Induction Of Inducible Nitric Oxide Synthase Research Articles

Anti-Inflammatory Effects of Jakyakgamcho-Tang in IL-4- and TNF-α-Stimulated Lung Epithelial Cells and Lipopolysaccharide-Stimulated Macrophages

Asthma is a chronic respiratory disease mediated by airway inflammation. Jakyakgamcho-tang (JGT), a traditional medicine, is widely subscribed to common diseases such as muscle pain and cramps in East Asian countries. Although the efficacy of JGT on peripheral neuropathy, gouty arthritis, and colitis has been reported, the effect of JGT on airway inflammation related to asthma is not clearly investigated. In this study, we aimed to evaluate the effects of JGT water extract (JGTW) on factors related to airway inflammation using the human bronchial epithelial BEAS-2B and the mouse monocyte-macrophage RAW264.7 cell lines. Furthermore, the constituents in JGTW were quantitatively and qualitatively studied for future reference of JGTW standardization. JGTW reduced the generation of several airway inflammation mediators such as eotaxins, regulated on activation normal T-cell expressed and secreted (RANTES), and matrix metalloproteinase-9, and expressions of adhesion molecules (ICAM-1 and VCAM-1), which attracts leukocytes to the site of inflammation in interleukin-4 + tumor necrosis factor-α (IT)-stimulated BEAS-2B cells. In lipopolysaccharide-stimulated RAW264.7 cells, JGTW effectively suppressed inducible nitric oxide synthase (iNOS) induction by inhibiting the MAPK and NF-κB signaling. In addition, JGTW treatment showed decreased inflammatory cells and Th2 cytokines in bronchoalveolar lavage fluid and decreased IgE levels in plasma in the OVA-induced asthmatic mice model. In the ultra-performance liquid chromatography-diode array detector-tandem mass spectrometry analysis, 24 phytochemicals were identified in JGTW, and paeoniflorin (63.971 mg/g) and glycyrrhizin (11.853 mg/g) were found to be the most abundant. These findings suggest that JGTW has anti-inflammatory effects on airway inflammation by regulating inflammatory response-related factors, possibly through MAPK and NF-κB in pulmonary epithelial cells and macrophages.

9,10-Phenanthrenequinone provokes dysfunction of brain endothelial barrier through down-regulating expression of claudin-5

Chronic exposure to diesel exhaust particle (DEP) is considered to provoke dysfunction of the blood-brain barrier, but the detailed molecular mechanism remains unclear. In this study, we investigated the toxic effects of five DEP components against human vascular cells and found that, among them, 9,10-phenanthrenequinone (9,10-PQ), a major tricyclic quinone in DEP, most potently elicits the cellular toxicities. Additionally, treatment with 9,10-PQ at its cytolethal concentrations (more than 2 μM) facilitated the production of reactive oxygen species (ROS), caspase activation, and DNA fragmentation in human brain microvascular endothelial (HBME) cells, inferring that high concentrations of 9,10-PQ elicit the cell apoptosis through the ROS-dependent mechanism. Measurement of trans-endothelial electrical resistance and paracellular permeability showed that treatment with sublethal concentrations (less than 1 μM) of 9,10-PQ elevates permeability across HBME cell monolayer. Immunofluorescence observation and Western blotting analysis also revealed that the 9,10-PQ treatment remarkably down-regulated the intercellular localization and expression of claudin-5 (CLDN5), a tight junctional protein that plays a key role in function of the blood-brain barrier, and the down-regulation was markedly recovered by pretreatment with a proteasome inhibitor Z-Leu-Leu-Leu-CHO. This result may indicate that sublethal concentrations of 9,10-PQ facilitate the dysfunction of the endothelial cell barrier through lowering in the expression and proteasomal proteolysis of CLDN5. The treatment with 9,10-PQ promoted nitric oxide (NO) production presumably through the induction of inducible NO synthase. In addition, the 9,10-PQ-mediated down-regulation of CLDN5 was ameliorated and deteriorated by pretreating with a scavenger and donor, respectively, of NO. Similarly to the 9,10-PQ treatment, treatment with a donor of peroxynitrite, a highly reactive oxidant formed by the reaction of NO and superoxide anion, resulted in the marked reduction of CLDN5 expression and elevation of 26S proteasome-based proteolytic activities. Thus, it is suggested that the formation of NO and peroxynitrite participates in the mechanism of brain endothelial cell barrier dysfunction elicited by 9,10-PQ.

