Glutamine Reduces TNF-α by Enhancing Glutathione Synthesis in Lipopolysaccharide-Stimulated Alveolar Epithelial Cells of Rats

To investigate the role of glutathione (GSH) synthesis in the regulation on nuclear factor (NF)-κB activity and tumor necrosis factor-alpha (TNF-α) release by glutamine (GLN) in lipopolysaccharide (LPS)-stimulated alveolar type II (AT-II) epithelial cells of rat lungs. Primary cultured AT-II cells were pre-treated with various doses of GLN for 2, 8, 16, 24 h. At the 8 h time point before LPS stimulation, various doses of l-buthionine-(S,R)-sulfoximine (BSO), an inhibitor of GSH synthesis, were added with 10 mM GLN. Then the cells were stimulated with 1 μg/ml LPS for 24 h. The cells were obtained for GSH measurement. TNF-α level in the supernatant was determined by enzyme-linked immunosorbent assay. NF-κB activity was assessed by electrophoretic mobility shift assay. Eight hours before LPS exposure was the best time point for GLN’s enhancing GSH synthesis. LPS could significantly decrease the GSH level, increase NF-κB activation and TNF-α release in AT-II cells. Supplementation of GLN could increase the GSH level and attenuate the release of TNF-α in LPS-stimulated AT-II cells in a dose-dependant manner. And NF-κB activation also could be prevented by GLN. BSO could block the effect of GLN. As a precursor of GSH, glutamine could prevent the NF-κB activation and attenuate the release of TNF-α in LPS-stimulated AT-II cells and the effect may be mediated via GSH synthesis.     

META060 inhibits multiple kinases in the NF-κB pathway and suppresses LPS – mediated inflammation in vitro and ex vivo

Objective:  We investigated whether a novel candidate META060 targeted the inflammatory signal transduction without affecting constitutive COX-2 enzymatic activity in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. We also investigated its bioavailability in humans and its anti-inflammatory effect ex vivo. Methods:  We measured prostaglandin E₂, nitric oxide, TNFα and IL-6 by ELISA, COX-2 protein by Western blot, NF-κB nuclear binding by electrophoretic mobility shift assays, and NF-κB activation by luciferase assay. Kinase inhibitions were measured by cell-free assays. Bioavailability was tested in 4 human subjects consuming 940 mg META060. LPS-activated TNFα and IL-6 were measured in peripheral blood mononuclear cells (PBMC) isolated from 1 subject up to 6 hours post administration. Results:  META060 dose-dependently inhibited prostaglandin E₂ and nitric oxide formation, COX-2 abundance, and NF-κB activation. In cell-free assays, META060 inhibited multiple kinases in the NF-κB signaling pathway, including BTK, PI3K, and GSK3. META060 was detected in the plasma of the subjects; isolated PBMC were resistant to LPS-stimulated TNFα and IL-6 production. Conclusion:  Without inhibiting COX-2 enzyme, META060 reduces the inflammation by inhibiting multiple kinases involved in NF-κB pathway, and may have potential as a safe anti-inflammatory therapeutic. Received 28 July 2008; returned for revision 30 October 2008; received from final revision 4 November 2008; accepted by J. Di Battista 13 November 2008     

Effects of Trefoil Peptide 3 on Expression of TNF-α, TLR4, and NF-κB in Trinitrobenzene Sulphonic Acid Induced Colitis Mice

The trefoil factor (TFF) peptides are major secretory products of mucus cells of the gastrointestinal tract. There were evidences that administration of recombinant human TFF3 is effective in treatment of models of colitis, but the mechanism of the effects of rTFF3 is not fully understood. The main aims of this study is to evaluate effects of intraperitoneal injection recombinant human TFF3 on the expression of tumour necrosis factor α (TNF-α), toll-like receptor 4(TLR4), and nuclear factor κB (NF-κB) in trinitrobenzene sulphonic acid (TNBS) induced colitis mice. Distal colitis was induced in BALB/C mice by intracolonic administration of TNBS in ethanol. Treated with administration rhTFF3 for treatment group(5 mg/ml; approximately 0.5 mg/mouse), and normal saline for control for 5 consecutive days. Colonic damage score, tissue myeloperoxidase (MPO) activity, TLR4, NF-κB mRNA expression, and tissue TNF-α, TLR4, NF-κB production were determined, respectively. Once daily application of hTFF3 for 5 days after TNBS/ethanol had been injected, both microscopic and macroscopic injury and inflammatory index had been reduced compared with controls. In addition, decreased tissue TNF-α, TLR4, NF-κB production, and TLR4, NF-κB mRNA expression had been found. This study has shown that hTFF3 may have therapeutic potential in the treatment of inflammatory bowel disease, and one of the mechanisms may related to inhibit the TLR4/NF-κB signaling pathways.     

