a role for PGE(2) in IL-1beta-induced mucin synthesis in NCI-H292 cells. To investigate the roles of molecules upstream of COX-2 in mucin regulation, we examined the role of mitogen-activated protein kinases (MAPKs). Cells activated by IL-1beta showed increased extracellular signal-regulated kinase (ERK)1/2 and p38 phosphorylation, and IL-1beta-induced MUC2 and MUC5AC production was blocked by the ERK pathway inhibitor PD98059 or the p38 inhibitor SB203580. The inhibition of both MAPKs reduced IL-1beta-induced COX-2 expression and PGE(2) synthesis. Furthermore, the addition of PGE(2) to cells overcame the inhibitory effects of both MAPK inhibitors in IL-1beta-induced mucin production. These results indicate that in human pulmonary epithelial cells, IL-1beta activates ERK or p38 to induce COX-2 production, which in turn induces MUC2 and MUC5AC production.     

Inhibition of LPS-induced NO and PGE2 production by asiatic acid via NF-kappa B inactivation in RAW 264.7 macrophages: possible involvement of the IKK and MAPK pathways

In the present study, we investigated the effect of asiatic acid (the aglycon of asiaticoside) and asiaticoside isolated from the leaves of Centella asiatica (Umbelliferae) on LPS-induced NO and PGE(2) production in RAW 264.7 macrophage cells. Asiatic acid more potently inhibited LPS-induced NO and PGE(2) production than asiaticoside. Consistent with these observations, the protein and mRNA expression levels of inducible iNOS and COX-2 enzymes were inhibited by asiatic acid in a concentration-dependent manner. In addition, asiatic acid dose-dependently reduced the production of IL-6, IL-1 beta and TNF-alpha in LPS-stimulated RAW 264.7 macrophage cells. Furthermore, asiatic acid inhibited the NF-kappaB activation induced by LPS, and this was associated with the abrogation of I kappa B-alpha degradation and with subsequent decreases in nuclear p65 and p50 protein levels. Moreover, the phosphorylations of IKK, p38, ERK1/2, and JNK in LPS-stimulated RAW 264.7 cells were suppressed by asiatic acid in a dose-dependent manner. These results suggest that the anti-inflammatory properties of asiatic acid might be the results from the inhibition of iNOS, COX-2, IL-6, IL-1 beta, and TNF-alpha expressions through the down-regulation of NF-kappaB activation via suppression of IKK and MAP kinase (p38, ERK1/2, and JNK) phosphorylation in RAW 264.7 cells.     

Eugenolol and glyceryl-isoeugenol suppress LPS-induced iNOS expression by down-regulating NF-kappaB AND AP-1 through inhibition of MAPKS and AKT/IkappaBalpha signaling pathways in macrophages

Eugenol and isoeugenol, two components of clover oil, have been reported to possess several biomedical properties, such as anti-inflammatory, antimicrobial and antioxidant effects. This study aims to examine the anti-inflammatory effects of eugenol, isoeugenol and four of their derivatives on expression of inducible nitric oxide synthase (iNOS) activated by lipopolysaccharide (LPS) in mouse macrophages (RAW 264.7), and to investigate molecular mechanisms underlying these effects. We found that two derivatives, eugenolol and glyceryl-isoeugenol, had potent inhibitory effects on LPS-induced upregulation of nitrite levels, iNOS protein and iNOS mRNA. In addition, they both suppressed the release of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) induced by LPS. Moreover, they both attenuated the DNA binding of NF-kB and AP-1, phosphorylation of inhibitory kB-alpha (IkB-alpha), and nuclear translocation of p65 protein induced by LPS. Finally, we demonstrated that glyceryl-isoeugenol suppressed the phosphorylation of ERK1/2, JNK and p38 MAPK, whereas eugenolol suppressed the phosphorylation of ERK1/2 and p38 MAPK. Taken together, these results suggest that that eugenolol and glyceryl-isoeugenol suppress LPS-induced iNOS expression by down-regulating NF-kB and AP-1 through inhibition of MAPKs and Akt/IkB-alpha signaling pathways. Thus, this study implies that eugenolol and glyceryl-isoeugenol may provide therapeutic benefits for inflammatory diseases.