Expression of cyclooxygenase-2 and pro-inflammatory cytokines induced by 2,2',4,4',5,5'-hexachlorobiphenyl (PCB 153) in human mast cells requires NF-kappa B activation

Mast cells are critical for initiating innate immune and inflammatory responses by releasing a number of pro-inflammatory mediators. The potential immunomodulatory properties of hydrogenated aromatic hydrocarbons have been the subject of extensive investigation, as the immune system is a sensitive target for hydrogenated aromatic hydrocarbon toxicity. In this report, the effects of polychlorinated biphenyl (PCB) on the expression of cyclooxygenase-2 and pro-inflammatory cytokines such as interleukin-1beta (IL-1beta), IL-6 and tumor necrosis factor (TNF)-alpha in the human leukemic mast cell line were investigated. TNF-alpha and IL-1beta expressed their respective mRNA in the presence or absence of PCB, while cyclooxygenase-2 (COX-2) and IL-6 mRNA expression were highly induced by PCB after 2 h. Moreover, pre-treatment with the nuclear factor (NF)-kappaB pathway inhibitor, pyrrolidine dithiocarbamate, suppressed COX-2, TNF-alpha and IL-1beta induction and reduced the IL-6 mRNA levels induced by PCB. The NF-kappaB activity was determined by electrophoretic mobility shift analysis (EMSA) using an oligonucleotide containing a consensus NF-kappaB binding sequence. Stimulating the cells with PCB activated NF-kappaB. However, pre-treating them with a NF-kappaB pathway inhibitor, pyrrolidine dithiocarbamate, suppressed PCB-induced NF-kappaB activation. This suggests that PCB induces cycloxoygenase-2 and pro-inflammatory cytokine expression, and that this induction occurs through NF-kappaB.     

Identification of a signaling cascade for interleukin-8 production by Helicobacter pylori in human gastric epithelial cells

Infecting gastric epithelial cells with Helicobacter pylori (H. pylori) has been shown to induce interleukin-8 (IL-8) production, but the signal transduction mechanism leading to IL-8 production is not defined clearly. In the present study, we investigated the molecular mechanism responsible for H. pylori-induced IL-8 release in human gastric epithelial cells. IL-8 levels in culture supernatants were determined by an enzyme linked-immunosorbent assay. Extracellular signal-regulated kinase (ERK) activity was tested using an in vitro kinase assay, which measured the incorporation of [gamma-33P]ATP into a synthetic peptide that is a specific ERK substrate. ERK phosphorylation and IkappaBalpha degradation by H. pylori infection were assessed by western blotting. In MKN45 cells, H. pylori-induced IL-8 release in a time-dependent manner. This IL-8 release was abolished by treatment with intracellular Ca2+ chelators (BAPTA-AM and TMB-8) but not by EGTA or nifedipine. The Ca2+ ionophore A23187 also induced IL-8 release to an extent similar to that of H. pylori infection. Calmodulin inhibitors (W7 and calmidazolium) and tyrosine kinase inhibitors (genistein and ST638) completely blocked IL-8 release by H. pylori and A23187. PD98059, an ERK pathway inhibitor, completely abolished H. pylori-induced IL-8 release. Moreover, BAPTA-AM, calmidazolium, and genistein, but not nifedipine, suppressed the ERK activation induced by H. pylori infection. PD98059 as well as MG132, an NF-kappaB pathway inhibitor, blocked both IL-8 production and degradation of IkappaBalpha induced by H. pylori infection, whereas only PD98059 inhibited ERK activity in response to H. pylori. There was no significant difference between IL-8 production induced by the cagA positive wild-type strain and the cagA negative isogenic mutant strain of H. pylori; therefore, CagA is not involved in the IL-8 production pathway. H. pylori-induced IL-8 production is dominantly regulated by Ca2+/calmodulin signaling, and ERK plays an important role in signal transmission for the efficient activation of H. pylori-induced NF-kappaB activity, resulting in IL-8 production.     