The Role of HSP70 in the Protective Effects of NVP-AUY922 on Multiple Organ Dysfunction Syndrome in Endotoxemic Rats.

Sepsis is defined as a life-threatening organ dysfunction syndrome with high morbidity and mortality caused by bacterial infection. The major characteristics of sepsis are systemic inflammatory responses accompanied with elevated oxidative stress, leading to multiple organ dysfunction syndrome (MODS), and disseminated intravascular coagulation (DIC). As a molecular chaperon to repair unfolded proteins, heat shock protein 70 (HSP70) maintains cellular homeostasis and shows protective effects on inflammatory damage. HSP 90 inhibitors were reported to exert anti-inflammatory effects via activation of the heat shock factor-1 (HSF-1), leading to induction of HSP70. We evaluated the beneficial effect of HSP 90 inhibitor NVP-AUY 922 (NVP) on multiple organ dysfunction syndrome induced by lipopolysaccharide (LPS) and further explored the underlying mechanism. NVP (5 mg/kg, i.p.) was administered 20 h prior to LPS initiation (LPS 30 mg/kg, i.v. infusion for 4 h) in male Wistar rats. Results demonstrated that pretreatment with NVP significantly increased survival rate and prevented hypotension at 6 h after LPS injection. Plasma levels of ALT, CRE and LDH as well as IL-1β and TNF-α were significantly reduced by NVP at 6 h after LPS challenge. The induction of inducible NO synthase in the liver, lung and heart and NF-κB p-p65 and caspase 3 protein expression in the heart were also attenuated by NVP. In addition, NVP markedly induced HSP70 and HO-1 proteins in the liver, lung and heart after LPS injection. These results indicated that NVP possessed the anti-inflammatory and antioxidant effects on LPS-induced acute inflammation, which might be associated with HSP70 and HO-1, leading to prevent MODS in sepsis. NVP might be considered as a novel therapeutic strategy in the prevention of sepsis-induced MODS.

The Janus Kinase Inhibitor Ruxolitinib Prevents Terminal Shock in a Mouse Model of Arenavirus Hemorrhagic Fever.

Arenaviruses such as Lassa virus cause arenavirus hemorrhagic fever (AVHF), but protective vaccines and effective antiviral therapy remain unmet medical needs. Our prior work has revealed that inducible nitric oxide synthase (iNOS) induction by IFN-γ represents a key pathway to microvascular leak and terminal shock in AVHF. Here we hypothesized that Ruxolitinib, an FDA-approved JAK inhibitor known to prevent IFN-γ signaling, could be repurposed for host-directed therapy in AVHF. We tested the efficacy of Ruxolitinib in MHC-humanized (HHD) mice, which develop Lassa fever-like disease upon infection with the monkey-pathogenic lymphocytic choriomeningitis virus strain WE. Anti-TNF antibody therapy was tested as an alternative strategy owing to its expected effect on macrophage activation. Ruxolitinib but not anti-TNF antibody prevented hypothermia and terminal disease as well as pleural effusions and skin edema, which served as readouts of microvascular leak. As expected, neither treatment influenced viral loads. Intriguingly, however, and despite its potent disease-modifying activity, Ruxolitinib did not measurably interfere with iNOS expression or systemic NO metabolite levels. These findings suggest that the FDA-approved JAK-inhibitor Ruxolitinib has potential in the treatment of AVHF. Moreover, our observations indicate that besides IFN-γ-induced iNOS additional druggable pathways contribute essentially to AVHF and are amenable to host-directed therapy.

Inhibitory effects of cynaropicrin and related sesquiterpene lactones from leaves of artichoke (Cynara scolymus L.) on induction of iNOS in RAW264.7 cells and its high-affinity proteins.

The methanolic extract of the leaves of artichoke (Cynara scolymus L.) was found to inhibit nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Among the constituents of the extract, six sesquiterpene lactones (cynaropicrin, grosheimin, 11β,13-dihydrocynaropicrin, 3β-hydroxy-8α-[(S)-3-hydroxy-2-methylpropionyloxy]guaia-4(15),10(14),11(13)-trien-1α,5α,6βH-12,6-olide, 3β-hydroxy-8α-[2-methoxymethyl-2-propenoyloxy]guaia-4(15),10(14),11(13)-trien-1α,5α,6βH-12,6-olide, and deacylcynaropicrin) inhibited NO production and/or inducible nitric oxide synthase (iNOS) induction. The acyl group having an α,β-unsaturated carbonyl group at the 8-position and the α-methylene-γ-butyrolactone moiety were important for the strong inhibitory activity. Our results suggested that these sesquiterpene lactones inhibited the LPS-induced iNOS expression via the suppression of the JAK-STAT signaling pathway in addition to the κNF-κB signaling pathway. With regard to the target molecules of the sesquiterpene lactones, high-affinity proteins of cynaropicrin were purified from the cell extract. ATP/ADP translocase 2 and tubulin were identified and suggested to be involved in the cytotoxic effects of cynaropicrin, although the target molecules for the inhibition of iNOS expression were not clarified.