Hyaluronan inhibits cytokine production by lipopolysaccharide-stimulated U937 macrophages through down-regulation of NF-κB via ICAM-1

Objective: This study investigated the inhibitory mechanism of hyaluronan (HA) on lipopolysaccharide (LPS)-stimulated production of proinflammatory cytokines in U937 macrophages. Methods: HA was added to U937 macrophage cultures in the presence of LPS, with or without pretreatment with anti-intercellular adhesion molecule-1 (ICAM-1) antibody. Secreted levels of tumor necrosis factor α (TNFα), interleukin (IL)-1β, and IL-6 were determined by enzyme-linked immunosorbent assay. The phosphorylation of nuclear factor (NF)-κB, IκBα, and mitogen-activated protein kinases (MAPKs) was analyzed by immunoblotting. Results: LPS stimulated production of TNFα, IL-1β, and IL-6. In contrast to 800 kDa HA, 2700 kDa HA at 1 mg/ml inhibited LPS-induced cytokine production. Anti-ICAM-1 antibody blocked the effects of HA on the LPS actions on U937 cells. LPS activated NF-κB and MAPK pathways, whereas HA down-regulated p65 NF-κB and IκBα phosphorylation by LPS without affecting MAPKs. Inhibition studies revealed the requirement of NF-κB for LPS-stimulated cytokine production. Anti-ICAM-1 antibody reversed the inhibitory effects of HA on phosphorylation of p65 NF-κB and IκBα. Conclusion: HA of intrinsic molecular weight suppresses LPS-stimulated production of proinflammatory cytokines via ICAM-1 through down-regulation of NF-κB and IκB. Exogenous HA injected into arthritic joints could act as an anti-NF-κB agent by the mechanism demonstrated in the present study. Received 23 September 2006; returned for revision 12 October 2006; accepted J. Di Battista 18 December 2006     

Preventive Effects of Valnemulin on Lipopolysaccharide-Induced Acute Lung Injury in Mice

Valnemulin reportedly regulates inflammatory responses in addition to its in vitro antibacterial activity. In this study, we established a mouse model of lipopolysaccharide (LPS)-induced inflammatory lung injury and investigated the effect of valnemulin (100 mg/kg) on acute lung injury (ALI) 8 h after LPS challenge. We prepared bronchoalveolar lavage fluid (BALF) for measuring protein concentrations, cytokine levels, and superoxidase dismutase (SOD) activity, and collected lungs for assaying wet-to-dry weight (W/D) ratios, myeloperoxidase (MPO) activity, cytokine mRNA expression, and histological change. We found that the pre-administration of valnemulin significantly decreases the W/D ratio of lungs, protein concentrations, and the number of total cells, neutrophils, macrophages, and leukomonocytes, and histologic analysis indicates that valnemulin significantly attenuates tissue injury. Furthermore, valnemulin significantly increases LPS-induced SOD activity in BALF and decreases lung MPO activity as well. In addition, valnemulin also inhibits the production of tumor necrosis factor-α, interleukin-6, and interleukin-1β, which is consistent with mRNA expression in lung. The results showed that valnemulin had a protective effect on LPS-induced ALI in mice.     

