Cyclooxygenase-2 Expression In RAW Research Articles

Remission Effects of Dietary Soybean Isoflavones on DSS-Induced Murine Colitis and an LPS-Activated Macrophage Cell Line.

Inflammatory bowel diseases (IBDs), including ulcerative colitis and Crohn’s disease, are chronic disorders of the gastrointestinal tract, although the exact causes of IBD remain unknown. Present treatments for IBDs have poor tolerability and insufficient therapeutic efficacy, thus, alternative therapeutic approaches are required. Soybean-derived isoflavones have multiple bioactivities such as anti-inflammation. However, the low water solubility of soybean isoflavones limits their bioavailability and practical use. Therefore, in order to study the preventive effects of water-soluble soybean isoflavones on colonic inflammatory status, we examined soybean-derived isoflavone glycosides (SIFs) in a dextran sodium sulfate (DSS)-induced murine colitis model and in lipopolysaccharide (LPS)-activated RAW264.7 macrophages. Oral administration of SIF (0.5 w/v%) attenuated DSS-induced colitis in terms of body weight decrease, colon shortening, epithelial apoptosis, histological score, mRNA levels of inflammatory cytokines, and immune cell infiltration in colon tissues. In the in vitro assessment, we observed the inhibitory effects of SIF on the production of nitric oxide and prostaglandin E2, via suppression of inducible nitric oxide synthase and cyclooxygenase-2 expression in RAW264.7 macrophages in response to LPS. Furthermore, we confirmed that the expression of inflammatory cytokines and chemokines were decreased by pre-treatment with SIF in LPS-activated RAW264.7 macrophages. Moreover, we demonstrated that SIF suppressed inflammatory mediators involved in nuclear factor-κB signaling pathway via inhibitory κB kinase phosphorylation and degradation of inhibitory κB. Our results suggested that SIF may be beneficial for the remission of colonic inflammatory status including IBDs.

Quantitative analysis of the five marker compounds in Mori Cortex Radicis and its anti-inflammatory effects

Mori Cortex Radicis (MCR, Moraceae), which is root bark of Morus alba L., is used traditionally in the treatment of jaundice, hematemesis, edema, and pollakisuria in Korea. In this study, the anti-inflammatory effects of MCR extract and the five components, neochlorogenic acid (1), chlorogenic acid (2), cryptochlorogenic acid (3), caffeic acid (4), and p-coumaric acid (5) were investigated using RAW 264.7 cells. The simultaneous analysis of the compounds 1 – 5 in MCR extract was performed using high-performance liquid chromatography coupled with photodiode array detector. All analytes were separated on a Gemini C18 column and the column oven temperature was set at 40 ° C with the detection wavelength was set at 320nm. The mobile phase was a gradient comprising 1.0% (v/v) aqueous acetic acid (A) and 1.0% (v/v) acetic acid in acetonitrile (B). The flow rate was 1.0 mL/min and the injection volume was 10 mL. We determined the effects of MCR extract and the compounds 1-5 on the production of nitric oxide (NO), prostaglandin E2 (PGE2), and mRNA expression of cyclooxygenase-2 (COX-2) in RAW 264.7 cells. The HPLC method showed good linearity with a correlation coefficient (r2) ≥0.9999 over the concentration range 0.31 – 20.00 mg/mL. The limit of detection and the quantification of the compounds 1-5 were 12.23 – 61.46 ng/mL and 40.76 – 204.87 ng/mL, respectively. The concentration of the compounds 1-5 was 0.16 – 1.40 mg/g. MCR extract suppressed the production of NO and PGE2 in RAW 264.7 cells in a dose-dependent manner. None of the five components of MCR extract had any influence on the production of NO. However, compounds 4 and 5 were inhibited the production of PGE2 and mRNA expression of COX-2 in RAW 264.7 cells. Our results suggest that MCR extract may offer potential as a therapeutic agent for the treatment of inflammation. The method we have established will help to improve the quality control of MCR extracts.

Esculetin suppresses lipopolysaccharide-induced inflammatory mediators and cytokines by inhibiting nuclear factor-κB translocation in RAW 264.7 macrophages.