Animal and Cellular Studies Demonstrate Some of the Beneficial Impacts of Herring Milt Hydrolysates on Obesity-Induced Glucose Intolerance and Inflammation.

The search for bioactive compounds from enzymatic hydrolysates has increased in the last few decades. Fish by-products have been shown to be rich in these valuable molecules; for instance, herring milt is a complex matrix composed of lipids, nucleotides, minerals, and proteins. However, limited information is available on the potential health benefits of this by-product. In this context, three industrial products containing herring milt hydrolysate (HMH) were tested in both animal and cellular models to measure their effects on obesity-related metabolic disorders. Male C57Bl/6J mice were fed either a control chow diet or a high-fat high-sucrose (HFHS) diet for 8 weeks and received either the vehicle (water) or one of the three HMH products (HMH1, HMH2, and HMH3) at a dose of 208.8 mg/kg (representing 1 g/day for a human) by daily oral gavage. The impact of HMH treatments on insulin and glucose tolerance, lipid homeostasis, liver gene expression, and the gut microbiota profile was studied. In parallel, the effects of HMH on glucose uptake and inflammation were studied in L6 myocytes and J774 macrophages, respectively. In vivo, daily treatment with HMH2 and HMH3 improved early time point glycemia during the oral glucose tolerance test (OGTT) induced by the HFHS diet, without changes in weight gain and insulin secretion. Interestingly, we also observed that HMH2 consumption partially prevented a lower abundance of Lactobacillus species in the gut microbiota of HFHS diet-fed animals. In addition to this, modulations of gene expression in the liver, such as the upregulation of sucrose nonfermenting AMPK-related kinase (SNARK), were reported for the first time in mice treated with HMH products. While HMH2 and HMH3 inhibited inducible nitric oxide synthase (iNOS) induction in J774 macrophages, glucose uptake was not modified in L6 muscle cells. These results indicate that milt herring hydrolysates reduce some metabolic and inflammatory alterations in cellular and animal models, suggesting a possible novel marine ingredient to help fight against obesity-related immunometabolic disorders.

The Proton Pump Inhibitor Lansoprazole Has Hepatoprotective Effects in In Vitro and In Vivo Rat Models of Acute Liver Injury.

The proton pump inhibitor lansoprazole (LPZ) is clinically used to reduce gastric acid secretion, but little is known about its possible hepatoprotective effects. This study aimed to investigate the hepatoprotective effects of LPZ and its potential mechanisms using in vitro and in vivo rat models of liver injury. For the in vitro model of liver injury, primary cultured rat hepatocytes were treated with interleukin-1β in the presence or absence of LPZ. The influence of LPZ on inducible nitric oxide synthase (iNOS) induction and nitric oxide (NO) production and on the associated signaling pathways was analyzed. For the in vivo model, rats were treated with D-galactosamine (GalN) and lipopolysaccharide (LPS). The effects of LPZ on survival and proinflammatory mediator expression (including iNOS and tumor necrosis factor-α) in these rats were examined. LPZ inhibited iNOS induction partially through suppression of the nuclear factor-kappa B signaling pathway in hepatocytes, thereby reducing potential liver injury from excessive NO levels. Additionally, LPZ increased survival by 50% and decreased iNOS, tumor necrosis factor-α, and cytokine-induced neutrophil chemoattractant-1 mRNA expression in the livers of GalN/LPS-treated rats. LPZ also inhibited nuclear factor-kappa B activation by GalN/LPS. LPZ inhibits the induction of several inflammatory mediators (including cytokines, chemokines, and NO) partially through suppression of nuclear factor-kappa B, resulting in the prevention of fulminant liver failure. The therapeutic potential of LPZ for liver injuries warrants further investigation.

