Morin inhibits interleukin-1β-induced nitric oxide and prostaglandin E2 production in human chondrocytes

It is well known that the inflammatory cytokines play important roles in osteoarthritis (OA). In the present study, we investigated the anti-inflammatory properties of morin in chondrocytes. The nitric oxide (NO) production was determined by Griess method, the prostaglandin E2 (PGE(2)) production was detected by Enzyme-linked immunosorbent assay (ELISA). The expression of inducible NO synthase (iNOS) and cyclooxygenase (COX)-2 were investigated by quantitative real-time PCR and western blot. In addition, western blotting and immunofluorescence staining were performed to investigate the protein level of inhibitor of nuclear factor-κB (IκB-α) and the translocation of nuclear factor kappa B (NF-κB). For the in vivo study, morin was administered by intra-articular injection in rats, and the gene expression of iNOS and COX-2 was assessed. We showed that morin inhibited the production of NO and PGE(2) as well as the expression of iNOS and COX-2 in interleukin-1-beta (IL-1β)-induced chondrocytes. In addition, morin suppressed the degradation of IκB-α as well as the translocation of NF-κB. In vivo study, morin exerted anti-inflammatory properties in an IL-1β-induced rat OA model. Our data suggest that morin possess potential value in the treatment of OA.     

Diclofenac enhances proinflammatory cytokine-induced nitric oxide production through NF-κB signaling in cultured astrocytes

Recently, the number of reports of encephalitis/encephalopathy associated with influenza virus has increased. In addition, the use of a non-steroidal anti-inflammatory drug, diclofenac sodium (DCF), is associated with a significant increase in the mortality rate of influenza-associated encephalopathy. Activated astrocytes are a source of nitric oxide (NO), which is largely produced by inducible NO synthase (iNOS) in response to proinflammatory cytokines. Therefore, we investigated whether DCF enhances nitric oxide production in astrocytes stimulated with proinflammatory cytokines. We stimulated cultured rat astrocytes with three cytokines, interleukin-1β, tumor necrosis factor-α and interferon-γ, and then treated the astrocytes with DCF or acetaminophen (N-acetyl-p-aminophenol: APAP). iNOS and NO production in astrocyte cultures were induced by proinflammatory cytokines. The addition of DCF augmented NO production, but the addition of APAP did not. NF-κB inhibitors SN50 and MG132 inhibited iNOS gene expression in cytokine-stimulated astrocytes with or without DCF. Similarly, NF-κB p65 Stealth small interfering RNA suppressed iNOS gene expression in cytokine-stimulated astrocytes with or without DCF. LDH activity and DAPI staining showed that DCF induces cell damage in cytokine-stimulated astrocytes. An iNOS inhibitor, l-NMMA, inhibited the cytokine- and DCF-induced cell damage. In conclusion, this study demonstrates that iNOS and NO are induced in astrocyte cultures by proinflammatory cytokines. Addition of DCF further augments NO production. This effect is mediated via NF-κB signaling and leads to cell damage. The enhancement of DCF on NO production may explain the significant increase in the mortality rate of influenza-associated encephalopathy in patients treated with DCF.     

Hydrangenol inhibits lipopolysaccharide-induced nitric oxide production in BV2 microglial cells by suppressing the NF-κB pathway and activating the Nrf2-mediated HO-1 pathway