Although previous studies have demonstrated that the natural coumarin compound esculetin possesses various pharmacological properties, the molecular mechanism of esculetin-mediated anti-inflammatory potential is not fully understood. In this study, we determined the effects of esculetin on lipopolysaccharide (LPS)-induced inflammatory responses of murine RAW 264.7 macrophages. The results indicate that esculetin inhibits LPS-induced nitric oxide and prostaglandin E2 production in a concentration-dependent manner, and inhibits inducible nitric oxide synthase and cyclooxygenase-2 expression in RAW 264.7 cells. Esculetin also significantly suppresses the production of inflammatory cytokines, including tumor necrosis factor-α and interleukin-1β, which was concomitant with a decrease in their expression levels. Furthermore, it was observed that esculetin attenuated the LPS-mediated nuclear factor-kappa B (NF-κB) translocation associated with the blocking of inhibitor of NF-κB (IκB)-α degradation as well as reactive oxygen species (ROS) production, without any significant cytotoxicity. These data suggest that, by blocking NF-κB activation, esculetin suppresses LPS-elicited inflammatory events, and this is mediated, at least in part, by inhibiting the generation of ROS. Collectively, these findings provide mechanistic insights into the anti-inflammatory action of esculetin in macrophages.

HPLC-PDA Analysis and Anti-inflammatory Effects of Mori Cortex Radicis

Mori Cortex Radicis (MCR, Moraceae) is used traditionally in the treatment ofjaundice, hematemesis, edema, and pollakisuria in Korea. In this study, the antiinflammatory effects of MCR extract were investigated using RAW 264.7 cells. The simultaneous analysis of five components present (neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, and p-coumaric acid) in the MCR extract was performed using high-performance liquid chromatography (HPLC) coupled with photodiode array (PDA) detection. We determined the effects of MCR extract and its components on the production of nitric oxide (NO), prostaglandin E2 (PGE2), and mRNA expression of cyclooxygenase-2 (COX-2) in RAW 264.7 cells. MCR extract suppressed the production of NO and PGE2 in RAW 264.7 cells in a dose-dependent manner. None of the five components of the MCR extract had any influence on the production of NO. However, caffeic acid and p-coumaric acid inhibited the production of PGE2 and mRNA expression of COX-2 in RAW 264.7 cells. Our results suggest that MCR extract may offer potential as a therapeutic agent for the treatment of inflammation. The method we have established will help to improve the quality control of MCR extracts.

Taurine exerts anti-osteoclastogenesis activity via inhibiting ROS generation, JNK phosphorylation and COX-2 expression in RAW264.7 cells

Taurine is one of the abundant amino acids present in mammalian cells. It exerts various physiological actions such as wound healing, radioprotection, neuroprotection and anti-anxiety. In the present study, we sought to determine if taurine could inhibit osteoclastogenesis and explore the potential role of cyclooxygenase-2 (COX-2) and Jun N-terminal kinase (JNK) with reactive oxygen species (ROS). The level of intracellular ROS generated by lipopolysaccharide (LPS) was measured with DCFH-DA staining and fluorescence microscopic analysis was also performed in response to taurine in RAW264.7 cells. The expression of COX-2 and phosphorylation status of JNK by LPS was analyzed by Western blot analysis in the presence of taurine. Osteoclastogenesis was induced by LPS in the absence or presence of taurine and TRAP assay was performed to confirm the formation of osteoclast cells. ROS production was significantly enhanced by LPS and taurine treatment inhibited the ROS generation in a dose-dependent manner. The fluorescence microscopic analysis clearly showed the inhibition of ROS staining by taurine. Western blot analysis indicated that taurine significantly inhibited LPS induced COX-2 protein expression and it also inhibited phosphorylation of JNK. Taurine at the same concentration inhibited osteoclastogenesis induced by LPS, suggesting that taurine prevent osteoclast differentiation by inhibiting ROS generation. Inhibition of COX-2 expression and JNK phoshorylation could be an important mechanism by which taurine exerts anti-osteoclastogeneis.