羟基红花黄色素A对脑缺血大鼠皮层炎症信号转导途径相关因子的抑制作用

羟基红花黄色素A(hydroxysafflor yellow A，HSYA)是红花中的单体有效成分。本研究采用线栓法制备大鼠永久性脑缺血模型，观察HSYA对永久性脑缺血大鼠炎症信号转导途径相关因子的抑制作用。于缺血后3、 6、 12和24 h取大脑皮层。Western blotting 检测细胞核及胞浆核转录因子κB(NF-κB) p65及胞浆磷酸化IκB-α(pIκB-α)蛋白水平表达；Trans AM试剂盒检测NF-κB DNA结合活性；RT-PCR检测炎性因子TNF-α、 IL-1β、 IL-6和IL-10转录水平表达。结果表明，大鼠脑缺血后多次静脉注射HSYA(10 mg·kg^-1)，能显著抑制p65核转位以及IκB-α的磷酸化，降低NF-κB DNA结合活性，并降低促炎因子TNF-α、 IL-1β和IL-6 mRNA表达，升高抗炎因子IL-10 mRNA表达水平。提示HSYA的抗脑缺血作用可能与其抑制炎症信号途径中NF-κB激活及炎性因子转录水平表达有关。     

Inhibition by licochalcone A, a novel flavonoid isolated from liquorice root, of IL-1beta-induced PGE2 production in human skin fibroblasts

Licochalcone A, a novel flavonoid isolated from the root of Glycyrrhiza inflata, has been reported to exhibit anti-inflammatory activity in animal models. In this study, we examined the effect of licochalcone A on the production of chemical mediators such as prostaglandin (PG)E2 and cytokines by interleukin (IL)-1beta in human skin fibroblasts. Licochalcone A (IC50 15.0 nM) inhibited PGE2 production, but not IL-6 and IL-8 production, in response to IL-1beta. NS-398 (IC50 1.6 nM), a COX-2 selective inhibitor, also suppressed the PGE2 production. Furthermore, licochalcone A and NS-398 suppressed PGF(2alpha) production by IL-1beta. However, licochalcone A (1 microM) had no effect on increased levels of cyclooxygenase (COX)-2 mRNA and protein in cells. Dexamethasone (100 nM) not only inhibited PGE2, PGF(2alpha), IL-6 and IL-8 production but also strongly suppressed the expression of COX-2 mRNA and protein. Licochalcone A had no effect on COX-1-dependent PGE2 production, whereas indometacin (100 nM), a dual inhibitor of COX-1 and COX-2, was very effective. These results suggest that licochalcone A induces an anti-inflammatory effect through the inhibition of COX-2-dependent PGE2 production. Furthermore, it appears that the inhibitory effect of licochalcone A on PGE2 production in response to IL-1beta is quite different from that of the steroid.     

Changes in mRNA expression induced by sustained noradrenaline stimulation are different for alpha1-adrenoceptor subtypes in HEK293 cells

1. The aim of the present study was to investigate noradrenaline (NA)-induced regulation of alpha1-adrenoceptor (AR) mRNA expression in human embryonic kidney (HEK) 293 cells stably expressing cloned alpha1-AR subtypes with similar receptor densities. Stable transfection was performed by calcium phosphate precipitation. Receptor expression was detected by radioligand binding assay. The mRNA expression was measured by RNase protection assay. 2. alpha1-Adrenoceptor subtype mRNA respond in distinct ways following prolonged exposure to NA. The mRNA level of the alpha 1A-AR subtype was unchanged, the mRNA level of the alpha 1B-AR subtype was increased and the mRNA level of the alpha 1D-AR subtype declined time dependently. The protein kinase C (PKC) inhibitor calphostin C or RO 31-8220 abolished the NA-induced downregulation of alpha 1D-AR mRNA. Phorbol myristate acetate (PMA), a PKC activator, similarly repressed the effects of NA on alpha 1D-AR. However, calphostin C, RO 31-8220 or PMA had no effect on the induction of alpha 1B-AR mRNA by NA. The Ca2+-ATPase inhibitor thapsigargin or the calcium chelator 1,2-bis-(o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid tetra (acetoxymethyl) ester (BAPTA/AM) had no effect on the repression of alpha 1D-AR mRNA, but did inhibit the induction of alpha 1B-AR mRNA by NA. Noradrenaline significantly decelerated the degradation of alpha 1B-AR mRNA, but had no effect on the degradation of alpha 1D-AR mRNA. 3. Thus, the mRNA expression of three alpha1-AR subtypes in HEK293 cells is differentially regulated through distinct signal transduction pathways under sustained NA stimulation. The upregulation of alpha 1B-AR mRNA is via the Ca2+ pathway, whereas the downregulation of alpha 1D-AR mRNA is via the PKC pathway.     

