红毛五加多糖单一组分AHP-Ⅲ对巨噬细胞的免疫调节作用及其机制

探讨红毛五加多糖(Acanthopanax giraldii Hams polysaccharide)单一组分AHP-Ⅲ(Acanthopanax giraldii Hams polysaccharideⅢ)对小鼠巨噬细胞RAW 264.7的激活作用及机制。不同浓度AHP-Ⅲ作用RAW 264.7细胞,中性红试验检测细胞吞噬能力;ELISA和Griess法检测其IL-6、TNF-α和NO的释放量;RT-qPCR检测iNOS、TNF-α和IL-6 mRNA相对表达水平;Western blot检测NF-κB信号通路相关蛋白磷酸化水平。在实验浓度范围内,AHP-Ⅲ可显著增强RAW 264.7细胞的吞噬能力(P<0.05);促进RAW 264.7分泌NO、TNF-α和IL-6(P<0.05或P<0.001);并显著增加RAW 264.7细胞中IL-6、TNF-α和iNOS mRNA的表达量,呈剂量依赖性;Western blot结果表明,AHP-Ⅲ作用RAW 264.7细胞后,NF-κB中的p65、IKKβ、IκBα磷酸化水平明显升高。结果显示红毛五加多糖AHP-Ⅲ对小鼠巨噬细胞RAW 264.7具有显著激活作用。     

Effects of globin digest and its active ingredient Trp-Thr-Gln-Arg on galactosamine/lipopolysaccharide-induced liver injury in ICR mice

AimsWe investigated the effects of globin digest (GD) and its active ingredient Trp-Thr-Gln-Arg (WTQR) on galactosamine/lipopolysaccharide (GalN/LPS)-induced liver injury in imprinting control region (ICR) mice.Main methodsThe effects of WTQR and GD on the liver injury were examined by measuring the survival rate, serum aminotransferase activities, hepatic components, antioxidant enzyme activities, histopathological analysis, serum levels and hepatic gene expression of tumor necrosis factor-alpha (TNF-α), macrophage inflammatory protein-2 (MIP-2), and nitric oxide (NO) or inducible nitric oxide synthase (iNOS), and nuclear factor-kappa B (NF-κB) p65 content in GalN/LPS-treated ICR mice. RAW264 mouse macrophages were used to confirm the anti-inflammatory effects of WTQR and GD on the macrophages.Key findingsWTQR and GD increased the survival rate, suppressed the serum aminotransferase activities, serum levels and hepatic gene expression of TNF-α, MIP-2, and NO or iNOS, and nuclear NF-κB p65 content in GalN/LPS-treated mice; decreased the oxidized glutathione content, increased the superoxide dismutase activity, and decreased the histopathological grade values of the hepatocyte necrosis and lobular inflammation in GalN/LPS-injured liver; and suppressed the release levels and gene expression of TNF-α, MIP-2, and NO or iNOS, and nuclear NF-κB p65 content in LPS-stimulated RAW264 macrophages. WTQR and GD may improve the antioxidant defense system and inflammatory status in GalN/LPS-injured liver.SignificanceThese findings indicate that WTQR and GD have hepatoprotective effects on GalN/LPS-induced liver injury in ICR mice.     

β-Glucan from Lentinus edodes inhibits nitric oxide and tumor necrosis factor-α production and phosphorylation of mitogen-activated protein kinases in lipopolysaccharide-stimulated murine RAW 264.7 macrophages

Lentinan (LNT), a β-glucan from the fruiting bodies of Lentinus edodes, is well known to have immunomodulatory activity. NO and TNF-α are associated with many inflammatory diseases. In this study, we investigated the effects of LNT extracted by sonication (LNT-S) on the NO and TNF-α production in LPS-stimulated murine RAW 264.7 macrophages. The results suggested that treatment with LNT-S not only resulted in the striking inhibition of TNF-α and NO production in LPS-activated macrophage RAW 264.7 cells, but also the protein expression of inducible NOS (iNOS) and the gene expression of iNOS mRNA and TNF-α mRNA. It is surprising that LNT-S enhanced LPS-induced NF-κB p65 nuclear translocation and NF-κB luciferase activity, but severely inhibited the phosphorylation of JNK1/2 and ERK1/2. The neutralizing antibodies of anti-Dectin-1 and anti-TLR2 hardly affected the inhibition of NO production. All of these results suggested that the suppression of LPS-induced NO and TNF-α production was at least partially attributable to the inhibition of JNK1/2 and ERK1/2 activation. This work discovered a promising molecule to control the diseases associated with overproduction of NO and TNF-α.     