Chemical composition, antioxidant and anti-inflammatory properties for ethanolic extracts from Pleurotus eryngii fruiting bodies harvested at different time

Pleurotus eryngii, a popular edible mushroom in Taiwan, is usually cultivated using sawdust medium packing bags through several procedures including culture medium confection, bagging and sterilization, spawn inoculation, fostering mycelia, full growth of mycelia, and inducing fruiting body formation. In this study, P. eryngii commercial products harvested at the 10th, 12th and 15th days after inducing the fruiting body formation were extracted with ethanol, individually. Through determination of chemical composition, antioxidant and anti-inflammatory properties of these extracts, the optimal harvest time of P. eryngii fruiting bodies with higher functional attributes was revealed. The earlier harvested sample extracts had higher effects for scavenging 2,2-diphenyl-1-picrylhydrazyl radicals, reducing power, chelating power, and β-carotene bleaching inhibition, as well as down-regulating lipopolysaccharide-stimulated nitric oxide, prostaglandin E2, inducible nitric oxide synthase, and cyclooxygenase-2 expression in RAW264.7 macrophages. These functional responses were closely related to levels of phytochemical components including phenolic acids, flavonoids, tocopherols and carotenoids.

Lipopolysaccharide (LPS) promotes osteoclast differentiation and activation by enhancing the MAPK pathway and COX-2 expression in RAW264.7 cells

Bone degradation is a serious complication of chronic inflammatory diseases such as septic arthritis, osteomyelitis and infected orthopedic implant failure. At present, effective therapeutic treatments for lipopolysaccharide (LPS)-induced bone destruction are limited to antibiotics and surgical repair in chronic inflammatory diseases. The present study aimed to evaluate the mechanism of LPS on osteoclast differentiation and activation. RAW264.7 cells were non-induced, or induced by the receptor activator of nuclear factor-κB (RANK) ligand (RANKL) and macrophage-colony stimulating factor (M-CSF), and then treated with LPS. Following treatment, the number of osteoclasts and cell viability were measured. The expression of osteoclast-related genes including tartrate-resistant acid phosphatase (TRAP), matrix metalloproteinase-9 (MMP-9), cathepsin K (CK), carbonic anhydrase II (CAII) and cyclooxygenase-2 (COX-2) was determined by RT-PCR. Protein levels of RANK, tumor necrosis factor receptor-associated factor 6 (TRAF6), COX-2 and mitogen-activated protein kinases (MAPK) were measured using western blotting assays. LPS promoted osteoclast differentiation of RAW264.7 cells and differentiated osteoclasts. LPS significantly increased mRNA expression of osteoclast-related genes in RAW264.7 cells. Differentiated osteoclasts were treated with LPS (100 ng/ml) and the results showed a significantly increased mRNA expression of osteoclast-related genes and protein levels of RANK, TRAF6 and COX-2. Furthermore, LPS at 100 ng/ml significantly promoted the MAPK pathway including increasing the phosphorylation of c-Jun N-terminal kinases (JNK) and the phosphorylation of the extracellular signal-regulated kinase (ERK1/2). In conclusion, LPS promoted osteoclast differentiation and activation by enhancing RANK signaling and COX-2 expression. LPS also promoted osteoclast differentiation via activation of the JNK and ERK1/2 cell proliferation pathways.

Suppression of Heme Oxygenase-1 by Prostaglandin E2-Protein Kinase A-A-Kinase Anchoring Protein Signaling Is Central for Augmented Cyclooxygenase-2 Expression in Lipopolysaccharide-Stimulated RAW 264.7 Macrophages