Effect of the Lipid Peroxidation Product 4-Hydroxynonenal on Neuroinflammation in Microglial Cells: Protective Role of Quercetin and Monochloropivaloylquercetin

The lipid peroxidation-derived aldehyde 4-hydroxynonenal (HNE) has been implicated in a number of oxidative stress-induced inflammatory pathologies such as neurodegenerative diseases and aging. In this regard, we investigated the effects of HNE on neuroinflammatory responses by measuring cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) induction with cytokine production. In addition, we measured nuclear factor erythroid 2-related factor 2 (Nrf-2)/Kelch-like ECH-associated protein 1 (Keap1) signaling proteins, and antioxidant enzymes heme oxygenase-1 (HO-1) and nicotinamide adenine dinucleotide phosphate dehydrogenase, quinone 1 (NQO1), and compared the results with quercetin and monochloropivaloylquercetin (MPQ) pretreated microglial cells. Cytotoxicity was determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and production of cytokines was determined by cytokine array. Furthermore, intracellular Nfr2/Keap1 signaling proteins, HO-1, NQO1, and COX-2 expression were analyzed by western blot in 2.5 μM HNE treated BV-2 cells. Inducible nitric oxide synthase (iNOS) and COX-2 mRNA levels were measured with reverse transcription-quantitative polymerase chain reaction. HNE induced both COX-2 mRNA and protein levels, iNOS mRNA expression, and cytokine production. In addition, HNE markedly increased Keap1 levels and decreased cytoplasmic Nrf-2 expression with antioxidant enzyme HO-1 levels. Quercetin and monochloropivaloylquercetin treatment alleviated neuroinflammatory responses in microglial cells, by decreasing COX-2 mRNA expression. Monochloropivaloylquercetin decreased cytoplasmic Keap1 levels and increased nuclear translocation of Nrf-2 resulted in induction of HO-1 and NQO1 expression. These results suggest that HNE could be a link between oxidative stress and inflammation in BV-2 microglia cells. In particular, monochloropivaloylquercetin alleviated inflammation, probably by decreasing the expression of proinflammatory genes and strengthening the antioxidant defense system.

Arsenite suppresses NO production evoked by lipopolysaccharide and poly(I:C) via the suppression of interferon-β expression in RAW264.7 cells.

Immunological functions are disturbed in humans who have been chronically exposed to arsenic via contaminated groundwater. Little is known about the specific mechanisms underlying the impairment of immunological defense system caused by arsenic. The activation of macrophage cells upon infection with bacteria and viruses plays important roles in the defense against these pathogens. Here we show that exposure to arsenite (As(III)) suppresses nitric oxide (NO) production in murine RAW264.7 macrophage cells stimulated with lipopolysaccharide (LPS) and poly(I:C), the compounds mimicking bacterial and viral infection, respectively. As(III) suppressed the LPS- or poly(I:C)-evoked induction of inducible NO synthase (iNOS) without affecting the transactivation of NF-κB. As the interferon (IFN)-β/STAT1 pathway is also involved in the induction of iNOS in addition to NF-κB, we examined the effects of As(III) on the expression and secretion of IFN-β, the expression of the components of IFN-α/β receptor, the phosphorylation of STAT1, and the levels of cytokines involved in STAT1 activation. The results showed that the expression and secretion of IFN-β were specifically suppressed by As(III) treatment in RAW264.7 cells stimulated with LPS or poly(I:C). These results suggest that As(III) suppresses the expression and secretion of IFN-β, leading to the reduced STAT1 activation and consequently the reduced iNOS induction in macrophage cells. Our data suggest an important role of the arsenic-induced suppression of IFN-β on the disturbances in immunological defense against both bacteria and viruses.

L-Carnitine has a liver-protective effect through inhibition of inducible nitric oxide synthase induction in primary cultured rat hepatocytes

Background: L-Carnitine has protective effects on various injured organs. However, it has not been reported whether L-carnitine influences the induction of inducible nitric oxide synthase (iNOS) expression during inflammation. Nitric oxide (NO) produced by iNOS is an inflammatory indicator in organs which become inflamed, including the liver.Objective: This study aimed to examine whether L-carnitine influences the induction of iNOS gene expression in inflammatory cytokine-stimulated hepatocytes and the mechanisms involved in the action. Methods: L-Carnitine was added into the primary cultures of rat hepatocytes stimulated by interleukin-1β (an in vitro liver injury model). The production of NO and induction of iNOS and its signaling pathway were analyzed.Results: Transfection experiments with iNOS promoter-luciferase constructs revealed how L-carnitine inhibited iNOS mRNA synthesis activity and reduced its stability. In support of this observation, L-carnitine reduced iNOS mRNA and iNOS protein expression levels, resulting in reduced NO production. L-Carnitine blocked two essential pathways for iNOS induction: IκB kinase (IκB degradation/NF-κB activation) and phosphatidylinositol 3-kinase/Akt (type I IL-1 receptor upregulation).Conclusions: L-Carnitine inhibited the induction of inflammatory mediator iNOS, partially through inhibition of NF-κB activation, which demonstrated L-carnitine has protective effects in an in vitro liver injury model. L-Carnitine may have therapeutic potential for organ injuries, including the liver.Keywords: L-carnitine, hepatic encephalopathy, inducible nitric oxide synthase, liver injury, primary cultured hepatocytes, nuclear factor-κB, type I interleukin-1 receptor