Anti-inflammatory activity of myricetin from Diospyros lotus through suppression of NF-κB and STAT1 activation and Nrf2-mediated HO-1 induction in lipopolysaccharide-stimulated RAW264.7 macrophages

Diospyros lotus is traditionally used for the treatment of diabetes, diarrhea, tumor, and hypertension. The purpose of this study was to investigate the anti-inflammatory effect and underlying molecular mechanisms of myricetin in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. Myricetin dose-dependently suppressed the production of pro-inflammatory mediators (NO, iNOS, PGE₂, and COX-2) in LPS-stimulated RAW264.7 macrophages. Myricetin administration decreased the production of NO, iNOS, TNF-α, IL-6, and IL-12 in mice. Myricetin decreased NF-κB activation by suppressing the degradation of IκBα, nuclear translocation of p65 subunit of NF-κB, and NF-κB DNA binding activity in LPS-stimulated RAW264.7 macrophages. Moreover, myricetin attenuated the phosphorylation of STAT1 and the production of IFN-β in LPS-stimulated RAW264.7 macrophages. Furthermore, myricetin induced the expression of HO-1 through Nrf2 translocation. In conclusion, these results suggest that myricetin inhibits the production of pro-inflammatory mediators through the suppression of NF-κB and STAT1 activation and induction of Nrf2-mediated HO-1 expression in LPS-stimulated RAW264.7 macrophages.     

Classical NF-κB activation impairs skeletal muscle oxidative phenotype by reducing IKK-α expression

Background: Loss of quadriceps muscle oxidative phenotype (OXPHEN) is an evident and debilitating feature of chronic obstructive pulmonary disease (COPD). We recently demonstrated involvement of the inflammatory classical NF-κB pathway in inflammation-induced impairments in muscle OXPHEN. The exact underlying mechanisms however are unclear. Interestingly, IκB kinase α (IKK-α: a key kinase in the alternative NF-κB pathway) was recently identified as a novel positive regulator of skeletal muscle OXPHEN. We hypothesised that inflammation-induced classical NF-κB activation contributes to loss of muscle OXPHEN in COPD by reducing IKK-α expression.Methods: Classical NF-κB signalling was activated (molecularly or by tumour necrosis factor α: TNF-α) in cultured myotubes and the impact on muscle OXPHEN and IKK-α levels was investigated. Moreover, the alternative NF-κB pathway was modulated to investigate the impact on muscle OXPHEN in absence or presence of an inflammatory stimulus. As a proof of concept, quadriceps muscle biopsies of COPD patients and healthy controls were analysed for expression levels of IKK-α, OXPHEN markers and TNF-α.Results: IKK-α knock-down in cultured myotubes decreased expression of OXPHEN markers and key OXPHEN regulators. Moreover, classical NF-κB activation (both by TNF-α and IKK-β over-expression) reduced IKK-α levels and IKK-α over-expression prevented TNF-α-induced impairments in muscle OXPHEN. Importantly, muscle IKK-α protein abundance and OXPHEN was reduced in COPD patients compared to controls, which was more pronounced in patients with increased muscle TNF-α mRNA levels.Conclusion: Classical NF-κB activation impairs skeletal muscle OXPHEN by reducing IKK-α expression. TNF-α-induced reductions in muscle IKK-α may accelerate muscle OXPHEN deterioration in COPD.     

Inhibitory effect of sulphated polysaccharide porphyran on nitric oxide production in lipopolysaccharide-stimulated RAW264.7 macrophages

Porphyran, extracted from an edible red alga (Porphyra yezoensis), is a sulphated polysaccharide with a wide variety of biological activities including anti-tumour, antioxidant and immuno-modulating activities. In this study, we examined the effect of porphyran on nitric oxide (NO) production in mouse macrophage cell line RAW264.7 cells. Although no significant activity of porphyran to induce NO or tumour necrosis factor-α (TNF-α) production in RAW264.7 cells was observed at the concentration range tested (10-500 μg/ml), it was found for the first time that porphyran inhibited NO production and expression of inducible nitric oxide synthase (iNOS) in RAW264.7 cells stimulated with lipopolysaccharide (LPS). In the presence of 500 μg/ml porphyran, NO production and expression of iNOS in LPS-treated RAW264.7 cells were completely suppressed. On the other hand, porphyran showed only a marginal effect on the secretion of TNF-α from LPS-stimulated RAW264.7 cells. Electrophoretic mobility shift assay (EMSA) using infrared dye labelled oligonucleotide with nuclear factor-κB (NF-κB) consensus sequence suggested that porphyran inhibited the LPS-induced NF-κB activation. The LPS-inducible nuclear translocation of p65, and the phosphorylation and degradation of IκB-α were also inhibited by the pre-treatment with porphyran. Our results obtained in in vitro analysis suggest that porphyran suppresses NO production in LPS-stimulated macrophages by the blocking of NF-κB activation.     

