autoregulatory role of endothelium-derived nitric oxide (NO) on Lipopolysaccharide-induced vascular inducible NO synthase expression and function

Nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) is responsible for sepsis-induced hypotension and plays a major contributory role in the ensuing multiorgan failure. The present study aimed to elucidate the role of endothelial NO in lipopolysaccharide (LPS)-induced iNOS expression, in isolated rat aortic rings. Exposure to LPS (1 mug/ml, 5 h) resulted in a reversal of phenylephrine precontracted tone in aortic rings (70.7 +/- 3.2%). This relaxation was associated with iNOS expression and NF-kappaB activation. Positive immunoreactivity for iNOS protein was localized in medial and adventitial layers of LPS-treated aortic rings. Removal of the endothelium rendered aortic rings resistant to LPS-induced relaxation (8.9 +/- 4.5%). Western blotting of these rings demonstrated an absence of iNOS expression. However, treatment of endothelium-denuded rings with the NO donor, diethylamine-NONOate (0.1 mum), restored LPS-induced relaxation (61.6 +/- 6.6%) and iNOS expression to levels comparable with arteries with intact endothelium. Blockade of endothelial NOS (eNOS) activation using geldanamycin and radicicol, inhibitors of heat shock protein 90, in endothelium-intact arteries suppressed both LPS-induced relaxation and LPS-induced iNOS expression (9.0 +/- 8.0% and 2.0 +/- 6.2%, respectively). Moreover, LPS treatment (12.5 mg/kg, intravenous, 15 h) of wild-type mice resulted in profound elevation of plasma [NO(x)] measurements that were reduced by approximately 50% in eNOS knock-out animals. Furthermore, LPS-induced changes in vascular reactivity and iNOS expression evident in wild-type tissues were profoundly suppressed in tissues taken from eNOS knockout animals. Together, these data suggest that eNOS-derived NO, in part via activation of NF-kappaB, regulates iNOS-induction by LPS. This study provides the first demonstration of a proinflammatory role of vascular eNOS in sepsis.     

Nitric oxide suppresses LPS-induced inflammation in a mouse asthma model by attenuating the interaction of IKK and Hsp90

A feature of allergic airway disease is the observed increase of nitric oxide (NO) in exhaled breath. Gram-negative bacterial infections have also been linked with asthma exacerbations. However, the role of NO in asthma exacerbations with gram-negative bacterial infections is still unclear. In this study, we examined the role of NO in lipopolysaccharide (LPS)-induced inflammation in an ovalbumin (OVA)-challenged mouse asthma model. To determine whether NO affected the LPS-induced response, a NO donor (S-nitroso-N-acetylpenicillamine, SNAP) or a selective inhibitor of NO synthase (1400W) was injected intraperitoneally into the mice before the LPS stimulation. Decreased levels of proinflammatory cytokines were demonstrated in the bronchoalveolar lavage fluid from mice treated with SNAP, whereas increased levels of cytokines were found in the 1400W-treated mice. To further explore the molecular mechanism of NO-mediated inhibition of proinflammatory responses in macrophages, RAW 264.7 cells were treated with 1400W or SNAP before LPS stimulation. LPS-induced inflammation in the cells was attenuated by the presence of NO. The LPS-induced IκB kinase (IKK) activation and the expression of IKK were reduced by NO through attenuation of the interaction between Hsp90 and IKK in the cells. The IKK decrease in the lung immunohistopathology was verified in SNAP-treated asthma mice, whereas IKK increased in the 1400W-treated group. We report for the first time that NO attenuates the interaction between Hsp90 and IKK, decreasing the stability of IKK and causing the down-regulation of the proinflammatory response. Furthermore, the results suggest that NO may repress LPS-stimulated innate immunity to promote pulmonary bacterial infection in asthma patients.     

Activation of acid sphingomyelinase and its inhibition by the nitric oxide/cyclic guanosine 3',5'-monophosphate pathway: key events in Escherichia coli-elicited apoptosis of dendritic cells