Curcumin protects liver inflammation by suppressing expression of inducible nitric oxide synthase in primary cultured rat hepatocytes

Background: Curcumin has beneficial effects on organ metabolism. However, there is little evidence that curcumin affects inflammatory mediators, such as tumor necrosis factor (TNF)-α and nitric oxide (NO). In an inflamed liver, proinflammatory cytokines stimulate liver cells, followed by the induction of inducible NO synthase (iNOS). Excessive NO produced by iNOS is one of the factors in liver injury. Therefore, inhibiting iNOS induction for preventing liver injury is important.Objective: This study aimed to investigate liver protective effects of curcumin by examining interleukin (IL)-1β-stimulated hepatocytes.Methods: Primary cultured rat hepatocytes were treated with IL-1β in the presence or absence of curcumin. Induction of NO production and iNOS, and the signaling pathway of iNOS were analyzed.Results: Simultaneous addition of IL-1β and curcumin decreased expression levels of iNOS protein and mRNA, resulting in inhibition of NO production. Curcumin also reduced mRNA expression of TNF-α and IL-6. Curcumin inhibited two essential signaling pathways for iNOS induction, NF-κB activation and type I IL-1 receptor upregulation. Transfection experiments revealed that curcumin reduced iNOS mRNA levels at the promoter activation and mRNA stabilization steps. Delayed administration of curcumin after IL-1β addition also inhibited iNOS induction.Conclusions: Curcumin affects induction of inflammatory mediators, such as iNOS and TNF-α, in part through the inhibition of NF-κB activation in hepatocytes. Curcumin may have therapeutic potential for organ injuries, including the liver.Key words: curcumin, inducible nitric oxide synthase, liver injury, primary cultured hepatocytes, nuclear factor-κB, type I interleukin-1 receptor, tumor necrosis factor-α.

Advanced glycation end products reduce the calcium transient in cardiomyocytes by increasing production of reactive oxygen species and nitric oxide.

Advanced glycation end products (AGE) are central to the development of cardiovascular complications associated with diabetes mellitus. AGE may alter cellular function through cross‐linking of cellular proteins or by activating the AGE receptor (RAGE). However, the signalling molecules involved during AGE stimulation in cardiomyocytes remain unclear. Here, we investigated the effects of AGE treatment on intracellular calcium homeostasis of isolated cardiomyocytes and studied the activation of signalling molecules involved in this process. Treatment of cardiomyocytes with AGE for 24 h resulted in a dose‐dependent reduction in calcium transient amplitude, reaching a maximum 50% reduction at a dose of 1 mg·mL−1. This was accompanied with a 32% reduction in sarcoplasmic reticulum calcium content but without any detectable changes in the expression of major calcium channels. Mechanistically, we observed a significant increase in the production of reactive oxygen species (ROS) in AGE‐treated cardiomyocytes and enhancement of NADPH oxidase activity. This was accompanied with activation of p38 kinase and nuclear translocation of NF‐κB, and subsequently induction of inducible NO synthase (iNOS) expression, leading to excessive nitric oxide production. Overall, our data reveal the molecular signalling that may underlie the alteration of intracellular calcium homeostasis in cardiac myocytes due to AGE stimulation. This may provide new insights into the pathophysiological mechanisms of the development of diabetic cardiomyopathy.

Involvement of inducible nitric oxide synthase and mitochondrial dysfunction in the pathogenesis of enterovirus 71 infection.