We previously demonstrated the anti-inflammatory effect of water extract of Hydrangea macrophylla in lipopolysaccharide (LPS)-stimulated macrophage cells. Here, we investigated whether hydrangenol, a bioactive component of H. macrophylla, attenuates the expression of nitric oxide (NO) and its associated gene, inducible NO synthase (iNOS), in LPS-stimulated BV2 microglial cells. Our data showed that low dosages of hydrangenol inhibited LPS-stimulated NO release and iNOS expression without any accompanying cytotoxicity. Hydrangenol also suppressed LPS-induced nuclear translocation of nuclear factor-κB (NF-κB) subunits, consequently inhibiting DNA-binding activity of NF-κB. Additionally, the NF-κB inhibitors, pyrrolidine dithiocarbamate (PDTC) and PS-1145, significantly attenuated LPS-induced iNOS expression, indicating that hydrangenol-induced NF-κB inhibition might be a key regulator of iNOS expression. Furthermore, our data showed that hydrangenol suppresses NO production by inducing heme oxygenase-1 (HO-1). The presence of cobalt protoporphyrin, a specific HO-1 inducer, potently suppressed LPS-induced NO production. Hydrangenol also promoted nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) and subsequently increased its binding activity at the specific antioxidant response element sites. Additionally, transient knockdown of Nrf2 significantly downregulated hydrangenol-induced HO-1 expression, indicating that hydrangenol-induced Nrf2 is an upstream regulator of HO-1. Taken together, these data suggest that hydrangenol attenuates NO production and iNOS expression in LPS-stimulated BV2 microglial cells by inhibiting NF-κB activation and by stimulating the Nrf2-mediated HO-1 signaling pathway. Therefore, hydrangenol is a promising therapeutic agent for treatment of LPS-mediated inflammatory diseases.     

Britanin suppresses LPS-induced nitric oxide,PGE2 and cytokine production via NF-κB and MAPK inactivation in RAW 264.7 cells

Little is known about the biological properties of britanin, which is isolated from the flowers of Inula japonica (Inulae Flos). Based on our previous studies that Inulae Flos had anti-inflammation and anti-asthmatic activities, we tried to find the bioactive compounds from it. In this study, the anti-inflammatory effects of britanin on the inflammatory mediators as well as on nuclear factor (NF)-кB and mitogen-activated protein (MAP) kinase activation were evaluated in RAW 264.7 cells. Britanin inhibited the production of nitric oxide (NO) and prostaglandin E2 (PGE₂) along with the expression of inducible NO synthase (iNOS) and cyclooxygenase (COX)-2 in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. In addition, britanin reduced the release of pro-inflammatory cytokines, such as TNF-α, IL-1β, and IL-6. Furthermore, the phosphorylations of MAP kinases (p38 and JNK) in LPS-stimulated RAW 264.7 cells were suppressed by britanin. Moreover, britanin inhibited the NF-κB activation induced by LPS, which was associated with the abrogation of IκBα degradation and subsequent decreases in nuclear p65 levels. This study suggests that the anti-inflammatory activities of britanin might be attributed to the inhibition of iNOS and COX-2 and cytokine expression at least in part, through the attenuation of the phosphorylations of MAP kinases and NF-κB activation via IκBα degradation in macrophages. We conclude that britanin may have potential for the treatment of inflammatory diseases through the down-regulation of MAP kinases and NF-κB mediated activation of macrophages.     

Nuclear factor-κB activation mediates inducible nitric oxide synthase expression in carrageenin-induced rat pleurisy

We studied the involvement of nuclear factor-kappaB (NF-kappaB) in the regulation of inducible nitric oxide synthase expression in carrageenin-induced rat pleurisy. Injection of 0.2 ml of 1% lambda-carrageenin into the pleural cavity of male Wistar rats caused after 6 h: (a) exudate formation and leukocyte migration into the pleural cavity; (b) inducible NO synthase protein expression and accumulation of NO2- plus NO3- in pleural exudate; (c) increase in p50/p65 nuclear level as well as NF-kappaB/DNA binding activity. Treatment of rats with pyrrolidine dithiocarbamate (10, 30, and 100 mg/kg) and N-alpha-p-tosyl-L-lysine chloromethylketone (30 mg/kg), two inhibitors of NF-kappaB activation, given subcutaneously concomitantly with carrageenin, caused a significant inhibition of all the parameters assayed. These results suggest that in carrageenin-induced rat pleurisy the activation of NF-kappaB plays a key role in inducible NO synthase protein expression and in the development of inflammatory response.     