Depletion of dendritic cells (DCs) via apoptosis contributes to sepsis-induced immune suppression. The mechanisms leading to DC apoptosis during sepsis are not known. In this study we report that immature DCs undergo apoptosis when treated with high numbers of Escherichia coli. This effect was mimicked by high concentrations of LPS. Apoptosis was accompanied by generation of ceramide through activation of acid sphingomyelinase (A-SMase), was prevented by inhibitors of this enzyme, and was restored by exogenous ceramide. Compared with immature DCs, mature DCs expressed significantly reduced levels of A-SMase, did not generate ceramide in response to E. coli or LPS, and were insensitive to E. coli- and LPS-triggered apoptosis. However, sensitivity to apoptosis was restored by addition of exogenous A-SMase or ceramide. Furthermore, inhibition of A-SMase activation and ceramide generation was found to be the mechanism through which the immune-modulating messenger NO protects immature DCs from the apoptogenic effects of E. coli and LPS. NO acted through formation of cGMP and stimulation of the cGMP-dependent protein kinase. The relevance of A-SMase and its inhibition by NO/cGMP were confirmed in a mouse model of LPS-induced sepsis. DC apoptosis was significantly higher in inducible NO synthase-deficient mice than in wild-type animals and was significantly reduced by treatment ex vivo with NO, cGMP, or the A-SMase inhibitor imipramine. Thus, A-SMase plays a central role in E. coli/LPS-induced DC apoptosis and its inhibition by NO, and it might be a target of new therapeutic approaches to sepsis.     

Thrombin peptide TP508 prevents nitric oxide mediated apoptosis in chondrocytes in the endochondral developmental pathway

TP508 is a 23-amino acid peptide derived from human prothrombin that increases cartilage matrix production and reduces alkaline phosphatase activity without changing chondrocyte proliferation. This study tested the hypothesis that TP508 acts by blocking the onset of apoptosis associated with hypertrophy. Rat costochondral resting zone chondrocytes and human auricular chondrocytes were cultured in DMEM containing 50microM ascorbic acid and 10% FBS. Apoptosis was induced by treatment of confluent cultures with chelerythrine, tamoxifen, or inorganic phosphate (Pi) for 24h. One half of the cultures received TP508 (0, 0.7, or 7microg/ml). Apoptosis was assessed as a function of DNA fragmentation ([3H]-thymidine labeled DNA fragments), TUNEL staining, and cell viability using the MTT assay, as well as by assessing the Bcl-2/Bax mRNA and protein ratios and caspase-3 activity. The universal NO synthase inhibitor l-NMMA was used to assess the effect of NO production on chondrocyte apoptosis and specific NO synthase subspecies were identified using iNOS inhibitor 1400W and nNOS inhibitor vinyl-l-NIO, as well as l-NAME, which inhibits both iNOS and eNOS. Finally, we assessed if TP508 would block NO production induced by the apoptogens. Chelerythrine, tamoxifen and Pi-induced apoptosis and this was reversed by TP508. All apoptogens increased NO production and this was reduced by TP508. TP508 reduced NO levels to the same extent as 1400W but not to the same extent as l-NAME, suggesting that its effects are mediated primarily by iNOS. In addition, TP508 reduced the effect of chelerythrine to the same extent as 1400W and l-NAME, again indicating that it acts via inhibition of an iNOS pathway. TP508 also regulated Bcl-2/Bax mRNA in a time and dose-dependent manner. The Bcl-2/Bax mRNA ratio was 0.11 in the absence of TP508 at 1h and 4.95 at 7microg/ml TP508; by 3h the ratio was approximately 1 in both groups. The Bcl-2/Bax protein ratio also increased by 63% at 1h. TP508 did not affect caspase-3 activity. TP508 also caused a dose-dependent increase in protein kinase C (PKC) activity within 9min that was maximal at 270min. These results show that TP508 prevents apoptosis in growth plate chondrocytes via inhibition of iNOS-dependent NO and suggest a possible role for PKC in the mechanism.     

Inducible nitric oxide synthase is key to peroxynitrite-mediated,LPS-induced protein radical formation in murine microglial BV2 cells

Microglia are the resident immune cells in the brain. Microglial activation is characteristic of several inflammatory and neurodegenerative diseases including Alzheimer's disease, multiple sclerosis, and Parkinson's disease. Though lipopolysaccharide (LPS)-induced microglial activation in models of Parkinson's disease is well documented, the free radical-mediated protein radical formation and its underlying mechanism during LPS-induced microglial activation are not known. Here we have used immuno-spin trapping and RNA interference to investigate the role of inducible nitric oxide synthase (iNOS) in peroxynitrite-mediated protein radical formation in murine microglial BV2 cells treated with LPS. Treatment of BV2 cells with LPS resulted in morphological changes, induction of iNOS, and increased protein radical formation. Pretreatments with FeTPPS (a peroxynitrite decomposition catalyst), L-NAME (total NOS inhibitor), 1400W (iNOS inhibitor), and apocynin significantly attenuated LPS-induced protein radical formation and tyrosine nitration. Results obtained with coumarin-7-boronic acid, a highly specific probe for peroxynitrite detection, correlated with LPS-induced tyrosine nitration, which demonstrated involvement of peroxynitrite in protein radical formation. A similar degree of protection conferred by 1400W and L-NAME led us to conclude that only iNOS, and no other forms of NOS, is involved in LPS-induced peroxynitrite formation. Subsequently, siRNA for iNOS, the iNOS-specific inhibitor 1400W, the NF-κB inhibitor PDTC, and the p38 MAPK inhibitor SB202190 was used to inhibit iNOS directly or indirectly. Inhibition of iNOS precisely correlated with decreased protein radical formation in LPS-treated BV2 cells. The time course of protein radical formation also matched the time course of iNOS expression. Taken together, these results prove the role of iNOS in peroxynitrite-mediated protein radical formation in LPS-treated microglial BV2 cells.     

