Saccharated colloidal iron enhances lipopolysaccharide-induced nitric oxide production in vivo

We investigated the effects of iron on the production of nitric oxide (NO), inducible NO synthase (iNOS), and plasma cytokines induced by lipopolysaccharide (LPS) in vivo. Male Wistar rats were preloaded with a single intravenous injection of saccharated colloidal iron (Fesin, 70 mg iron/kg body weight) or normal saline as a control, and then given an intraperitoneal injection of LPS (5.0 mg/kg body weight). Rats, preloaded with iron, had evidence of both iron deposition and strong iNOS induction in liver Kupffer cells upon injection of LPS; phagocytic cells in the spleen and lung had similar findings. LPS-induced NO production in iron-preloaded rats was significantly higher than control rats as accessed by NO-hemoglobin levels measured by ESR (electron spin resonance) and NOx (nitrate plus nitrite) levels. Western blot analysis showed that iron preloading significantly enhanced LPS-induced iNOS induction in the liver, but not in the spleen or lung. LPS-induced plasma levels of IL-6, IL-1beta, and TNF-alpha were also significantly higher in iron-preloaded rats as shown by ELISA, but IFN-gamma levels were unchanged. We conclude that colloidal-iron phagocytosed by liver Kupffer cells enhanced LPS-induced NO production in vivo, iNOS induction in the liver, and release of IL-6, IL-1beta, and TNF-alpha.     

Effects of esculetin on lipopolysaccharide (LPS)-induced acute lung injury via regulation of RhoA/Rho Kinase/NF-кB pathways in vivo and in vitro

The purpose of the present study was to investigate the protective effect of esculetin (ES) in lipopolysaccharide (LPS)-induced acute lung injury (ALI) and the lung epithelial A549 cells. Mice were intragastrically administered with ES (20 and 40 mg/kg) 1 h prior to LPS challenge. ES pretreatment at doses of 20 and 40 mg/kg effectively attenuated LPS-induced lung histopathological change, myeloperoxidase or MPO activity, inflammatory cells infiltration, pulmonary wet-to-dry weight ratio, and the generation of pro-inflammatory cytokines including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) in vivo and in vitro. Furthermore, we demonstrated that ES blocked the activation of NF-кB and RhoA/Rho kinase pathways in LPS-induced mice and A549 cells. The results suggested that ES exhibited protective effect on ALI and might attribute partly to the inhibition of NF-кB and RhoA/Rho kinase pathways in vivo and in vitro.     

Inhibitory mechanisms of highly purified vitamin B2 on the productions of proinflammatory cytokine and NO in endotoxin-induced shock in mice

Inhibitory effects of highly purified vitamin B2 (riboflavin-5'-sodium phosphate, >97%) on the interleukin (IL)-6, macrophage inflammatory protein (MIP)-2 and nitric oxide (NO) in LPS-induced shock mice were evaluated. Vitamin B2 at 20 mg/kg (protective effect on mice mortality induced by LPS), intravenously administered 6 h after LPS injection, significantly decreased the plasma elevated levels of IL-6 and MIP-2 at 9 and 12 h. In addition, vitamin B2 lowered the tissue concentration and the mRNA expression of IL-6 in lung and those of MIP-2 in liver at 9 h. Vitamin B2 also reduced concentration of MIP-2 concentration in lung, and inhibited mRNA expression in kidney, respectively. Vitamin B2 decreased the plasma elevated NO levels in accordance with a reduction in expression of inducible NO synthase (iNOS) both at 21 and 24 h. Accordingly, the reduction in elevated plasma cytokine levels and NO based on the inhibitory effect on local cytokine mRNA expression and iNOS would be responsible for the anti-septic effect of vitamin B2.     