Thrombin induces inducible nitric oxide synthase expression via the MAPK, MSK1, and NF-κB signaling pathways in alveolar macrophages

In this study, we investigated the roles of mitogen activated protein kinase (MAPK), mitogen stress-activated protein kinase 1 (MSK1), and nuclear factor-κB (NF-κB) signaling pathways in thrombin-induced inducible nitric oxide synthase (iNOS) expression in alveolar macrophages (NR8383). Treatment of NR8383 cells with thrombin caused an increase in iNOS expression in a concentration- and time-dependent manner. Treatment of NR8383 cells with SB203580 (4-(4-Fluorophenyl)-2-[4-(methylsulfinyl)phenyl]-5-(4-pyridyl)-1H-imidazole, a p38 MAPK inhibitor), PD98059 (2'-amino-3'-methoxyflavone, a MAPK kinase (MEK) inhibitor), and SP600125 (anthra[1-9-cd]pyrazol-6(2H)-one, a JNK inhibitor) all inhibited thrombin-induced iNOS expression. Stimulation of cells with thrombin caused an increase in p38 MAPK, ERK, and JNK phosphorylation. Treatment of cells with Ro 31-8220 (an MSK1 inhibitor) and MSK1 small interfering RNA (MSK1 siRNA) both inhibited thrombin-induced iNOS expression. Thrombin caused time-dependent activation of MSK1 Ser531 phosphorylation, which was inhibited by SB203580 and PD98059, but not by SP600125. Treatment of cells with pyrrolidine dithiocarbamate (PDTC, an NF-κB inhibitor) inhibited thrombin-induced iNOS expression in a concentration-dependent manner. Treatment of NR8383 cells with thrombin induced κB-luciferase activity and p65 Ser276 phosphorylation. Thrombin-induced increases in p65 Ser276 phosphorylation and κB-luciferase activity were inhibited by SB203580, PD98059, Ro 31-8220, and MSK1 siRNA. Taken together, these results suggest that the signaling pathways of MAPK, MSK1, and NF-κB play important roles in thrombin-induced iNOS expression in alveolar macrophages.     

Acrolein,an I-κBα-independent downregulator of NF-κB activity,causes the decrease in nitric oxide production in human malignant keratinocytes

Acrolein, a reactive electrophilic α, β-unsaturated aldehyde, is known to be an alkylating chemical carcinogen. The effect of acrolein on the activation of NF-κB in human malignant epidermal keratinocytes was examined to elucidate the molecular mechanism associated with this NF-κB-acrolein regulation and its consecutive sequence, nitric oxide (NO) production. Acrolein significantly downregulated the cellular NF-κB activity up to 60% compared with control as well as the lipopolysaccharide (LPS)-induced NO production in a dose response manner at concentrations of 10~30 μM. To investigate the regulatory mechanism associated with this NF-κB-acrolein downregulation, the relative level of phosphorylation of I-κBα (serines-32 and -36), a principle regulator of NF-κB activation, represented by acrolein, was quantified. Acrolein inhibited NF-κB activity without altering cellular levels of the phosphorylated and nonphosphorylated forms of I-κBα, implying that the downregulatory effect of acrolein on cellular NF-κB activity in human skin cells is an I-κBα-independent activation pathway. The results suggests that acrolein causes the decrease in nitric oxide production as an I-κBα-independent downregulator of NF-κB activity in human malignant keratinocytes, and acrolein-induced carcinogenesis may be associated with the modulation of cellular NF-κB activity.     

Salvianic acid A inhibits induction of inflammatory mediators by blocking Nuclear Factor-κB activation in macrophages

Objective To investigate the anti-inflammation effect and possible mechanism of Salvianic acid A(SAA)in mouse peritoneal macrophages.Methods Peritoneal macrophages were obtained from BALB/c mice.LPS induced nitric oxide(NO),tumor necrosis factor-alpha(TNF-α)and interleukin-6(IL-6)in supernatant,protein expression of inducible nitric oxide synthase(iNOS),matrix metalloproteinase-9(MMP-9)and activation of nuclear factor-kappa B(NF-κB)in the extract were measured.Results SAA strongly inhibited the excessive production of NO,TNF-α and IL-6 in LPS-induced peritoneal macrophages in a concentration-dependent manner and blocked the expression of iNOS and MMP-9.Treatment with LPS alone increased the translocation of NF-κB(p65)from cytosol to the nucleus,but the SAA inhibited the translocation of NF-κB(p65).Conclusions The results showed that SAA had strong anti-inflammatory effects in LPS-stimulated peritoneal macrophages.The important mechanism is due to its inhibition of NF-κB activation.     