In situ glomerular expression of activated NF-κB in human lupus nephritis and other non-proliferative proteinuric glomerulopathy

Nuclear Factor-κB (NF-κB) has been suggested to play a role in the cellular and molecular mechanisms underlying glomerular injury. We investigated the potential role of NF-κB activation in the pathogenesis of glomerular injury in 31 patients with class III–V lupus nephritis (LN), 14 patients with non-proliferative proteinuric glomerulopathy and six normal controls. The expression of NF-κB subunits p65 and p50, and the NF-κB regulated proinflammatory mediators tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6) and intercellular adhesion molecule-1 (ICAM-1) as well as CD68 and synaptopodin was examined by Southwestern histochemistry (SWH) or immunohistochemistry. In contrast to non-proliferative glomerulopathy and normal controls, NF-κB activation (both p65 and p50) was enhanced in glomerular endothelial, mesangial cells or infiltrating cells in class IV LN, along with upregulation of TNF-α, IL-1β, IL-6 and ICAM-1 expression. Glomerular endothelial and mesangial activation of NF-κB and mesangial ICAM-1 expression correlated with disease activity and the level of glomerular macrophage infiltration. Podocyte NF-κB overactivation (predominantly p65) paralleled podocyte expression of TNF-α and IL-1β in patients with LN and non-proliferative glomerulopathy. Podocyte staining scores of NF-κB and p65 were positively correlated with the severity of proteinuria in LN and non-proliferative glomerulopathy. These results suggest a pathogenic role for NF-κB in glomerular injury by multiple mechanisms.     

Verapamil modulates LPS-induced cytokine production via inhibition of NF-kappa B activation in the liver

Objective: To investigate the effect of verapamil on Lipopolysaccharide (LPS)-induced cytokines [tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-10 (IL-10)] and nuclear factor kappa B (NF-κ B) in the liver. Methods and Materials: Adult male Sprague-Dawley rats were randomly divided into seven groups of eight rats each: control rats treated with saline (0.9 % NaCl); rats treated with saline and then challenged intraperitoneally with LPS (10 mg/kg); rats treated intraperitoneally with different levels of verapamil (1, 2.5, 5, 10 mg/kg) and then challenged with LPS (10 mg/kg); and rats treated only with verapamil (10 mg/kg). TNF-α, IL-6, IL-10 and NF-κ B in the liver tissues were investigated as well as the serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) one hour after LPS injection. Results: LPS alone stimulated production of TNF-α, IL-6 and IL-10, and activated NF-κ B in the liver. Pretreatment with verapamil before LPS challenge reduced acute liver injury, down-regulated production of LPS-induced pro-inflammatory cytokines (TNF-α and IL-6), up-regulated production of anti-inflammatory cytokines (IL-10) and inhibited NF-κ B activation in the liver in a dose-dependent manner. Conclusion: Verapamil can attenuate acute liver injury by down-regulating the production of TNF-α and IL-6 and up-regulating IL-10 in the liver, possibly via inhibition of NF-κ B. Received 8 August 2005; returned for revision 25 September 2005; returned for final revision 18 November 2005; accepted by M. Katori 6 December 2005     

Taurine Attenuates Liver Injury by Downregulating Phosphorylated p38 MAPK of Kupffer Cells in Rats with Severe Acute Pancreatitis

This study was undertaken to clarify the effects of taurine on liver injury in rats with severe acute pancreatitis (SAP). Rats were randomly assigned to three groups: a sham operation (SO), a SAP (established by infusion of 5% taurocholate), and a SAP given taurine (Taur). At 12 and 24 h post-operation, taurine pretreatment significantly attenuated hepatic tissue injury induced by SAP, and concurrently, serum alanine aminotransferase, aspartate transaminase, and amylase levels were significantly reduced by taurine pretreatment. Compared with the SO group, the total and phosphorylated p38 mitogen-activated protein kinase (p38 MAPK) expression and nuclear factor-κB (NF-κB) activity of Kupffer cells (KCs) were significantly higher in the SAP group, but taurine pretreatment inhibited the total and phosphorylated p38 MAPK expression and NF-κB activity of KCs in the SAP group. The increase of tumor necrosis factor-α and interleukin-lβ in cultured supernate of the SAP rat-derived KCs was also significantly inhibited by taurine pretreatment. These results suggest that taurine pretreatment ameliorated liver injury in rats with SAP mainly by inhibiting phosphorylated p38 MAPK and NF-κB activity in KCs, which may play an important role in liver injury.     