Enterovirus71 (EV71) is recognized as the main causative agent of severe hand, foot and mouth disease (HFMD). However, the pathogenesis of EV71 infection has not been well characterized. Clinical evidence indicated that inducible nitric oxide synthase (iNOS) induction in the lung of HFMD patients contributes to the severe symptoms of pulmonary edema. In the present study, we recruited 142 subjects including HFMD patients and controls, and serum level of nitric oxide (NO) was determined. Next, cellular and animal model were used to further investigate the roles of iNOS and mitochondria damage during EV71 infection. Serum NO level in HFMD patients with mild or severe symptoms was higher than that in controls, and there was a trend towards an increase in the serum NO level of severe cases relative to mild cases. EV71 infection caused apoptosis and increased levels of NO, iNOS, superoxide dismutase (SOD) activity and malondialdehyde (MDA), and degraded mitochondrial membrane potential (ΔΨm) in vitro. Pathological alterations of mitochondrial morphology were observed in vitro and in vivo. Furthermore, the expression of iNOS levels in target organs including brain, spinal cord, skeletal muscle, lung and heart were increased with the progression of the pathogenesis of EV71 infection in mice. Taken together, iNOS and mitochondrial damage participate in the pathogenesis of EV71 infection.

Effects of SPAK on vascular reactivity and nitric oxide production in endotoxemic mice

Vasoplegia impedes therapeutic interventions to restore vascular tone, leading to severe hypotension, poor tissue perfusion, and multiple organ failure in septic shock. High levels of circulating nitric oxide (NO) play a crucial role in endotoxin-induced vascular hyporeactivity. Proinflammatory cytokines have been implicated in the induction of inducible NO synthase and overproduction of NO. Anti-inflammatory therapy can diminish NO formation and improve vascular hyporeactivity in septic shock. STE20/SPS1-realted proline/alanine-rich kinase (SPAK) has been reported to activate mitogen-activated protein kinase and contribute to intestinal inflammation. Thus, we evaluated the roles of SPAK in NO production and vascular hyporeactivity in endotoxemic animals. Male wild-type and SPAK deficiency mice were intraperitoneally administered vehicle or Escherichia coli lipopolysaccharide (LPS, 50mg/kg). The changes of systolic blood pressure and plasma nitrate and nitrite levels were measured during the experimental period. Thoracic aortas were exercised to assess vascular reactivity and SPAK expression. In the present study, mice in endotoxin model showed severe hypotension and hyporeactivity to serotonin, phenylephrine (PE), and acetylcholine in the aortic rings. Phosphorylated SPAK expression in the aorta and NO levels in the plasma were also increased in animals with endotoxic shock. However, deletion of SPAK not only reduced the elevation of NO levels but also improved vascular hyporeactivity to serotonin and PE in endotoxemic mice. Taken together, SPAK could be involved in the NO overproduction and vascular hyporesponsiveness to vasoconstrictors in endotoxic shock. Thus, inhibition of SPAK could be useful in the prevention of endotoxin-induced vascular hyporeactivity.

Involvement of HSP70 and HO-1 in the protective effects of raloxifene on multiple organ dysfunction syndrome by endotoxemia in ovariectomized rats.

Accumulating evidence demonstrates that raloxifene, a selective estrogen receptor modulator, possesses anti-inflammatory action. This study evaluates the preventive effects of long-term treatment of raloxifene on acute inflammation and multiple organ dysfunction syndrome (MODS) in ovariectomized (OVX) rats with endotoxemia and its underlying mechanism of action. Adult female rats were OVX bilaterally to induce estrogen insufficiency. OVX rats were administered with raloxifene (1 mg/kg, gavage, once daily) for 8 weeks, beginning 1 week after surgery, followed by induction of sepsis via intravenous infusion of lipopolysaccharides (LPS; 30 mg/kg) for 4 hours. LPS-activated RAW 264.7 cells were used to investigate the mechanism of raloxifene. Ovariectomy amplified the endotoxemia-induced hypotensive effect, MODS, and superoxide anion production in the myocardium. The levels of inducible nitric oxide synthase, high mobility group box 1, and nuclear factor-κB p65 protein increased in OVX rats 6 hours after LPS initiation. Raloxifene mitigated MODS, together with reduced inducible nitric oxide synthase induction and fewer superoxide anions in organs. Raloxifene induced high levels of heat shock protein 70 (HSP70) and heme oxygenase 1 (HO-1), which are associated with an increase in the transcription factor heat shock factor-1 and Nrf-2, respectively. Pretreatment with quercetin, an inhibitor of HSP70, or SnPP, an inhibitor of HO-1, reversed the protective effects of raloxifene in septic OVX rats and LPS-activated macrophages. Long-term treatment with raloxifene reduces the severity of sepsis in OVX rats, attributed from up-regulation of HSP70 and HO-1 to exert the antioxidant and anti-inflammatory capacities. These findings provide new insights into bacterial infection during menopause and the molecular mechanism of raloxifene.

