Inhibition of lipopolysaccharide-induced expression of inducible nitric oxide synthase by butein in RAW 264.7 cells

Butein has been reported to exert anti-inflammatory effect but the possible mechanism involved is still unclear. Here, we report the inhibitory effect of butein on nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) gene expression. Butein also inhibited the induction of tumor necrosis factor-alpha and cyclooxygenase 2 by LPS. To further investigate the mechanism responsible for the inhibition of iNOS gene expression by butein, we examined the effect of butein on LPS-induced nuclear factor-kappaB (NF-kappaB) activation. The LPS-induced DNA binding activity of NF-kappaB was significantly inhibited by butein, and this effect was mediated through inhibition of the degradation of inhibitory factor-kappaB and phosphorylation of Erk1/2 MAP kinase. Furthermore, increased binding of the osteopontin alphavbeta3 integrin receptor by butein may explain its inhibitory effect on LPS-mediated NO production. Taken together, these results suggest that butein inhibits iNOS gene expression, providing possible mechanisms for its anti-inflammatory action.     

Differential regulation of NO availability from macrophages and endothelial cells by the garlic component S-allyl cysteine

Garlic has been used as a traditional medicine for prevention and treatment of cardiovascular diseases. However, the molecular mechanism of garlic's pharmacological action has not been clearly elucidated. We examined here the effect of garlic extract and its major component, S-allyl cysteine (SAC), on nitric oxide (NO) production by macrophages and endothelial cells. The present study demonstrates that these reagents inhibited NO production through the suppression of iNOS mRNA and protein expression in the murine macrophage cell line RAW264.7, which had been stimulated with LPS and IFNgamma. The garlic extract also inhibited NO production in peritoneal macrophages, rat hepatocytes, and rat aortic smooth muscle cells stimulated with LPS plus cytokines, but it did not inhibit NO production in iNOS-transfected AKN-1 cells or iNOS enzyme activity. These reagents suppressed NF-kappaB activation and murine iNOS promoter activity in LPS and IFNgamma-stimulated RAW264.7 cells. In contrast, these reagents significantly increased cGMP production by eNOS in HUVEC without changes in activity, protein levels, and cellular distribution of eNOS. Finally, garlic extract and SAC both suppressed the production of hydroxyl radical, confirming their antioxidant activity. These data demonstrate that garlic extract and SAC, due to their antioxidant activity, differentially regulate NO production by inhibiting iNOS expression in macrophages while increasing NO in endothelial cells. Thus, this selective regulation may contribute to the anti-inflammatory effect and prevention of atherosclerosis by these reagents.     

Acetylbritannilatone suppresses NO and PGE2 synthesis in RAW 264.7 macrophages through the inhibition of iNOS and COX-2 gene expression

In order to elucidate the mechanism of anti-inflammatory effect of 1-o-acetylbritannilatone (ABL) isolated from Inula Britannica-F, we investigated ABL for its ability to inhibit the inflammatory factor production in RAW 264.7 macrophages. The studies showed that ABL not only inhibited LPS/IFN-gamma-mediated nitric oxide (NO) production and inducible nitric synthase (iNOS) expression, but also decreased LPS/IFN-gamma-induced prostaglandin E2 (PGE2) production and cyclo-oxygenase-2 (COX-2) expression in a concentration-dependent manner. EMSA demonstrated that ABL inhibited effectively the association of NF-kappaB, which is necessary for the expression of iNOS and COX-2, with its binding motif in the promoter of target genes. These data suggest that ABL suppress NO and PGE2 synthesis in RAW 264.7 macrophages through the inhibition of iNOS and COX-2 gene expression, respectively. The anti-inflammatory effect of ABL involves blocking the binding of NF-kappaB to the promoter in the target genes and inhibiting the expression of iNOS and COX-2.     