The protective effect of 5-O-methylvisammioside on LPS-induced depression in mice by inhibiting the over activation of BV-2 microglia through Nf-κB/IκB-α pathway

Background5-O-methylvisammioside (MeV), also known as 4′-O-β-D-glucosyl-5-O-methylvisamminol, is a conventional marker compound for quality control of roots of Saposhnikovia diviaricata (Radix Saposhnikoviae), which exhibits anti-inflammatory and neuroprotective activities.PurposeAccording to the activity of MeV, we speculated that MeV may have antidepressant effect on LPS induced depression, and further explored its mechanism.Study DesignFirst, to explore the effect and mechanism of MeV on LPS-induced depression in mice, and then to further explore the effect and mechanism of MeV on LPS-activated BV-2 microglia.MethodsBy the OFT, EPM, TST and FST behavioral tests, to explore the effect of MeV pretreatment on the behavior of LPS-induced depression mice. ELISA and Griess method were used to detect the changes of the serum TNF-α and IL-6 levels, the hippocampus SOD and MDA levels, and the NO, SOD, MDA, TNF-α and IL-6 levels in the culture medium of LPS-stimulated BV-2 microglia. Western blot was used to analyze the protein expression in the Nf-κB/IκB-α and BDNF/TrkB pathway in the hippocampus of mice and BV-2 microglia.ResultsMeV (4 mg/kg, i.p.) pretreatment significantly improves the activity and exploration ability of LPS-induced depression mice, and reduces the immobility time. MeV inhibited the production of pro-inflammatory cytokines in the serum of mice induced by LPS, such as IL-6 and TNF-α. MeV also increased the levels of SOD and reduces the expression of MDA in the hippocampus, thus promoting the alleviation of depressive symptoms in mice. Western blotting analysis showed that the antidepressant activity of MeV was related to the decrease of Nf-κB nuclear transport, the inhibition of IκB-α phosphorylation, and the increase of BDNF and TrkB expression. MeV (40 μM) significantly reduced the contents of NO, MDA, TNF-α and IL-6 in the culture medium of LPS-stimulated BV-2 microglia, and increased the content of SOD.ConclusionMeV can regulate the neurotrophic factors in the mouse brain, reduce the content of inflammatory factors by the Nf-κB/IκB-α pathway, improve oxidative stress, and inhibit the excessive activation of LPS-stimulated BV -2 microglia. It effectively reversed the depression-like behAavior induced by LPS in mice.     

Inhibitory effect of vitamin E on proinflammatory cytokines-and endotoxin-induced nitric oxide release in alveolar macrophages

Nitric oxide is thought to be an important modulator of various functions in normal and inflamed airways. In the present study, we evaluated the effects of high vitamin E (250 mg and 1250 mg alpha-tocopheryl acetate (TA)/kg diet/10 days) on nitric oxide (NO(.)) release by alveolar macrophages (AMs) in response to lipopolysaccharide (LPS), interleukin-1beta (IL-1beta) and tumor necrosis factor (TNF-alpha). LPS and IL-1beta treatment (1-10 microg/ml) enhanced NO(.) release in AMs from control animals fed on 50 mg vitamin E/kg diet in a concentration dependent manner. However, this enhancement of NO(.) was attenuated in the AMs of animals fed with 250 mg or 1250 mg vitamin E/kg diet. TNF-alpha had no effect in eliciting the release of NO(.) in AMs obtained either from control or from hyper vitamin E fed animals. Further, LPS (1-10 microg/ml) enhanced the inducible nitric oxide synthase (iNOS) activity of AMs of control group and TA-fed animals almost to equal extent. Similarly, LPS-induced formation of N-nitrosamine (N-nitroso-L-[(14)C]-proline) in AMs of control and TA-supplemented animals were not different statistically. On the other hand, in vitro addition of vitamin E (200 microM) in AMs of control animals, when triggered with 10 microg LPS/ml, caused a significant decrease in N-nitroso-L-[(14)C]-proline formation. It seems that high doses of TA in diet may play a role in reducing the lipopolysaccharide and proinflammatory cytokines-induced NO(.)-mediated damage by AMs.     