Gigantol isolated from the whole plants of Cymbidium goeringii inhibits the LPS-induced iNOS and COX-2 expression via NF-kappaB inactivation in RAW 264.7 macrophages cells

During our efforts to find bioactive natural products with anti-inflammatory activity, we isolated gigantol from the whole plants of Cymbidium goeringii (Orchidaceae) by activity-guided chromatographic fractionation. Gigantol was found to have potent inhibitory effects on LPS-induced nitric oxide (NO) and prostaglandin E (2) (PGE (2)) production in RAW 264.7 cells. Consistent with these findings, gigantol suppressed the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) at the protein and mRNA levels in RAW 264.7 cells in a concentration-dependent manner. Our data also indicate that gigantol is a potent inhibitor of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta) and interleukin-6 (IL-6) release and influenced the mRNA expression levels of these cytokines in a dose-dependent manner. Furthermore, a reporter gene assay for nuclear factor kappa B (NF-kappaB) and an electromobility shift assay (EMSA) demonstrated that gigantol effectively inhibited the activation of NF-kappaB, which is necessary for the expression of iNOS, COX-2, TNF-alpha, IL-1beta and IL-6. Thus, our studies suggest that gigantol inhibits LPS-induced iNOS and COX-2 expression by blocking NF- kappaB activation.     

Protein kinase C (PKC) dependent induction of tissue factor (TF) by mesangial cells in response to inflammatory mediators and release during apoptosis

1. In inflammatory kidney diseases procoagulatory activity (PCA) becomes evident. Glomerular fibrin deposits and capillary microthrombi are histopathological hallmarks in most forms of glomerulonephritis. 2. Therefore in this study the expression of tissue factor (TF) as the main inducer of thrombogenesis was examined in cultured human mesangial cells (MC) in response to proinflammatory stimuli such as interleukin-1 (IL-1 beta), tumour necrosis factor alpha (TNF-alpha) and lipopolysaccharide (LPS). Also main signalling pathways were investigated. 3. IL-1 beta, TNF-alpha and LPS induced TF in MC in a time and dose dependent manner on mRNA and protein levels. Highest activity was found after 12 h of stimulation. Induction of TF was completely blockable by BAPTA-AM, a chelator of intracellular [Ca(2+)](i) as well as calphostin, a protein kinase C (PKC) inhibitor. Activation of the protein kinase A (PKA) pathway had no influence on basal TF expression, but down-regulated cytokine-induced TF. The PKA blocker, KT5720, increased TF formation significantly. Since TF exerts its activity primarily on the surface of cells and after release of encrypted receptors we further tested TF activity in MC supernatants. IL-1 beta did not significantly increase TF activity in supernatants of intact cells. However, when MC were rendered apoptotic by oxidative metabolites, IL-1 beta treated MC released highly stimulated TF activity into the supernatants, suggesting that a paracrine activation of the coagulatory cascade can take place under such conditions. 4. Inflammatory mediators up-regulate TF expression in MC by a PKC dependent pathway whereas PKA can serve as a negative feed-back link. Apoptosis of inflammatory MC may trigger to spread PCA.     

Inhibition of TNF-alpha,IL-1beta,and IL-6 productions and NF-kappa B activation in lipopolysaccharide-activated RAW 264.7 macrophages by catalposide,an iridoid glycoside isolated from Catalpa ovata G. Don (Bignoniaceae)

Catalposide, the major iridoid glycoside isolated from the stem bark of Catalpa ovata G. Don (Bignoniaceae), was found to inhibit the productions of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and interleukin-6 (IL-6), and the activation of nuclear factor kappaB (NF-kappaB) in RAW 264.7 macrophages activated with lipopolysaccharide (LPS). Catalposide also inhibited the expressions of TNF-alpha, IL-1beta, and IL-6 genes and the nuclear translocation of p65 subunit of NF-kappaB in LPS-activated RAW 264.7 cells. Flow cytometric analysis revealed that catalposide suppressed the binding of FITC-conjugated LPS to CD14 on the surface of cells, probably resulting in the inhibitory effects on TNF-alpha, IL-1beta, and IL-6 productions and NF-kappaB activation. These findings suggest that catalposide could be an attractive candidate for adjunctive therapy in gram-negative bacterial infections.     

C-reactive protein augments interleukin-8 secretion in human peripheral blood monocytes