Interleukin 4 induces the expression of inducible nitric oxide synthase in eosinophils

We investigated the effects of the Th2-like cytokines IL-4 and IL-13 and of IL-10 on the induction of iNOS and NO production in rat eosinophils. Addition of mIL-4 to the eosinophil culture increased iNOS activity and nitrite production but did not improve the stimulatory effect of IFN-gamma and LPS. In contrast to eosinophils, addition of mIL-4 to macrophage cultures inhibited the iNOS expression and nitrite production induced by IFN-gamma plus LPS. Addition of mIL-13 to the eosinophil cultures did not significantly change iNOS activity and nitrite production in cells stimulated or not with IFN-gamma plus LPS. On the other hand, IL-13 inhibited iNOS activity in IFN-gamma plus LPS-stimulated macrophages. In the presence of IL-10, iNOS activity in non-stimulated eosinophil or macrophage cultures was not significantly altered, but the enzyme expression was inhibited in IFN-gamma plus LPS-stimulated eosinophils or macrophages. The production of nitrite by eosinophils stimulated by IFN-gamma plus LPS was inhibited by the presence of IL-10 in the medium. In conclusion, eosinophils might exhibit differential modulation of the L-arginine/iNOS pathway depending on the profile of Th2 cytokines produced during allergic diseases. IL-4 appears to be an important Th2 cytokine involved in the induction of the L-arginine/iNOS pathway in eosinophils.     

Activation of phosphoinositide 3-kinase in response to inflammation and nitric oxide leads to the up-regulation of cyclooxygenase-2 expression and subsequent cell proliferation in mesangial cells

In this study, we showed that nitric oxide (NO) donors induced the mesangial cell proliferation and cyclooxygenase-2 (COX-2) protein expression in murine mesangial cells. An inflammatory condition [lipopolysaccharide (LPS) plus interferon-gamma (IFN-gamma)] could also induce cell proliferation and significantly enhance inducible nitric oxide synthase (iNOS) and COX-2 expression. Phosphoinositide 3-kinase (PI3K) inhibitor, LY294002, inhibited these responses. LPS/IFN-gamma-induced COX-2 expression in mesangial cells could be inhibited by iNOS inhibitor, aminoguanidine. Selective COX-2 inhibitor, NS398, was capable of inhibiting NO donor- or LPS/IFN-gamma-induced mesangial cell proliferation. Both NO donor and LPS/IFN-gamma markedly activated the PI3K activity and the phosphorylation of Akt and nuclear factor (NF)-kappaB DNA binding activity in mesangial cells, which could be inhibited by LY294002 and transfection of dominant-negative vectors of PI3K/p85 and Akt. These results indicate that a PI3K/Akt-dependent pathway involved in the NO-regulated COX-2 expression and cell proliferation in mesangial cells under inflammatory condition.     

The effect of analgesic drugs on the instillation/abortion time of hypertonic saline induced mid-trimester abortion

Three drugs, a non-steroid anti-inflammatory drug sodium salicylate, an analgesic propoxyphene hydrochloride and an antipyretic-analgesic acetaminophen were administered orally to patients undergoing mid-trimester hypertonic saline induced abortion to determine analgesic potency and the effect on the instillation/abortion time interval (I/ATI). Sodium salicylate (structurally related to acetylsalicylic acid, aspirin) and propoxyphene hydrochloride did not prolong the I/ATI. Acetaminophen, reportedly free of anti-inflammatory activity, prolonged the I/ATI. Clinically, acetaminophen and propoxyphene appeared to provide better analgesia than sodium salicylate. The hypothesized relationship of the three drugs to suppression of prostaglandin biosynthesis is presented.     

Anti-inflammatory activity of c-phycocyanin in lipopolysaccharide-stimulated RAW 264.7 macrophages

C-phycocyanin (C-PC), found in blue green algae, is often used as a dietary nutritional supplement. C-PC has been found to have an anti-inflammatory activity and exert beneficial effect in various diseases. However, little is known about its mechanism of action. Overproduction of nitric oxide (NO) derived from inducible nitric oxide synthase (iNOS) plays an important role in the pathogenesis of inflammation. The aim of this study was to determine whether C-PC inhibits production of nitrite, an index of NO, and iNOS expression in lipopolysaccharide (LPS)-treated RAW 264.7 macrophages. Our results indicated that C-PC significantly inhibited the LPS-induced nitrite production and iNOS protein expression accompanied by an attenuation of tumor necrosis factor-alpha (TNF-alpha) formation but had no effect on interleukin-10 production in macrophages. Furthermore, C-PC also suppressed the activation of nuclear factor-kappaB (NF-kappaB) through preventing degradation of cytosolic IkappaB-alpha in LPS-stimulated RAW 264.7 macrophages. Thus, the inhibitory activity of C-PC on LPS-induced NO release and iNOS expression is probably associated with suppressing TNF-alpha formation and nuclear NF-kappaB activation, which may provide an additional explanation for its anti-inflammatory activity and therapeutic effect.     