Up-regulation of IL-18BP,but not IL-18 mRNA in rat liver by LPS

Interleukin (IL)-18 is a pro-inflammatory cytokine that plays a critical role in inflammation leading to liver damage, through promotion of Fas-mediated apoptosis. Inhibition of IL-18 activity protects against LPS-induced lethality in mice and against liver damage induced by LPS after sensitisation of mice with Proprionibacterium acnes. A specific, potent, endogenous inhibitor of IL-18 (IL-18BP) has been identified in mice and humans, and IL-18BP mRNA is expressed constitutively in liver. The objectives of this study were to compare changes in IL-1beta and IL-18 mRNA expression in the liver of rats in response to peripheral injection of LPS, using real-time PCR, and also to investigate whether IL-18BP mRNA expression is affected by this treatment. LPS rapidly up-regulated IL-1beta mRNA expression, but IL-18 mRNA expression was unaffected by LPS treatment. Unlike IL-18, IL-18BP mRNA was up-regulated dramatically by approximately 12-fold above nai;ve levels, peaking 3 h after LPS injection. This ability of LPS to up-regulate expression of the endogenous IL-18 inhibitor may indicate a mechanism by which the inflammatory response to LPS is regulated.     

Pretreatment with granulocyte-colony stimulating factor decreases lipopolysaccharide-induced interferon-gamma production in mice in association with the production of interleukin-18

We studied the effects of pretreatment with granulocyte-colony stimulating factor (G-CSF) on the production of pro- and anti-inflammatory cytokines induced by lipopolysaccharide (LPS). Mice received G-CSF or control saline once a day for 7 days or once at 1 h before the injection of LPS. Cytokines were measured by enzyme-linked immunosorbent assay or antibody-based electrochemiluminescence assay and cytokine mRNA was measured by RNAse protection assay. Mice pretreated with G-CSF for 7 days before LPS had lower serum levels of LPS-induced interferon-gamma (IFN-gamma) and higher levels of interleukin (IL)-6 and IL-10 than controls. G-CSF-pretreated mice also had lower mRNA levels of IFN-gamma and higher mRNA levels of IL-6 and IL-10 in the spleen and/or liver than controls. G-CSF-pretreated mice had serum levels of tumor necrosis factor, IL-12 p70 and IL-12 p40 similar to controls. G-CSF-pretreated mice had lower levels of spleen IL-18 than controls-serum IL-18 being undetectable in mice after LPS-and lower levels of IL-18 mRNA in the spleen. Mice pretreated with G-CSF 1 h before LPS had lower levels of serum IFN-gamma and spleen IL-18 than controls. G-CSF pretreatment alters the expression of LPS-induced cytokines with a decrease in pro-inflammatory IFN-gamma and an increase in anti-inflammatory IL-6 and IL-10. G-CSF decrease of IL-18 production may be a major mechanism explaining the effects of G-CSF on the production of IFN-gamma.     

IL-18-binding protein protects against lipopolysaccharide- induced lethality and prevents the development of Fas/Fas ligand-mediated models of liver disease in mice.