PurposeProstaglandin (PG) E2 is an immunomodulatory lipid mediator generated mainly via the cyclooxygenase-2 (COX-2) pathway from arachidonic acid at sites of infection and inflammation. A positive feedback loop of PGE2 on COX-2 expression is critical for homeostasis during toll-like receptor (TLR)-mediated inflammatory processes. The mechanism of PGE2-regulated COX-2 expression remains poorly understood. The low-molecular-weight stress protein heme oxygenase-1 (HO-1) contributes to the anti-inflammatory, anti-oxidant and anti-apoptotic response against environmental stress.MethodsWe explored the involvement of HO-1 on PGE2 regulation of LPS-induced COX-2 expression in RAW 264.7 macrophages.ResultsLPS-induced COX-2 expression in RAW 264.7 macrophages was enhanced by exogenous PGE2 or cyclic AMP (cAMP) analogue and was suppressed by a COX inhibitor (indomethacin), a protein kinase A (PKA) inhibitor (KT5720), and A kinase anchoring protein (AKAP) disruptors (Ht31 and RIAD). This result suggests that the stimulatory effects of endogenous and exogenous PGE2 on COX-2 expression are mediated by a cAMP-PKA-AKAP-dependent pathway. The induction of HO-1 was observed in LPS-stimulated RAW 264.7 macrophages. This induction was suppressed by exogenous PGE2 and enhanced by blockage of the endogenous PGE2 effect by the PKA inhibitor or AKAP disruptors. In addition, HO-1 induction by the HO activator copper protoporphyrin suppressed LPS-induced COX-2 expression, which was restored by the addition of exogenous PGE2. The induction of HO-1 inhibited LPS-induced NF-κB p-65 nuclear expression and translocation.ConclusionsAKAP plays an important role in PGE2 regulation of COX-2 expression, and the suppression of HO-1 by PGE2-cAMP-PKA-AKAP signaling helps potentiate the LPS-induced COX-2 expression through a positive feedback loop in RAW 264.7 macrophages.

Dichloromethane fraction of Laminaria japonica ethanolic extract inhibits lipopolysaccharide-induced nitric oxide synthase and cyclooxygenase-2 expression in RAW 264.7 cells via NF-κB pathway.

Strong anti-inflammatory activity has been found in Laminaria japonica dichloromethane fraction (LDF); however, the molecular mechanisms underlying its anti-inflammatory activity are not reported. Our results indicated that LDF inhibited LPS-induced nitric oxide and prostaglandin E(2) production in a dose-dependent manner and suppressed the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) in RAW 264.7 cells. Also, levels of pro-inflammatory cytokines such as tumor necrosis factor-α, interleukin (IL)-1β and IL-6 were remarkably reduced by LDF in LPS-treated RAW 264.7 cells. LDF greatly inhibited promoter activity of nuclear factor-κB (NF-κB) and translocation of NF-κB subunits by prevention of the degradation of inhibitor κB-α in LPS-treated RAW 264.7 cells (p < 0.05). Moreover, LDF inhibited activation of mitogen-activated protein kinases and AKT in LPS-treated RAW 264.7 cells. These results indicate that the LDF downregulates iNOS and COX-2 expressions through the suppression of NF-κB pathway associated with inhibition of multiple signaling proteins.

Aminoethyl-chitosan inhibits LPS-induced inflammatory mediators, iNOS and COX-2 expression in RAW264.7 mouse macrophages

Chitosan, naturally occuring biopolymer, has received considerable attention as a phamaceutical applications because of biocompatible, biodegradable and less toxic nature. In this study, chemically modified chitosans, aminoethyl-chitosans (AECs), were prepared in order to improve both water-solubility and bioactivity, and the effects of AECs on lipopolysaccharides (LPS)-induced inflammation in RAW264.7 mouse macrophages were investigated. AEC90 derived from 90% deacetylated chitosan showed strong inhibition capacity with regard to nitric oxide (NO) production than that of AEC50 derived from 50% deacetylated chitosan. Therefore further studies were coducted using AEC90. The production of prostaglandin E2 (PGE2) also inhibited by pretreatment of AEC90 in a dose-dependent manner which suggest the possibility of down-regulating their respective genes, inducible nitric oxide synthases (iNOS) and cyclooxygenase-2 (COX-2). Reverse transcrition-polymerase chain reaction (RT-PCR) and Western blot analysis revealed that AEC90 can affect both transcriptional and translational levels of iNOS and COX-2 expression via NF-κB pathway. Furthermore, AEC90 also suppressed the production of proinflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6), and the transcriptional and translational levels of such cytokines were also inhibited by AEC90 treatment.