Effects of Protein-Energy Malnutrition on NF-KappaB Signalling in Murine Peritoneal Macrophages

Protein-energy malnutrition (PEM) is an important public health problem affecting millions of people worldwide. PEM decreases resistance to infection, impairing a number of physiological processes. In unstimulated cells, NF-κB is kept from binding to its consensus sequence by the inhibitor IκBα, which retains NF-κB in the cytoplasm. Upon various signals, such as lipopolysaccharide (LPS), IκBα is rapidly degraded and NF-κB is induced to translocate into the nucleus, where it activates expression of various genes that participate in the inflammatory response, including those involved in the synthesis of TNF-α. TRAF-6 is a cytoplasmic adapter protein that links the stimulatory signal from Toll like receptor-4 to NF-κB. The aim of this study was to evaluate the effect of malnutrition on induction of TNF-α by LPS in murine peritoneal macrophages. We evaluated peritoneal cellularity, the expression of MyD88, TRAF-6, IKK, IκBα and NF-κB, NF-κB activation and TNF-α mRNA and protein synthesis in macrophages. Two-month-old male BALB/C mice were submitted to PEM with a low-protein diet that contained 2% protein, compared to 12% protein in the control diet. When the experimental group had lost about 20% of the original body weight, it was used in the subsequent experiments. Malnourished animals presented anemia, leucopenia and severe reduction in peritoneal cavity cellularity. TNF-α mRNA and protein levels of macrophages stimulated with LPS were significantly lower in malnourished animals. PEM also decreased TRAF-6 expression and NF-κB activation after LPS stimulation. These results led us to conclude that PEM changes NF-kB signalling pathway in macrophages to LPS stimulus.     

Schisantherin A Exhibits Anti-inflammatory Properties by Down-Regulating NF<Emphasis Type="Italic">-</Emphasis>κB and MAPK Signaling Pathways in Lipopolysaccharide-Treated RAW 264.7 Cells

Schisantherin A, a dibenzocyclooctadiene lignan isolated from the fruit of Schisandra sphenanthera, has been used as an antitussive, tonic, and sedative agent under the name of Wuweizi in Chinese traditional medicine. In the present study, we carry out a screening program to identify the anti-inflammatory potentials of schisantherin A. We found that schisantherin A reduced lipopolysaccharide (LPS (1 mg/L))-induced levels of TNF-α, IL-6, NO, and PGE2 (p < 0.01 or p < 0.05), and also reduced levels of iNOS and COX-2 in RAW 264.7 macrophages in a concentration-dependent manner. We further investigated signal transduction mechanisms to determine how schisantherin A affects. RAW264.7 cells were pretreated with 0.5, 2.5, or 25 mg/L of schisantherin A 1 h prior to treatment with 1 mg/L of LPS. Thirty minutes later, cells were harvested and mitogen activated protein kinases (MAPKs) activation and IκBα was measured by Western blot. Alternatively, cells were fixed and nuclear factor-κB (NF-κB) activation was measured using immunocytochemical analysis. Signal transduction studies showed that schisantherin A significantly inhibited extracellular signal-regulated kinase (ERK), p38, and c-jun NH2-terminal kinase (JNK) phosphorylation protein expression. Schisantherin A also inhibited p65-NF-κB translocation into the nucleus by IκBα degradation. By using specific inhibitors of ERK, JNK and p38, we found that schisantherin A may inhibit TNF-α mostly through ERK pathway. Therefore, schisantherin A may inhibit LPS-induced production of inflammatory cytokines by blocking NF-κB and MAPKs signaling in RAW264.7 cells.     

Rebamipide inhibits tumor necrosis factor-α-induced interleukin-8 expression by suppressing the NF-κB signal pathway in human umbilical vein endothelial cells

Objective  

This study was designed to identify the inhibitory effect of rebamipide on tumor necrosis factor-α (TNF-α)-induced interleukin-8 (IL-8) production and nuclear factor-κB (NF-κB) activation in human umbilical vein endothelial cells (HUVECs).     