Enterovirus 71 2C protein inhibits TNF-α-mediated activation of NF-κB by suppressing IκB kinase β phosphorylation

Enterovirus 71 (EV71), a single, positive-stranded RNA virus, has been regarded as the most important neurotropic enterovirus after the eradication of the poliovirus. EV71 infection can cause hand, foot, and mouth disease or herpangina. Cytokine storm with elevated levels of proinflammatory and inflammatory cytokines, including TNF-α, has been proposed to explain the pathogenesis of EV71-induced disease. TNF-α-mediated NF-κB signaling pathway plays a key role in inflammatory response. We hypothesized that EV71 might also moderate host inflammation by interfering with this pathway. In this study, we tested this hypothesis and identified EV71 2C protein as an antagonist of TNF-α-mediated activation of NF-κB signaling pathway. Expression of 2C protein significantly reduced TNF-α-mediated NF-κB activation in 293T cells as measured by gene reporter and gel mobility shift assays. Furthermore, overexpression of TNFR-associated factor 2-, MEK kinase 1-, IκB kinase (IKK)α-, or IKKβ-induced NF-κB activation, but not constitutively active mutant of IKKβ (IKKβ SS/EE)-induced NF-κB activation, was inhibited by 2C protein. These data together suggested that the activation of IKKβ is most likely targeted by 2C; this notion was further strengthened by immunoblot detection of IKKβ phosphorylation and IκBα phosphorylation and degradation. Coimmunoprecipitation and colocalization of 2C and IKKβ expressed in mammalian cells provided compelling evidence that 2C interacts with IKKβ. Collectively, our data indicate that EV71 2C protein inhibits IKKβ activation and thus blocks NF-κB activation.     

Anti-inflammatory activities of inotilone from Phellinus linteus through the inhibition of MMP-9, NF-κB, and MAPK activation in vitro and in vivo

Inotilone was isolated from Phellinus linteus. The anti-inflammatory effects of inotilone were studied by using lipopolysaccharide (LPS)-stimulated mouse macrophage RAW264.7 cells and λ-carrageenan (Carr)-induced hind mouse paw edema model. Inotilone was tested for its ability to reduce nitric oxide (NO) production, and the inducible nitric oxide synthase (iNOS) expression. Inotilone was tested in the inhibitor of mitogen-activated protein kinase (MAPK)?[extracellular signal-regulated protein kinase (ERK), c-Jun NH(2)-terminal kinase (JNK), p38], and nuclear factor-κB (NF-κB), matrix-metalloproteinase (MMP)-9 protein expressions in LPS-stimulated RAW264.7 cells. When RAW264.7 macrophages were treated with inotilone together with LPS, a significant concentration-dependent inhibition of NO production was detected. Western blotting revealed that inotilone blocked the protein expression of iNOS, NF-κB, and MMP-9 in LPS-stimulated RAW264.7 macrophages, significantly. Inotilone also inhibited LPS-induced ERK, JNK, and p38 phosphorylation. In in vivo tests, inotilone decreased the paw edema at the 4(th) and the 5(th) h after Carr administration, and it increased the activities of catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx). We also demonstrated that inotilone significantly attenuated the malondialdehyde (MDA) level in the edema paw at the 5(th) h after Carr injection. Inotilone decreased the NO and tumor necrosis factor (TNF-α) levels on serum at the 5(th) h after Carr injection. Western blotting revealed that inotilone decreased Carr-induced iNOS, cyclooxygenase-2 (COX-2), NF-κB, and MMP-9 expressions at the 5(th) h in the edema paw. An intraperitoneal (i.p.) injection treatment with inotilone diminished neutrophil infiltration into sites of inflammation, as did indomethacin (Indo). The anti-inflammatory activities of inotilone might be related to decrease the levels of MDA, iNOS, COX-2, NF-κB, and MMP-9 and increase the activities of CAT, SOD, and GPx in the paw edema through the suppression of TNF-α and NO. This study presents the potential utilization of inotilone, as a lead for the development of anti-inflammatory drugs.     

Amelioration of colitis in mice by Leuconostoc lactis EJ-1 by M1 to M2 macrophage polarization

Dysregulation of immune responses to environmental antigens by the intestine leads to the chronic inflammatory disease, inflammatory bowel disease (IBD). Recent studies have thus sought to identify a dietary component that can inhibit lipopolysaccharide (LPS)-induced nuclear factor-kappa beta (NF-κB) signaling to ameliorate IBD. This study assessed if the lactic acid bacteria (LAB) from kimchi, suppresses the expression of tumor necrosis factor-alpha (TNF-α) in peritoneal macrophages induced by LPS. Leuconostoc lactis EJ-1, an isolate from LAB, reduced the expression of interleukin-6 (IL-6) and IL-1β in peritoneal macrophages induced by LPS. The study further tested whether EJ-1 alleviates colitis induced by 2,4,6-trinitrobenzene sulfonic acid (TNBS) in mice. TNBS significantly increased myeloperoxidase (MPO) expression, macroscopic colitis scores, and colon shortening. Oral administration of L. lactis EJ-1 resulted in an inhibited in TNBS-induced loss in body weight, colon shortening, MPO activity, and NF-κB and inducible nitric oxide synthase expression; it also led to a marked reduction in cyclooxygenase-2 expression. L. lactis EJ-1 also inhibited the TNBS-induced expression of TNF-α, IL-1β, and IL-6; however, it induced the expression of IL-10. The M2 macrophage markers arginase I, IL-10, and CD206 were elevated by EJ-1. Collectively, these results suggest that EJ-1 inhibits the NF-κB signaling and polarizes M1- to M2-macrophage transition, which help in ameliorating colitis.     