The 15-deoxy-delta12,14-prostaglandin J2 inhibits the inflammatory response in primary rat astrocytes via down-regulating multiple steps in phosphatidylinositol 3-kinase-Akt-NF-kappaB-p300 pathway independent of peroxisome proliferator-activated receptor gamma

Ligands for peroxisome proliferator-activated receptor gamma (PPARgamma), such as 15-deoxy-12,14-PGJ2 (15d-PGJ2), have been proposed as a new class of anti-inflammatory compounds because 15d-PGJ2 was able to inhibit the induction of inflammatory response genes such as inducible NO synthase (iNOS) and TNF (TNF-alpha) in a PPAR-dependent manner in various cell types. In primary astrocytes, the anti-inflammatory effects (inhibition of TNF-alpha, IL-1beta, IL-6, and iNOS gene expression) of 15d-PGJ2 are observed to be independent of PPARgamma. Overexpression (wild-type and dominant-negative forms) of PPARgamma and its antagonist (GW9662) did not alter the 15d-PGJ2-induced inhibition of LPS/IFN-gamma-mediated iNOS and NF-kappaB activation. The 15d-PGJ2 inhibited the inflammatory response by inhibiting IkappaB kinase activity, which leads to the inhibition of degradation of IkappaB and nuclear translocation of p65, thereby regulating the NF-kappaB pathway. Moreover, 15d-PGJ2 also inhibited the LPS/IFN-gamma-induced PI3K-Akt pathway. The 15d-PGJ2 inhibited the recruitment of p300 by NF-kappaB (p65) and down-regulated the p300-mediated induction of iNOS and NF-kappaB luciferase reporter activity. Coexpression of constitutive active Akt and PI3K (p110) reversed the 15d-PGJ2-mediated inhibition of p300-induced iNOS and NF-kappaB luciferase activity. This study demonstrates that 15d-PGJ2 suppresses inflammatory response by inhibiting NF-kappaB signaling at multiple steps as well as by inhibiting the PI3K/Akt pathway independent of PPARgamma in primary astrocytes.     

Octamer binding protein-1 is involved in inhibition of inducible nitric oxide synthase expression by exogenous nitric oxide in murine liver cells

Nitric oxide (NO) has diverse effects on immune responses and hepatic functions. In BNL CL.2 cells, the murine embryonic liver cells, inducible nitric oxide synthase (iNOS) mRNA expression appeared after 3 h of treatment with IFN-gamma and LPS. Interestingly, mRNA and protein expression of iNOS was down-regulated by sodium nitroprusside (SNP) and diethylamine dinitric oxide in a time- and dose-dependent manner, but not by H2O2. TNF-alpha gene expression was also dramatically reduced by SNP, but IL-6 gene expression was inhibited much less. IFN-gamma and LPS-induced chloramphenicol acetyltransferase activity of iNOS promoter constructs was inhibited by SNP. Electrophoretic mobility shift assay showed that SNP inhibited IFN-gamma plus LPS-induced Oct-1 binding activity, and the inhibition was reversed by DTT. Mutation in the Oct-1 site completely abolished iNOS promoter activity. In addition, supershift assay and Southwestern analysis demonstrated that the Oct-1 binding activity was inhibited by SNP. Taken together, these results indicate that NO suppresses IFN-gamma plus LPS-induced iNOS expression, and that Oct-1 is an important element in this process.     