Liposomal microparticle injection can induce myeloid-derived suppressor cells (MDSC)-like cells in vivo

Context: Myeloid-derived suppressor cells (MDSCs) are a subset of immature myeloid cells that function as immunosuppressive cells in various pathological conditions. Membrane-derived microvesicles are thought to be involved in MDSC induction. Earlier reports have described that injection of considerable amount of liposome into rat can suppress Con A-induced splenic T-cell proliferation. Liposome-internalized cells expressing CD11b/c suppress T-cell proliferation. Nitric oxide (NO) appears to be involved in the suppression. We speculated that, similarly to membrane-derived microvesicles, liposomal microparticles can induce MDSC-like cells in vivo.Objectives: To confirm our speculation we investigated dose-dependency of the suppressive effect, the effect of liposome on the induction of inducible NO synthase (iNOS), and anti-CD3 antibody-stimulated T-cell proliferation and cytokine production.Materials and methods: Liposome particles of 250 nm diameter were prepared and suspended in saline. Then, various amounts of liposomal suspension were injected intravenously into rats. After 24 h, rat spleens were removed and concanavalin A (or anti-CD3 antibody) stimulated-splenic T-cell proliferation and the production of iNOS, NO and cytokines were evaluated.Results: T-cell proliferation was suppressed dose-dependently by liposome injection. The immunosuppressive cell exerts its suppressive activity in a dose-dependent manner. The suppression was eliminated by iNOS inhibitor. iNOS was detected in liposome-loaded splenocytes. Anti-CD3 antibody-stimulated T-cell proliferation was also inhibited. Enhanced production of IL-10 was observed.Conclusions: Liposomal microparticles can induce MDSC-like cells in vivo. The lipids which comprise liposomes might serve an important role in the induction of MDSCs in vivo.

Post-translational Activation of Glutamate Cysteine Ligase with Dimercaprol: A NOVEL MECHANISM OF INHIBITING NEUROINFLAMMATION IN VITRO

Neuroinflammation and oxidative stress are hallmarks of various neurological diseases. However, whether and how the redox processes control neuroinflammation is incompletely understood. We hypothesized that increasing cellular glutathione (GSH) levels would inhibit neuroinflammation. A series of thiol compounds were identified to elevate cellular GSH levels by a novel approach (i.e. post-translational activation of glutamate cysteine ligase (GCL), the rate-limiting enzyme in GSH biosynthesis). These small thiol-containing compounds were examined for their ability to increase intracellular GSH levels in a murine microglial cell line (BV2), of which dimercaprol (2,3-dimercapto-1-propanol (DMP)) was found to be the most effective compound. DMP increased GCL activity and decreased LPS-induced production of pro-inflammatory cytokines and inducible nitric-oxide synthase induction in BV2 cells in a concentration-dependent manner. The ability of DMP to elevate GSH levels and attenuate LPS-induced pro-inflammatory cytokine production was inhibited by buthionine sulfoximine, an inhibitor of GCL. DMP increased the expression of GCL holoenzyme without altering the expression of its subunits or Nrf2 target proteins (NQO1 and HO-1), suggesting a post-translational mechanism. DMP attenuated LPS-induced MAPK activation in BV2 cells, suggesting the MAPK pathway as the signaling mechanism underlying the effect of DMP. Finally, the ability of DMP to increase GSH via GCL activation was observed in mixed cerebrocortical cultures and N27 dopaminergic cells. Together, the data demonstrate a novel mechanism of GSH elevation by post-translational activation of GCL. Post-translational activation of GCL offers a novel targeted approach to control inflammation in chronic neuronal disorders associated with impaired adaptive responses.

Nitric oxide and pH modulation in gynaecological cancer.