Involvement of phosphatidylcholine-phospholipase C and protein kinase C in peptidoglycan-induced nuclear factor-κB activation and cyclooxygenase-2 expression in RAW 264.7 macrophages

In this study, we examined the role of phosphatidylcholine–phospholipase C (PC–PLC) and protein kinase C (PKC) in peptidoglycan (PGN)-induced nuclear factor-κB (NF-κB) activation and cyclooxygenase-2 (COX-2) expression in RAW 264.7 macrophages. PGN-induced COX-2 expression was attenuated by a PC–PLC inhibitor (D609) and by PKC inhibitors (Go 6976 and Ro 31-8220), but not by a phosphatidylinositol–PLC (PI–PLC) inhibitor (U-73122). PGN caused an increase in PKC activity, and this effect was inhibited by D609, Go 6976, and Ro 31-8220, but not by U-73122. Furthermore, the PGN-mediated increases in κB-luciferase activity were also inhibited by D609 and Ro 31-8220. Our data demonstrate that PGN activates PC–PLC which induces PKC activation; this in turn initiates NF-κB activation, and ultimately induces COX-2 expression in RAW 264.7 macrophages.

Chlorogenic acid inhibits lipopolysaccharide-induced cyclooxygenase-2 expression in RAW264.7 cells through suppressing NF-κB and JNK/AP-1 activation

Chlorogenic acid (CGA) is a naturally occurring phenolic acid in human diet. Data obtained from in vivo and in vitro experiments demonstrate that CGA mostly presents anti-oxidant and anti-carcinogenic activities. Here we show that CGA also inhibits lipopolysaccharide (LPS)-induced inflammatory response[AU1] in RAW 264.7 cells. Our results indicated that CGA significantly decreased LPS-induced up-regulation of cyclooxygenase (COX-2) at protein and mRNA levels in RAW 264.7 cells and as a result it inhibited prostaglandin E 2 (PGE 2) release from LPS-treated RAW 264.7 cells. In the further experiments, LPS-induced activation of nuclear factor-kappaB (NF-κB) and c-Jun N-terminal kinase (JNK)-c-Jun-activator protein (AP-1) pathway were suppressed significantly by CGA. In addition, CGA did not affect phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and p38. In conclusion, CGA suppresses LPS-induced COX-2 expression via attenuating the activation of NF-κB and JNK/AP-1 signaling pathways suggesting that CGA, the polyphenol compound in our food, could exert anti-inflammatory effects through inhibiting PGE 2 production.

Bis-(3-hydroxyphenyl) diselenide inhibits LPS-stimulated iNOS and COX-2 expression in RAW 264.7 macrophage cells through the NF-κB inactivation

Previously, we reported that diaryl diselenide compounds have strong inhibitory effects on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in macrophages. In this study, we investigated the molecular mechanisms underlying NO suppression and prostaglandin E(2) (PGE(2)) production by diaryl diselenide compounds, bis-(2-hydroxyphenyl) diselenide (DSE-A), bis-(3-hydroxyphenyl) diselenide (DSE-B), bis-(4-hydroxyphenyl) diselenide (DSE-C), dipyridyl diselenide (DSE-D) and diphenyl diselenide (DSE-E). The effect of these compounds on NO suppression and PGE(2) production was investigated in RAW 264.7 macrophages. Our data indicate that of the above, DSE-B most potently inhibits NO and PGE(2) production, and that it also significantly reduces the releases of tumour necrosis factor (TNF)-alpha, interleukin(IL)-1beta and IL-6. Consistent with these observations, DSE-B also reduced the protein levels of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2), and the mRNA levels of iNOS, COX-2, TNF-alpha, IL-1beta and IL-6. Furthermore, DSE-B inhibited LPS-induced nuclear factor-kappaB (NF-kappaB) activation, which was associated with the prevention of the inhibitor kappaB-alpha (IkappaB-alpha) degradation and a subsequent reduction in nuclear p65 protein levels. Taken together, our data suggest that the anti-inflammatory properties of DSE-B are due to reduction in the expression of iNOS, COX-2, TNF-alpha, IL-1beta and IL-6 through the down-regulation of NF-kappaB binding activity.