IL-18-binding protein (IL-18BP) is a natural IL-18 inhibitor. Human IL-18BP isoform a was produced as fusion construct with human IgG1 Fc and assessed for binding and neutralizing IL-18. IL-18BP-Fc binds human, mouse, and rat IL-18 with high affinity (K(D) 0.3-5 nM) in a BIAcore-based assay. In vitro, IL-18BP-Fc blocks IL-18 (100 ng/ml)-induced IFN-gamma production by KG1 cells (EC(50) = 0.3 microg/ml). In mice challenged with an LD(90) of LPS (15 mg/kg), IL-18BP-Fc (5 mg/kg) administered 10 min before LPS blocks IFN-gamma production and protects against lethality. IL-18BP-Fc administered 10 min before LPS blocks IFN-gamma production induced by LPS (5 mg/kg) with ED(50) of 0.005 mg/kg. Furthermore, IL-18BP-Fc (5 mg/kg) abrogates LPS (5 mg/kg)-induced IFN-gamma production even when administered 6 days before LPS but shows no effect when administered 9 or 12 days before LPS. Given 10 min before LPS challenge to mice primed 12 days in advance with heat-killed Propionibacterium acnes, IL-18BP-Fc prevents LPS-induced liver damage and IFN-gamma and Fas ligand expression. Given at the moment of priming with P. acnes, IL-18BP-Fc decreases P. acnes-induced granuloma formation, macrophage-inflammatory protein-1alpha and macrophage-inflammatory protein-2 production and prevents sensitization to LPS. IL-18BP-Fc also prevents Con A-induced liver damage and IFN-gamma and Fas ligand expression as well as liver damage induced by Pseudomonas aeruginosa exotoxin A or by anti-Fas agonistic Ab. In conclusion, IL-18BP can be engineered and produced in recombinant form to generate an IL-18 inhibitor, IL-18BP-Fc, endowed with remarkable in vitro and in vivo properties of binding and neutralizing IL-18.     

Melatonin inhibits expression of the inducible NO synthase II in liver and lung and prevents endotoxemia in lipopolysaccharide-induced multiple organ dysfunction syndrome in rats.

We evaluated the role of melatonin in endotoxemia caused by lipopolysaccharide (LPS) in unanesthetized rats. The expression of inducible isoform of nitric oxide synthase (iNOS) and the increase in the oxidative stress seem to be responsible for the failure of lungs, liver, and kidneys in endotoxemia. Bacterial LPS (10 mg/kg b. w) was i.v. injected 6 h before rats were killed and melatonin (10-60 mg/kg b.w.) was i.p. injected before and/or after LPS. Endotoxemia was associated with a significant rise in the serum levels of aspartate and alanine aminotransferases, gamma-glutamyl-transferase, alkaline phosphatase, creatinine, urea, and uric acid, and hence liver and renal dysfunction. LPS also increased serum levels of cholesterol and triglycerides and reduced glucose levels. Melatonin administration counteracted these organ and metabolic alterations at doses ranging between 20 and 60 mg/kg b. w. Melatonin significantly decreased lung lipid peroxidation and counteracted the LPS-induced NO levels in lungs and liver. Our results also show an inhibition of iNOS activity in rat lungs by melatonin in a dose-dependent manner. Expression of iNOS mRNA in lungs and liver was significantly decreased by melatonin (60 mg/kg b. w., 58-65%). We conclude that melatonin inhibits NO production mainly by inhibition of iNOS expression. The inhibition of NO levels may account for the protection of the indoleamine against LPS-induced endotoxemia in rats.     

Lipopolysaccharide-induced liver apoptosis is increased in interleukin-10 knockout mice