Cavidine Ameliorates Lipopolysaccharide-Induced Acute Lung Injury <Emphasis Type="Italic">via</Emphasis> NF-κB Signaling Pathway <Emphasis Type="Italic">in vivo</Emphasis> and <Emphasis Type="Italic">in vitro</Emphasis>

Acute lung injury (ALI) is characterized by widespread inflammation in the lungs and alveolar-capillary destruction, causing high morbidity and mortality. Cavidine, isolated from Corydalis impatiens, have been exhibited to have potent anti-inflammatory effects in previous studies. The purpose of this study was to evaluate the protective effect of cavidine on lipopolysaccharide (LPS)-induced ALI and to enunciate the underlying in vivo and in vitro mechanisms. Mice were intraperitoneally administrated with cavidine (1, 3, or 10 mg/kg) at 1 and 12 h, prior to the induction of ALI by intranasal administration of LPS (30 mg/kg). Blood samples, lung tissues, and bronchoalveolar lavage fluid (BALF) were harvested after LPS challenge. Furthermore, we used LPS-induced lung epithelial cells A549 to examine the mechanism of cavidine to lung injury. The results showed that pretreatment with cavidine significantly decreased lung wet-to-dry weight (W/D) ratio, reduced pro-inflammatory cytokine levels including TNF-α and IL-6 in BALF and serum from LPS-stimulated mice, and attenuated lung histopathological changes. In addition, western blot results showed that cavidine inhibited the phosphorylation of nuclear factor-kappaB (NF-κB) p65 and IκBα induced by LPS. In conclusion, our results demonstrate that cavidine protects against LPS-induced acute lung injury in mice via inhibiting of pro-inflammatory cytokine TNF-α and IL-6 production and NF-κB signaling pathway activation. Taken together, cavidine may be useful for the prevention and treatment of pulmonary inflammatory diseases, such as ALI.     

Effects of Urinary Trypsin Inhibitor on Lipopolysaccharide-Induced Acute Lung Injury in Rabbits

This study was undertaken to clarify the effects of urinary trypsin inhibitor (UTI) on lipopolysaccharide (LPS)-induced acute lung injury. Rabbits were randomly assigned to one of seven groups: saline only, UTI, LPS, pre- or post-UTI-high (infusion of UTI of 25,000 U/kg followed by 25,000 U/kg over 2 h), pre- or post-UTI-low (infusion of UTI of 2,500 U/kg followed by 2,500 U/kg over 2 h). UTI was administered 30 min before (pre-groups) or 15 min after (post-groups) LPS administration. Rabbits were mechanically ventilated with 40% oxygen for 6 h. LPS decreased peripheral blood leukocyte counts and increased wet/dry weight ratio of lung, lung injury score, neutrophil infiltration in lung, and IL-8 production in systemic blood and bronchoalveolar lavage fluid (BALF). Rabbits treated by UTI were protected from LPS-induced lung injury, as determined by wet/dry weight ratio, neutrophil infiltration in lung, lung injury score, and IL-8 in BALF levels. UTI attenuated LPS-induced acute lung injury in rabbits mainly by inhibiting neutrophil and IL-8 responses, which may play a central role in sepsis-related lung injury.     

1-磷酸鞘氨醇受体2抑制脂多糖诱导的急性肺损伤

 目的: 探讨1-磷酸鞘氨醇受体2(S1P2R)对脂多糖(LPS)诱导的急性肺损伤(ALI)中的作用及机制。方法: 野生小鼠和S1pr2^-/-小鼠经气管滴注LPS,建立急性肺损伤动物模型。LPS注射24 h时观察肺组织的病理改变,测定支气管肺泡灌洗液(BALF)中的蛋白浓度、总细胞数、中性粒细胞的比值及TNF-α、IL-6细胞因子的表达。为了观察S1P2R在肺损伤中的作用机制,LPS注射10 min前野生小鼠和S1pr2^-/-小鼠经尾静脉注射一氧化氮合酶抑制剂L-NAME,LPS注射12 h时,再观察肺的病理组织学变化以及BALF中的蛋白浓度,总细胞数及TNF-α、IL-6细胞因子表达的变化。结果: 与野生小鼠比较,S1pr2^-/-小鼠恶化LPS诱导的急性肺损伤,BALF中的蛋白浓度、总细胞数,中性粒细胞比值及炎症细胞因子表达显著增加。而L-NAME的预处理显著抑制在S1pr2^-/-小鼠LPS诱导加重的急性肺损伤。结论: S1P2R通过抑制NO合成,维持血管屏障,从而抑制急性肺损伤。