C-reactive protein (CRP) is a powerful predictor and risk factor for cardiovascular diseases. The CXC- and CC-type chemokines interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) are important chemokines for leukocyte trafficking identified in atheromatous plaque expressed mainly by macrophages in humans. We assessed whether C-reactive protein could induce MCP-1 and IL-8 secretion. In human peripheral blood monocytes, C-reactive protein (12.5-50 microg/mL) increased IL-8, but not MCP-1 secretion in a time- (6-24 hours) and dose-dependent manner as detected by ELISA. C-reactive protein could augment the production of reactive oxygen species (ROS) as measured by chemiluminescence and inhibitors of NAD(P)H oxidase (DPI and PAO) and ROS scavengers (superoxide dismutase, catalase, and 1% dimethyl sulphoxide) abolished C-reactive protein-induced IL-8 secretion. Furthermore, relative quantity of IL-8 mRNA was significantly increased by C-reactive protein 50 microg/mLfor 12 hours, which could be inhibited by DPI 1 microM or superoxide dismutase (SOD) 250 U/mL. The inhibitors of ERK 1/2 (PD98059), p38 (SB203580) MAPK, and NF-kappaB (PDTC and MG132) significantly decreased C-reactive protein-induced IL-8 secretion in human monocytes. Also, agonists of peroxisome proliferator-activated receptor (PPAR) alpha (WY14643) and PPARgamma (troglitazone) could largely inhibit C-reactive protein responses. Thus, our data indicate that C-reactive protein at pathologic levels increases IL-8 secretion and mRNA via enhancing ROS derived mainly from NAD(P)H oxidase and the subsequent activation of ERK1/2, p38 MAPK, and NF-kappaB. The activation of PPARalpha/gamma can negatively regulate C-reactive protein-induced IL-8 production in human monocytes.     

mGluR1-mediated potentiation of NMDA receptors involves a rise in intracellular calcium and activation of protein kinase C

Potentiation of ionotropic glutamate receptor activity by metabotropic glutamate receptors (mGluRs) is thought to modulate activity at glutamatergic synapses in the hippocampus. However, the precise pathway by which this modulation occurs is not well understood. The present study tests the hypothesis that mGluR1-mediated potentiation of N-methyl-D-aspartate receptors (NMDARs) occurs via a phospholipase C (PLC)-initiated cascade. NMDAR functional activity was examined by whole-cell recording from Xenopus oocytes expressing recombinant NMDARs and mGluR1alpha. The mGluR1 agonist (1S,3R)-1-amino-cyclopentane-1,3-dicarboxylic acid (ACPD) significantly potentiated NMDA-elicited currents. mGluR1alpha-mediated potentiation of NMDA responses was eliminated by the PLC inhibitor U-73122. Buffering of intracellular Ca2+ by BAPTA-AM or depletion of intracellular Ca2+ by the Ca2+/ATPase inhibitor thapsigargin greatly reduced ACPD potentiation. ACPD potentiation was reduced by the specific protein kinase C (PKC) inhibitor Ro-32-0432 and eliminated by the broad spectrum kinase inhibitor staurosporine. ACPD produced no further potentiation after potentiation of NMDARs by the PKC-activating phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA). Thus, Group I mGluRs potentiate NMDA responses via activation of PLC; at least part of the potentiation is due to rise in intracellular Ca2+ and stimulation of PKC. Cytochalasin D, which disrupts the actin cytoskeleton, blocked ACPD-elicited chloride currents and ACPD-induced potentiation of NMDAR currents, consistent with a role for cytoskeletal protein(s) in the signaling pathway. As Group I mGluRs are localized to the perisynaptic region in juxtaposition to NMDARs at glutamatergic synapses, mGluR-mediated potentiation of NMDAR activity may play a role in synaptic transmission and plasticity including LTP.     

The anti-inflammatory effect of Gigukjiwhangwhangami through the inhibition of nuclear factor-kappaB activation in the peripheral blood mononuclear cells of patients with cerebral infarction

The Korean genuine medicine "Gigukjiwhangwhangami (GJWGM)" has long been used for various cerebrovascular diseases. However, the exact mechanism that accounts for the anti-inflammatory effect of GJWGM is not completely understood. The aim of the present study is to elucidate how GJWGM modulates the inflammatory reaction in lipopolysaccaride (LPS)-stimulated peripheral mononuclear cells from patients with cerebral infarction. Production of cytokine was measured by the ELISA and RT-PCR method. The level of nuclear factor-kappaB (NF-kappaB)/Rel A protein and NF-kappaB DNA binding activity were determined by the Western blot analysis and TF-EIA method. We showed that GJWGM inhibited the production of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1alpha, IL-6, and IL-8 induced by LPS in dose dependent manner (p<0.05). Maximal inhibition rate of TNF-alpha, IL-1beta, IL-6 and IL-8 production by GJWGM was about 54.34%, 41.37%, 44.04%, and 54.46%, respectively. GJWGM inhibited the TNF-alpha and IL-8 mRNA expression. In addition, we showed that the inhibitory mechanism of GJWGM is through the suppression of NF-kappaB pathway. Our study suggests that an important molecular mechanism by GJWGM reduce inflammation, which may explain its beneficial effect in the regulation of inflammatory reactions.