Downregulation of nuclear factor-kappa B (NF-κB) pathway by silibinin in human monocytes challenged with Paracoccidioides brasiliensis

AimsSilibinin is the major active component of silymarin, a polyphenolic plant flavonoid that has anti-inflammatory effects. The modulatory effect of silibinin on monocyte function against Paracoccidioides brasiliensis (Pb18) has not yet been demonstrated. The present study investigated whether the effect of silibinin on nuclear factor-kappa B (NF-κB) pathways may affect the production of tumor necrosis factor-alpha (TNF-α), interleukin-10 (IL-10), transforming growth factor beta (TGF-β1), prostaglandin E₂ (PGE₂), nitric oxide (NO) and fungicidal activity of human monocytes challenged in vitro with Pb18.Main methodsPeripheral blood monocytes from healthy individuals were treated with silibinin and challenged with Pb18 for 18 h. TNF-α, IL-10, TGF-β1 and PGE₂ expression were determined by immunoenzymatic assay (ELISA) and NO release was determined by the accumulation of nitrite in culture supernatants. Fungicidal activity of monocytes was analyzed after treatment with interferon-gamma plus silibinin and challenge with Pb18. NF-κB activation in cultured monocytes was evaluated by flow cytometry and ELISA.Key findingsSilibinin partially inhibited p65NF-κB activation as the number of cells expressing this factor was reduced and the concentration of nuclear p65NF-κB was low, compared to untreated controls. The addition of silibinin also resulted in suppression of TNF-α, IL-10, TGF-β1, PGE₂ and NO production but did not affect the fungicidal activity of monocytes against Pb18.SignificanceSilibinin exerts anti-inflammatory and anti-fibrotic effects on CD14± human monocytes challenged by Pb18 by partial inhibition of p65NF-κB activation.     

Omentin inhibits TNF-α-induced expression of adhesion molecules in endothelial cells via ERK/NF-κB pathway

In the present study, we investigated whether omentin affected the expression of intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in tumor necrosis factor-α (TNF-α) induced human umbilical vein endothelial cells (HUVECs). Our data showed that omentin decreased TNF-α-induced expression of ICAM-1 and VCAM-1 in HUVECs. In addition, omentin inhibited TNF-α-induced adhesion of THP-1 cells to HUVECs. Further, we found that omentin inhibited TNF-α-activated signal pathway of nuclear factor-κB (NF-κB) by preventing NF-κB inhibitory protein (IκBα) degradation and NF-κB/DNA binding activity. Omentin pretreatment significantly inhibited TNF-α-induced ERK activity and ERK phosphorylation in HUVECs. Pretreatment with PD98059 suppressed TNF-α-induced NF-κB activity. Omentin, NF-kB inhibitor (BAY11-7082) and ERK inhibitor (PD98059) reduced the up-regulation of ICAM-1 and VCAM-1 induced by TNF-α. These results suggest that omentin may inhibit TNF-α-induced expression of adhesion molecules in endothelial cells via blocking ERK/NF-κB pathway.     

The role of viral hemorrhagic septicemia virus (VHSV) NV gene in TNF-α- and VHSV infection-mediated NF-κB activation

The role of viral hemorrhagic septicemia virus (VHSV) NV gene in nuclear factor-κB (NF-κB) activation was investigated. Epithelioma papulosum cyprini (EPC) cells pre-treated with tumor necrosis factor (TNF)-α showed a strong resistance against VHSV infection, but cells treated with TNF-α after VHSV infection showed no resistance, suggesting that immediate early TNF-α-mediated responses inhibit VHSV replication. Activation of NF-κB is a key step in TNF-α-mediated immunomodulatory pathways. In this study, activation of NF-κB by TNF-α exposure was inhibited in EPC cells harboring NV gene expressing vectors, indicating that the NV gene of VHSV can suppress TNF-α-mediated NF-κB activation. Furthermore, the NV gene knock-out recombinant VHSV (rVHSV-ΔNV-EGFP) induced significantly higher NF-κB activity in EPC cells than wild-type VHSV, suggesting that VHSV adopted a strategy to suppress early activation of NF-κB in host cells through and NV gene.