Calcitonin gene-related peptide (CGRP) modulates LPS-induced IL-12 release and nitric oxide production from murine peritoneal macrophages

The secretion of IL-12 after stimulation of macrophages has been found to play an important modulator role in cell-mediated immune response and T-cell differentiation.In order to investigate the relationship between neural and immune system, we examined whether sensory neuropeptide CGRP would modulate IL-12 release and nitric oxide (NO) production in murine macrophages.Macrophages were obtained from the peritoneal     

The natural flavonoid galangin inhibits osteoclastic bone destruction and osteoclastogenesis by suppressing NF-κB in collagen-induced arthritis and bone marrow-derived macrophages

We investigated the effect of galangin, a natural flavonoid, on osteoclastic bone destruction in collagen-induced arthritis and examined the molecular mechanisms by which galangin affects osteoclastogenesis in bone marrow derived macrophages. In mice with collagen-induced arthritis, administration of galangin significantly reduced the arthritis clinical score, edema and severity of disease without toxicity. Interestingly, galangin treatment during a later stage of collagen-induced arthritis, using mice with a higher clinical arthritis score, still significantly slowed the progression of the disease. Extensive cartilage and bone erosive changes as well as synovial inflammation, synovial hyperplasia and pannus formation were dramatically inhibited in arthritic mice treated with galangin. Furthermore, galangin-treated arthritic mice showed a significant reduction in the concentrations of IL-1β, TNF-α and IL-17 . We found that galangin inhibited osteoclastogenic factors and osteoclast formation in bone marrow-derived macrophages and osteoblast co-cultured cells, and increased osteoprotegerin (OPG) levels in osteoblasts. Galangin and NF-κB siRNA suppressed RANKL-induced phosphorylation of the c-jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK), but not AKT and extracellular signal-regulated kinase 1/2 (ERK1/2). Also, the JNK inhibitor SP600125 and p38 inhibitor SB203580 reduced RANKL-induced expressions of phospho-c-Jun, c-fos and NFATc1 genes during osteoclast development. In addition, galangin suppressed RANKL-induced phosphorylation of NF-κB, phospho-IκBα, inflammatory cytokines and osteoclast formation in bone marrow-derived macrophages. Our data suggest that galangin prevented osteoclastic bone destruction and osteoclastogenesis in osteoclast precursors as well as in collagen-induced arthritis mice without toxicity via attenuation of RANKL-induced activation of JNK, p38 and NF-κB pathways.     

6,6'-Bieckol, isolated from marine alga Ecklonia cava, suppressed LPS-induced nitric oxide and PGE₂ production and inflammatory cytokine expression in macrophages: the inhibition of NFκB

Ecklonia cava is an edible brown alga that contains high levels of phlorotannins, which are unique marine polyphenolic compounds. In the present study, we investigated the anti-inflammatory effects and the underlying molecular mechanism of phlorotannin 6,6′-bieckol, which is an active component isolated from E. cava, on lipopolysaccharide (LPS)-stimulated primary macrophages and RAW 264.7 macrophage cells. 6,6′-Bieckol was found to inhibit nitric oxide (NO) and prostaglandin E₂ (PGE₂) production and to suppress the LPS-induced expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) at the mRNA and protein levels. In addition, 6,6′-bieckol downregulated the production and mRNA expression of the inflammatory cytokines TNF-α and IL-6. Moreover, pretreatment with 6,6′-bieckol decreased LPS-induced transactivation of nuclear factor-kappa B (NFκB) and nuclear translocation of p50 and p65 subunits of NFκB. Furthermore, chromatin immunoprecipitation assay revealed that 6,6′-bieckol inhibited LPS-induced NFκB binding to the TNF-α and IL-6 promoters. Taken together, these data suggest that the anti-inflammatory properties of 6,6′-bieckol are related to the down-regulation of iNOS, COX-2, and pro-inflammatory cytokines through the negative regulation of the NFκB pathway in LPS-stimulated macrophages.     