Rac1 regulates peptidoglycan-induced nuclear factor-κB activation and cyclooxygenase-2 expression in RAW 264.7 macrophages by activating the phosphatidylinositol 3-kinase/Akt pathway

Previously, we found that peptidoglycan (PGN), a cell wall component of the gram-positive bacterium Staphylococcus aureus, may activate the Ras/Raf-1/extracellular signal-regulated kinase (ERK) pathway, which in turn initiates IκB kinases α/β (IKKα/β) and nuclear factor-κB (NF-κB) activation, and ultimately induces cyclooxygenase-2 (COX-2) expression in RAW 264.7 macrophages. In this study, we further investigated the roles of Rac1, phosphatidylinositol 3-kinase (PI3K), and Akt in PGN-induced NF-κB activation and COX-2 expression in RAW 264.7 macrophages. PGN-induced COX-2 expression was attenuated by a Rac1 dominant negative mutant (RacN17), PI3K inhibitors (wortmannin and LY 294002), and an Akt inhibitor (1L-6-hydroxymethyl-chiro-inositol2-[(R)-2-O-methyl-3-O-octadecylcarbonate]). PGN-induced PGE2 release was also inhibited by RacN17. Treatment of RAW 264.7 macrophages with PGN caused the activation of Rac and Akt. PGN-induced Akt activation was inhibited by RacN17, LY 294002, and the Akt inhibitor. Stimulation of RAW 264.7 macrophages with PGN resulted in an increase in IKKα/β phosphorylation and p65 Ser536 phosphorylation; these effects were inhibited by RacN17, LY 294002, an Akt inhibitor, and an Akt dominant negative mutant (AktDN). The PGN-induced increases in κB-luciferase activity were also inhibited by RacN17, wortmannin, LY 294002, an Akt inhibitor, and AktDN. Treatment of macrophages with PGN induced the recruitment of p85α and Rac1 to Toll-like receptor 2 (TLR2) in a time-dependent manner. These results indicate that PGN may activate the Rac1/PI3K/Akt pathway through the recruitment of p85α and Rac1 to TLR2 to mediate IKKα/β activation and p65 phosphorylation, which in turn induces NF-κB transactivation, and ultimately causes COX-2 expression in RAW 264.7 macrophages.

Anti-inflammatory activity of 4-methoxyhonokiol is a function of the inhibition of iNOS and COX-2 expression in RAW 264.7 macrophages via NF-κB, JNK and p38 MAPK inactivation

Abstract The extracts or constituents from the bark of Magnolia ( M. ) obovata are known to have many pharmacological activities. 4-Methoxyhonokiol, a neolignan compound isolated from the stem bark of M. obovata, was found to exhibit a potent anti-inflammatory effect in different experimental models. Pretreatment with 4-methoxyhonokiol (i.p.) dose-dependently inhibited the dye leakage and paw swelling in an acetic-acid-induced vascular permeability assay and a carrageenan-induced paw edema assay in mice, respectively. In the lipopolysaccharide (LPS)-induced systemic inflammation model, 4-methoxyhonokiol significantly inhibited plasma nitric oxide (NO) release in mice. To identify the mechanisms underlying this anti-inflammatory action, we investigated the effect of 4-methoxyhonokiol on LPS-induced responses in a murine macrophage cell line, RAW 264.7. The results demonstrated that 4-methoxyhonokiol significantly inhibited LPS-induced NO production as well as the protein and mRNA expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Furthermore, 4-methoxyhonokiol inhibited LPS-mediated nuclear factor-κB (NF-κB) activation via the prevention of inhibitor κB (IκB) phosphorylation and degradation. 4-Methoxyhonokiol had no effect on the LPS-induced phosphorylation of extracellular signal-regulated kinase (ERK), whereas it attenuated the phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) and c-Jun NH 2 -terminal kinase (JNK) in a concentration-dependent manner. Taken together, our data suggest that 4-methoxyhonokiol is an active anti-inflammatory constituent of the bark of M. obovata , and that its anti-inflammatory property might be a function of the inhibition of iNOS and COX-2 expression via down-regulation of the JNK and p38 MAP kinase signal pathways and inhibition of NF-κB activation in RAW 264.7 macrophages.