Although IL-10 down-regulates pro-inflammatory cytokine secretion by hepatic Kupffer cells, the mechanisms underlying its hepatoprotective effects are not fully clear. This study tested the hypothesis that IL-10 protects the liver against pro-inflammatory cytokines by counteracting their pro-apoptotic effects. Wild type and IL-10 knockout mice were treated with bacterial lipopolysaccharide and sacrificed 1, 4, 8, and 12 h later. Plasma ALT activity was measured as a marker of liver injury. Liver pathology and TUNEL response were assessed by histology. Plasma levels and whole liver mRNA levels were measured for TNF-alpha, IL-1 beta, TGF-beta1, IL-10, and their respective receptors. Hepatic mRNA levels were measured for several pro-apoptotic adaptors/regulators, including FasL, Fas receptor, FADD, TRADD, Bad, Bak, Bax, and Bcl-X(S), and anti-apoptotic regulators, including Bcl-w, Bcl-X(L), Bcl-2, and Bfl-1. Caspase-3 activity in the liver was determined as well as immunohistochemistry for IL-1RII, TGF-betaRII and Fas receptor. At all time points the livers from IL-10 knockout mice displayed a significantly increased number of apoptotic nuclei compared to wild type mice. Changes in plasma cytokine levels and their liver mRNA levels were consistent with suppression by IL-10 of pro-inflammatory cytokine secretion. In addition, pro-inflammatory cytokine receptor mRNA levels (TNF-alpha, TGF-beta, and IL-1 beta) were markedly up-regulated by LPS at all time points in IL-10 knockout mice as compared to wild type mice. Expression of the pro-inflammatory cytokine receptor IL-1RII was similarly increased as shown by immunostaining. The mRNA levels of a typical pro-apoptotic cytokine, TRAIL, were increased and LPS also up-regulated the mRNA expression of other apoptotic factors to a larger extent in IL-10 knockout mice than in their wild type counterparts, suggestive of an IL-10 anti-apoptotic effect. In the livers of knockout mice, markedly increased caspase-3 activity was already evident at the 1-h time point following LPS administration, while in the wild type animals this increase was delayed. Immunostaining also indicated that LPS increased hepatic expression of the pro-apoptotic receptors Fas and TGF-betaRII in IL-10 knockout mice. The data presented in this study show that: (i) IL-10 modulates not only the secretion of pro-inflammatory cytokines, but also the receptors of these cytokines, and ii) IL-10 protects the liver against LPS-induced injury at least in part by counteracting pro-inflammatory cytokine-induced liver apoptosis.     

Increased resistance of LFA-1-deficient mice to lipopolysaccharide-induced shock/liver injury in the presence of TNF-alpha and IL-12 is mediated by IL-10: a novel role for LFA-1 in the regulation of the proinflammatory and anti-inflammatory cytokine balance

Challenge with low doses of LPS together with D-galactosamine causes severe liver injury, resulting in lethal shock (low dose LPS-induced shock). We examined the role of LFA-1 in low dose LPS-induced shock. LFA-1(-/-) mice were more resistant to low dose LPS-induced shock/liver injury than their heterozygous littermates, although serum levels of TNF-alpha and IL-12 were higher in these mice. C57BL/6 mice were not rescued from lethal effects of LPS by depletion of NK1(+) cells, granulocytes, or macrophages, and susceptibility of NKT cell-deficient mice was comparable to that of controls. High numbers of platelets were detected in the liver of LFA-1(+/-) mice after low dose LPS challenge, whereas liver accumulation of platelets was only marginal in LFA-1(-/-) mice. Following low dose LPS challenge, serum levels of IL-10 were higher in LFA-1(-/-) mice than in LFA-1(+/-) mice, and susceptibility to low dose LPS-induced shock as well as platelet accumulation in the liver of LFA-1(-/-) mice were markedly increased by IL-10 neutralization. Serum levels of IL-10 in LFA-1(+/-) mice were only marginally affected by macrophage depletion. However, in LFA-1(-/-) mice macrophage depletion markedly reduced serum levels of IL-10, and as a corollary, susceptibility of LFA-1(-/-) mice to low dose LPS-induced shock was markedly elevated despite the fact that TNF-alpha levels were also diminished. We conclude that LFA-1 participates in LPS-induced lethal shock/liver injury by regulating IL-10 secretion from macrophages and that IL-10 plays a decisive role in resistance to shock/liver injury. Our data point to a novel role of LFA-1 in control of the proinflammatory/anti-inflammatory cytokine network.     