Artemisia fukudo essential oil attenuates LPS-induced inflammation by suppressing NF-κB and MAPK activation in RAW 264.7 macrophages

In the present study, the chemical constituents of Artemisia fukudo essential oil (AFE) were investigated using GC–MS. The major constituents were α-thujone (48.28%), β-thujone (12.69%), camphor (6.95%) and caryophyllene (6.01%). We also examined the effects of AFE on the production of nitric oxide (NO), prostaglandin E₂ (PGE₂), tumour necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6, in lipopolysaccharide (LPS)-activated RAW 264.7 macrophages. Western blotting and RT-PCR tests indicated that AFE has potent dose-dependent inhibitory effects on pro-inflammatory cytokines and mediators. We investigated the mechanism by which AFE inhibits NO and PGE₂ by examining the level of nuclear factor-κB (NF-κB) activation within the mitogen-activated protein kinase (MAPK) pathway, which is an inflammation-induced signal pathway in RAW 264.7 cells. AFE inhibited LPS-induced ERK, JNK, and p38 phosphorylation. Furthermore, AFE inhibited the LPS-induced phosphorylation and degradation of Iκ-B-α, which is required for the nuclear translocations of the p50 and p65 NF-κB subunits in RAW 264.7 cells. Our results suggest that AFE might exert an anti-inflammatory effect by inhibiting the expression of pro-inflammatory cytokines. Such an effect is mediated by a blocking of NF-κB activation which consequently inhibits the generation of inflammatory mediators in RAW264.7 cells. AFE may be useful for treating inflammatory diseases.     

Neurotropin inhibits neuroinflammation via suppressing NF-κB and MAPKs signaling pathways in lipopolysaccharide-stimulated BV2 cells

Neurotropin (NTP) is a widely used drug in China and Japan mainly for the treatment of chronic pain and peripheral inflammation. Nevertheless, the effects of NTP on neuroinflammation have not been explored. In this study, we investigated the anti-inflammatory effects of NTP in lipopolysaccharide (LPS)-stimulated BV-2 microglial cells and its underlying mechanisms. BV-2 cells were pretreated with NTP for 12 h before exposure to LPS. The expression of pro-inflammatory cytokines (TNF-α and IL-6) were detected by RT-PCR and EILSA at mRNA and protein levels, respectively. Western blotting was conducted to measure the protein levels of major genes in MAPKs and NF-κB signaling pathways. Results demonstrated that NTP could attenuate the production of pro-inflammatory cytokines. Furthermore, NTP inhibited the activation of NF-κB signaling by decreasing the translocation of NF-κB p65 to the nucleus and suppressed the MAPKs signaling pathway via inhibition of the phosphorylation of p38, ERK and JNK. Taken together, these findings suggest that neurotropin exerts anti-inflammatory effects by suppressing the production of pro-inflammatory mediators via inhibition of NF-κB and MAPKs signaling pathways in LPS-stimulated BV-2 cells.     

(1→3)-β-d-Glucan stimulates nitric oxide generation and cytokine mRNA expression in macrophages

Beta-glucans are known for their potent ability to induce nonspecific inflammatory reactions and are believed to play a role in bioaerosol-induced respiratory symptoms seen in both occupational and residential environments. Here, the ability of a (1→3)-β-d-glucan (Curdlan) to stimulate nitric oxide generation and cytokine mRNA expression in rat alveolar macrophages (AMs) and the murine monocyte/macrophage cell line, RAW 264.7 was investigated. Exposure to (1→3)-β-d-glucan (20, 100 and 500 μg/ml) induced a dose-dependent increase in the expression of inducible nitric oxide synthase mRNA and a release of nitric oxide into the culture medium in both rat AMs and RAW 264.7 cells. The mRNA expression of a number of other inflammatory mediators such as interleukin-1β, interleukin-6, tumor necrosis factor-α and cyclooxygenase-2 was also increased by the exposure to β-glucan. The capability of (1→3)-β-d-glucan (500 μg/ml) to induce mRNA synthesis of these various mediators were comparable to that of endotoxin (1 μg/ml). These results imply that (1→3)-β-d-glucan stimulates the generation of nitric oxide, cytokines and prostaglandins in macrophages and suggest the possibility that this may contribute to bioaerosol-induced respiratory symptoms seen in exposed individuals.