Astaxanthin inhibits nitric oxide production and inflammatory gene expression by suppressing I(kappa)B kinase-dependent NF-kappaB activation

Astaxanthin, a carotenoid without vitamin A activity, has shown anti-oxidant and anti-inflammatory activities; however, its molecular action and mechanism have not been elucidated. We examined in vitro and in vivo regulatory function of astaxanthin on production of nitric oxide (NO) and prostaglandin E2 (PGE2) as well as expression of inducible NO synthase (iNOS), cyclooxygenase-2, tumor necrosis factor-alpha (TNF-alpha), and interleukin-1beta (IL-1beta). Astaxanthin inhibited the expression or formation production of these proinflammatory mediators and cytokines in both lipopolysaccharide (LPS)-stimulated RAW264.7 cells and primary macrophages. Astaxanthin also suppressed the serum levels of NO, PGE2, TNF-alpha, and IL-1beta in LPS-administrated mice, and inhibited NF-kappaB activation as well as iNOS promoter activity in RAW264.7 cells stimulated with LPS. This compound directly inhibited the intracellular accumulation of reactive oxygen species in LPS-stimulated RAW264.7 cells as well as H2O2-induced NF-kappaB activation and iNOS expression. Moreover, astaxanthin blocked nuclear translocation of NF-kappaB p65 subunit and I(kappa)B(alpha) degradation, which correlated with its inhibitory effect on I(kappa)B kinase (IKK) activity. These results suggest that astaxanthin, probably due to its antioxidant activity, inhibits the production of inflammatory mediators by blocking NF-kappaB activation and as a consequent suppression of IKK activity and I(kappa)B-alpha degradation.     

8-Hydroxyquinoline inhibits iNOS expression and nitric oxide production by down-regulating LPS-induced activity of NF-kappaB and C/EBPbeta in Raw 264.7 cells

In activated macrophage, large amounts of nitric oxide (NO) are generated by inducible nitric oxide synthase (iNOS), resulting in acute or chronic inflammatory disorders. In Raw 264.7 cells stimulated with lipopolysaccharide (LPS) to mimic inflammation, 8-hydroxyquinoline (8HQ) inhibited the LPS-induced expression of both iNOS protein and mRNA in a parallel dose-dependent manner. 8HQ did not enhance the degradation of iNOS mRNA. To investigate the mechanism by which 8HQ inhibits iNOS gene expression, we examined the activation of MAP kinases in Raw 264.7 cells. We did not observe any significant change in the phosphorylation of MAPKs between LPS alone and LPS plus 8HQ-treated cells. Moreover, 8HQ significantly inhibited the DNA-binding activity of nuclear factor-kappaB (NF-kappaB) and CCAAT/enhancer-binding protein beta (C/EBPbeta), but not activator protein-1 and cAMP response element-binding protein. Taken together, these results suggest that 8HQ acts to inhibit inflammation through inhibition of NO production and iNOS expression through blockade of C/EBPbeta DNA-binding activity and NF-kappaB activation.     

Sesquiterpene lactones inhibit inducible nitric oxide synthase gene expression in cultured rat aortic smooth muscle cells.

Nitric oxide (NO) is an important regulator and effector molecule in various inflammatory disease states. High output of NO during inflammation is generated by the inducible isoform of nitric oxide synthase (iNOS). Sesquiterpene lactones are derived from Mexican-Indian medicinal plants and are known to have potent anti-inflammatory properties. The mechanisms by which sesquiterpene lactones exert their anti-inflammatory effects are not fully understood. In the current studies we determined if the sesquiterpene lactones, parthenolide and isohelenin, modulate iNOS gene expression in cultured rat aortic smooth muscle cells (RASMC) treated with lipopolysaccharide and interferon-gamma. Treatment with parthenolide or isohelenin inhibited NO production and iNOS mRNA expression in a concentration-dependent manner. Transient transfection studies with an iNOS promoter-luciferase reporter plasmid demonstrated that parthenolide and isohelenin also inhibited activation of the iNOS promoter. Inhibition of iNOS promoter activation was associated with inhibition of both I-kappaBalpha degradation and nuclear translocation of NF-kappaB. Neither parthenolide nor isohelenin induced the heat shock response in RASMC. We conclude that sesquiterpene lactones inhibit iNOS gene expression by a mechanism involving stabilization of the I-kappaBalpha/NF-kappaB complex. This effect is not related to induction of the heat shock response. The ability of sesquiterpene lactones to inhibit iNOS gene expression may account, in part, for their anti-inflammatory effects.