Anti-inflammatory activity of 4-methoxyhonokiol is a function of the inhibition of iNOS and COX-2 expression in RAW 264.7 macrophages via NF-κB, JNK and p38 MAPK inactivation

The extracts or constituents from the bark of Magnolia ( M.) obovata are known to have many pharmacological activities. 4-Methoxyhonokiol, a neolignan compound isolated from the stem bark of M. obovata, was found to exhibit a potent anti-inflammatory effect in different experimental models. Pretreatment with 4-methoxyhonokiol (i.p.) dose-dependently inhibited the dye leakage and paw swelling in an acetic-acid-induced vascular permeability assay and a carrageenan-induced paw edema assay in mice, respectively. In the lipopolysaccharide (LPS)-induced systemic inflammation model, 4-methoxyhonokiol significantly inhibited plasma nitric oxide (NO) release in mice. To identify the mechanisms underlying this anti-inflammatory action, we investigated the effect of 4-methoxyhonokiol on LPS-induced responses in a murine macrophage cell line, RAW 264.7. The results demonstrated that 4-methoxyhonokiol significantly inhibited LPS-induced NO production as well as the protein and mRNA expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Furthermore, 4-methoxyhonokiol inhibited LPS-mediated nuclear factor-κB (NF-κB) activation via the prevention of inhibitor κB (IκB) phosphorylation and degradation. 4-Methoxyhonokiol had no effect on the LPS-induced phosphorylation of extracellular signal-regulated kinase (ERK), whereas it attenuated the phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) and c-Jun NH 2-terminal kinase (JNK) in a concentration-dependent manner. Taken together, our data suggest that 4-methoxyhonokiol is an active anti-inflammatory constituent of the bark of M. obovata, and that its anti-inflammatory property might be a function of the inhibition of iNOS and COX-2 expression via down-regulation of the JNK and p38 MAP kinase signal pathways and inhibition of NF-κB activation in RAW 264.7 macrophages.

Anti-inflammatory activities of ent-16α H,17-hydroxy-kauran-19-oic acid isolated from the roots of Siegesbeckia pubescens are due to the inhibition of iNOS and COX-2 expression in RAW 264.7 macrophages via NF-κB inactivation

To isolate the anti-inflammatory components in Siegesbeckia pubescens root, we performed activity-guided fractionation using a carrageenan-induced edema rat model. Antinociceptive effects were followed using acetic acid-induced abdominal constriction and hot plate tests in mice. Chloroform extract was subjected to silica gel and octadesyl silane (ODS) column chromatography, and a diterpene was isolated which was identified as ent-16α H,17-hydroxy-kauran-19-oic acid (siegeskaurolic acid). Pretreatment with siegeskaurolic acid (20 or 30 mg/kg/day, p.o.) exhibited anti-inflammatory and antinociceptive effects in these animal models. To investigate the mechanisms underlying this anti-inflammatory action, we investigated the effect of siegeskaurolic acid on lipopolysaccharide (LPS)-induced responses in a murine macrophage cell line, RAW 264.7. Siegeskaurolic acid was found to significantly inhibit the productions of nitric oxide (NO), prostaglandin E 2 (PGE 2), and tumor necrosis factor-α (TNF-α). Consistent with these findings, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) proteins, and iNOS, COX-2, and TNF-α mRNAs were found to be inhibited by siegeskaurolic acid. Furthermore, siegeskaurolic acid inhibited the nuclear factor-κB (NF-κB) activation induced by LPS, and this was associated with the prevention of inhibitor κB degradation (I κB), and subsequently with decreased nuclear p65 and p50 protein levels. Taken together, our data indicate that the anti-inflammatory and antinociceptive properties of siegeskaurolic acid may be due to iNOS, COX-2 and TNF-α inhibition via the down-regulation of NF-κB binding activity.