Nitric oxide plays several roles in cellular physiology, including control of the vascular tone and defence against pathogen infection. Neuronal, inducible and endothelial nitric oxide synthase (NOS) isoforms synthesize nitric oxide. Cells generate acid and base equivalents, whose physiological intracellular concentrations are kept due to membrane transport systems, including Na+/H+ exchangers and Na+/HCO 3 − transporters, thus maintaining a physiological pH at the intracellular (~7.0) and extracellular (~7.4) medium. In several pathologies, including cancer, cells are exposed to an extracellular acidic microenvironment, and the role for these membrane transport mechanisms in this phenomenon is likely. As altered NOS expression and activity is seen in cancer cells and because this gas promotes a glycolytic phenotype leading to extracellular acidosis in gynaecological cancer cells, a pro‐inflammatory microenvironment increasing inducible NOS expression in this cell type is feasible. However, whether abnormal control of intracellular and extracellular pH by cancer cells regards with their ability to synthesize or respond to nitric oxide is unknown. We, here, discuss a potential link between pH alterations, pH controlling membrane transport systems and NOS function. We propose a potential association between inducible NOS induction and Na+/H+ exchanger expression and activity in human ovary cancer. A potentiation between nitric oxide generation and the maintenance of a low extracellular pH (i.e. acidic) is proposed to establish a sequence of events in ovarian cancer cells, thus preserving a pro‐proliferative acidic tumour extracellular microenvironment. We suggest that pharmacological therapeutic targeting of Na+/H+ exchangers and inducible NOS may have benefits in human epithelial ovarian cancer.

Aromatic malononitriles stimulate the resistance of insulin-producing beta-cells to oxidants and inflammatory cytokines

We presently report that treatment with tyrphostin AG-126 (2-(3-hydroxy-4-nitrobenzylidene)malononitrile) and ten other aromatic malononitrile compounds (AMN) improves the resistance of insulin-producing βTC6, RIN-5AH, and MIN6 cells to oxidative stress and pro-inflammatory cytokines. On the molecular level AMN compounds promote nuclear accumulation of the Nrf2 transcription factor and expression of the cytoprotective genes heme ogygenase 1 (HO-1) and NAD(P)H/quinone oxidoreductase 1 (NQO1), inhibit cytokine-dependent inducible nitric oxide synthase (iNOS) induction, suppress intracellular production of reactive oxygen species in βTC6 and counteract to impairments of glucose-stimulated insulin secretion induced by pro-inflammatory cytokines in MIN6 cells. Nrf2 up-regulation and HO-1 induction by AG-126 are attenuated at the presence of siRNA against Nrf2 and brusatol, an inhibitor of the Nrf2 signaling pathway. Our present results indicate that in respect of inhibition of IL-1β-dependent iNOS induction, βTC6 cells are more sensitive to EMK 1071 (2-((5-methylthiophen-2-yl)methylene)malononitrile) and EMK 31 (2-(4-hydroxy-3-methoxybenzylidene)malononitrile) as compared to other analyzed AMN compounds. We suggest that the ability of AMN compounds to inhibit iNOS induction and other cytokine-induced transcriptional events might be a tool to achieve improved β-cell survival and functionality.

Requirement for endogenous heat shock factor 1 in inducible nitric oxide synthase induction in murine microglia.

BackgroundInducible nitric oxide synthase (iNOS) makes a great contribution to host defense and inflammation. In many settings, lipopolysaccharide (LPS) induces iNOS expression through activation of the inhibitor of κB-α (IκB-α)-nuclear factor-κB (NF-κB) cascade, whereas interferon-γ (IFN-γ) acts through Janus kinase (JAK)-signal transducer and activator of transcription 1 (STAT1) signals. Heat shock factor 1 (HSF1), a major regulator of heat shock protein transcription, has been shown to regulate the production of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), but it remains obscure whether and how HSF1 affects iNOS induction.MethodsWestern blot was used to measure the protein expression. The mRNA level was measured by real-time PCR. Silence of HSF1 was achieved by small interfering RNA. Nitric oxide (NO) content and NF-κB binding activity were assayed by commercial kits. Chromatin immunoprecipitation (ChIP) was used to measure the binding activity of NF-κB and STAT1 to iNOS promoters.ResultsHSF1 inhibition or knockdown prevented the LPS- and/or IFN-γ-stimulated iNOS protein expression in cultured microglia. HSF1 inhibition blocked iNOS mRNA transcription. These inhibitory effects of HSF1 inhibition on iNOS expression were confirmed in brain tissues from endotoxemic mice. Further analysis showed that HSF1 inhibition had no effect on IκB-α degradation and NF-κB or STAT1 phosphorylation in LPS/IFN-γ-stimulated cells. The nuclear transport of active NF-κB or STAT1 was also not affected by HSF1 inhibition, but HSF1 inhibition reduced the binding of NF-κB and STAT1 to their DNA elements. In addition, HSF1 inhibition reduced NF-κB and STAT1 bindings to iNOS promoter inside the LPS/IFN-γ-stimulated cells.ConclusionsThis preventing effect of HSF1 inhibition on iNOS mRNA transcription presents the necessary role of HSF1 in iNOS induction.