Prophylactic effects of sulforaphane on depression-like behavior and dendritic changes in mice after inflammation

Inflammation plays a role in the pathophysiology of depression. Sulforaphane (SFN), an isothiocyanate compound derived from broccoli, is a potent activator of the NF-E2-related factor-2 (Nrf2), which plays a role in inflammation. In this study, we examined whether the prevention effects of SFN in lipopolysaccharide (LPS) induced depression-like behavior in mice. Pretreatment with SFN significantly blocked an increase in the serum tumor necrosis factor-α (TNF-α) level and an increase in microglial activation of brain regions after a single administration of LPS (0.5 mg/kg). Furthermore, SFN significantly potentiated increased serum levels of IL-10 after LPS administration. In the tail-suspension test and forced swimming test, SFN significantly attenuated an increase of the immobility time after LPS administration. In addition, SFN significantly recovered to control levels for LPS-induced alterations in the proteins such as brain-derived neurotrophic factor, postsynaptic density protein 95 and AMPA receptor 1 (GluA1) and dendritic spine density in the brain regions. Finally, dietary intake of 0.1% glucoraphanin (a glucosinolate precursor of SFN) food during the juvenile and adolescence could prevent the onset of LPS-induced depression-like behaviors and dendritic spine changes in the brain regions at adulthood. In conclusion, these findings suggest that dietary intake of SFN-rich broccoli sprout has prophylactic effects on inflammation-related depressive symptoms. Therefore, supplementation of SFN-rich broccoli sprout could be prophylactic vegetable to prevent or minimize the relapse by inflammation in the remission state of depressed patients.     

LPS-induced biomarkers in mice: a potential model for identifying insulin sensitizers

The contribution of nutrient overload and associated inflammation to insulin resistance has highlighted several therapeutic targets including c-Jun N-terminal kinase (JNK) and S6 kinase (S6K). To investigate how a lipopolysaccharide (LPS)-mediated inflammatory response may modulate pathways implicated in insulin resistance, we characterized the LPS-induced changes in key biomarkers. Administration of 0.06-4 mg/kg LPS to C57BL/6 mice stimulated increases in plasma levels of TNFalpha, IL-12p40, IL-6 and MCP-1 and in JNK activity as measured by phosphorylated c-Jun in fat. For the first time, we show that LPS induces S6K activity by up to 6.1-fold, as measured by the phosphorylation of S6 ribosomal protein in liver, and increases by up to 1.8-fold, plasma levels of the novel pro-inflammatory cytokine osteopontin which is implicated in the pathogenesis of insulin resistance. These novel findings suggest that LPS administration may form the basis of an acute in vivo pharmacodynamic model for therapies targeting multiple pathways implicated in insulin resistance.     

Suppressive activity of macrolide antibiotics on nitric oxide production by lipopolysaccharide stimulation in mice

BACKGROUND: Low-dose and long-term administration of macrolide antibiotics into patients with chronic airway inflammatory diseases could favorably modify their clinical conditions. However, the therapeutic mode of action of macrolides is not well understood. Free oxygen radicals, including nitric oxide (NO), are well recognized as the important final effector molecules in the development and the maintenance of inflammatory diseases. PURPOSE: The influence of macrolide antibiotics on NO generation was examined in vivo. METHODS: Male ICR mice, 5 weeks of age, were orally administered with either roxithromycin, clarithromycin, azithromycin or josamycin once a day for 2-4 weeks. The mice were then injected intraperitoneally with 5.0 mg/kg lipopolysaccharide (LPS) and the plasma NO level was examined 6 h later. RESULTS: Although pre-treatment of mice with macrolide antibiotics for 2 weeks scarcely affected NO generation by LPS injection, the administration of macrolide antibiotics, except for josamycin, for 4 weeks significantly inhibited LPS-induced NO generation. The data in the present study also showed that pre-treatment of mice with macrolide antibiotics for 4 weeks significantly suppresses not only production of pro-inflammatory cytokines interleukin-1beta, interleukin-6, and tumor necrosis factor-alpha, but also inducible nitric oxide synthase mRNA expressions, which are enhanced by LPS injection. CONCLUSION: These results strongly suggest that suppressive activity of macrolide antibiotics on NO generation in response to LPS stimulation in vivo may, in part, account for the clinical efficacy of macrolides on chronic inflammatory diseases.