Inhibition of inducible nitric oxide synthase and cyclooxygenase-2 expression by flavonoids isolated from Tanacetum microphyllum

Plant flavonoids show anti-inflammatory activity both in vitro and in vivo. Some flavonoids have been reported previously to inhibit nitric oxide (NO) and prostaglandin E2 (PGE2) production by suppressing inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression. The present study focuses on the effect of various naturally occurring flavonoids (santin, ermanin, centaureidin and 5,3′-dihydroxy-4′-methoxy-7-methoxycarbonylflavonol) on modulation of lipopolysaccharide (LPS)-induced iNOS and COX-2 expression in RAW 264.7 cells. Western blotting showed that all flavonoids suppressed the induction of both iNOS and COX-2. Ermanin and 5,3′-dihydroxy-4′-methoxy-7-methoxycarbonylflavonol were the most potent inhibitors. This study suggests that inhibition of iNOS and COX-2 expression by flavonoids may be one of the mechanisms responsible for their anti-inflammatory effects, and that they may be potential agents for use in the treatment of inflammatory diseases.

Dipyridamole activation of mitogen-activated protein kinase phosphatase-1 mediates inhibition of lipopolysaccharide-induced cyclooxygenase-2 expression in RAW 264.7 cells

Dipyridamole is a nucleoside transport inhibitor and a non-selective phosphodiesterase inhibitor. However, the mechanisms by which dipyridamole exerts its anti-inflammatory effects are not completely understood. In the present study, we investigated the role of mitogen-activated kinase phosphatase-1 (MKP-1) in dipyridamole's anti-inflammatory effects. We show that dipyridamole inhibited interleukin-6 and monocyte chemoattractant protein-1 secretion, inducible nitric oxide synthase protein expression, nitrite accumulation, and cyclooxygenase-2 (COX-2) induction in lipopolysaccharide (LPS)-activated RAW 264.7 macrophages. Dipyridamole inhibited the nuclear factor kappa B (NF-κB) signaling pathway as demonstrated by inhibition of the inhibitor of NF-κB (IκB) phosphorylation, IκB degradation, p65 translocation from the cytosol to the nucleus, and transcription of the reporter gene. Dipyridamole also inhibited LPS-stimulated p38 mitogen-activated protein kinase (p38 MAPK) and IκB kinase-beta (IKK-β) activities in RAW 264.7 cells. A p38 MAPK inhibitor, SB 203580, inhibited LPS-stimulated COX-2 expression and IKK-β activation suggesting that LPS may activate the NF-κB signaling pathway via upstream p38 MAPK activation. Furthermore, dipyridamole stimulated transient activation of MKP-1, a potent inhibitor of p38 MAPK function. Knockdown of MKP-1 by transfecting MKP-1 siRNA or inhibition of MKP-1 by the specific inhibitor, triptolide, significantly reduced the inhibitory effects of dipyridamole on COX-2 expression induced by LPS. Taken together, these data suggest that dipyridamole exerts its anti-inflammatory effect via activation of MKP-1, which dephosphorylates and inactivates p38 MAPK. Inactivation of p38 MAPK in turn inhibits IKK-β activation and subsequently the NF-κB signaling pathway that mediates LPS-induced cyclooxygenase-2 expression in RAW 264.7 cells.

Proteasome inhibitor-induced cyclooxygenase-2 expression in Raw264.7 cells is potentiated by inhibition of c-Jun N-terminal kinase activation

Prostaglandins play regulatory roles in a variety of physiological and pathological processes in immune response and inflammation. MG132, proteasome inhibitor, is known to anti-tumor agent activity and anti-inflammation with inhibitory property of NF-κB. We investigated the effect of MG132 on the expression of cyclooxygenase-2 (COX-2), the rate-limiting enzyme in the synthesis of PGE 2, using macrophage cell line, Raw264.7. Our results showed that COX-2 expression is up-regulated by MG132 treatment and that this induction of COX-2 is regulated in part at the transcriptional level. In addition, we demonstrated the signal transduction pathway of mitogen-activated protein kinase (MAP kinase) in MG132-induced COX-2 expression. The p38 MAPK inhibitor (SB 203580) prevented MG132-induced COX-2 expression, whereas c-Jun N-terminal kinase (JNK) inhibitor (SP 600125) and MAPK kinase 4 (MKK4)-DN (dominant negative mutant) and MKK7-DN significantly enhanced COX-2 expression. These results suggest that MG132-induced COX-2 expression is associated with the activation of p38 MAPK and the inhibition of JNK signaling pathways.