Leptin suppresses Porphyromonas gingivalis lipopolysaccharide interference with salivary mucin synthesis

Leptin, a multifunctional hormone produced predominantly by adipocytes but also identified throughout the glandular tissue of alimentary tract, including salivary glands and oral mucosa, has emerged recently as an important regulator of mucosal inflammatory responses to bacterial infection. In this study, we report that leptin prevents (up to 88.4%) the reduction in mucin synthesis evoked in mucous cells of sublingual salivary gland by LPS of periodontopathic bacterium, Porphyromonas gingivalis. The effect of leptin, moreover, was reflected in a marked decrease in the LPS-induced apoptosis, expression of TNF-alpha, caspase-3 activity, and NO generation. The impedance by leptin of the LPS inhibitory effect on mucin synthesis was blocked by wortmannin, an inhibitor of PI3K, which also obviated the inhibitory effect of leptin on the LPS-induced upregulation in apoptosis, caspase-3 activity, and NO generation. A potentiation in the impedance by leptin of the LPS-induced apoptosis, caspase-3 activity, and NO generation was, however, attained with NOS-2 inhibitor, 1400W, that also caused further enhancement in the impedance by leptin of the LPS detrimental effect on mucin synthesis. Taken together, our data are the first to demonstrate the nature of the involvement of leptin in countering the pathological consequences of P. gingivalis infection on the synthesis of salivary mucins.     

NO and NOS isoforms in the development of apoptosis in renal ischemia/reperfusion

Nitric oxide (NO) and the expression of endothelial (eNOS) and inducible (iNOS) isoforms of nitric oxide synthase (NOS) are recognized as important mediators of physiological and pathological processes of renal ischemia/reperfusion (I/R) injury, but little is known about their role in apoptosis. The ability of the eNOS/NO system to regulate the iNOS/NO system and thus promote apoptosis was assessed during experimental renal I/R. Renal caspase-3 activity and the number of TUNEL-positive cells increased with I/R, but decreased when NOS/NO systems were blocked with L-NIO (eNOS), 1400W (iNOS), and N-nitro-l-arginine methyl ester (L-NAME; a nonselective NOS inhibitor). I/R increased renal eNOS and iNOS expression as well as NO production. The NO increase was eNOS- and iNOS-dependent. Blockage of NOS/NO systems with L-NIO or L-NAME also resulted in a lower renal expression of iNOS and iNOS mRNA; in contrast, eNOS expression was not affected by iNOS-specific blockage. In conclusion, two pathways define the role of NOS/NO systems in the development of apoptosis during experimental renal I/R: a direct route, through eNOS overexpression and NO production, and an indirect route, through expression/activation of the iNOS/NO system, induced by eNOS.     

Tumor necrosis factor-alpha in mechanic trauma plasma mediates cardiomyocyte apoptosis

Mechanical traumatic injury causes cardiomyocyte apoptosis and cardiac dysfunction. However, the signaling mechanisms leading to posttraumatic cardiomyocyte apoptosis remains unclear. The present study attempted to identify the molecular mechanisms responsible for cardiomyocyte apoptosis induced by trauma. Normal cardiomyocytes (NC) or traumatic cardiomyocytes (TC; isolated immediately after trauma) were cultured with normal plasma (NP) or traumatic plasma (TP; isolated 1.5 h after trauma) for 12 h, and apoptosis was determined by caspase-3 activation. Exposure of TC to NP failed to induce significant cardiomyocyte apoptosis. In contrast, exposure of NC to TP resulted in a greater than twofold increase in caspase-3 activation (P < 0.01). Incubation of cardiomyocytes with cytomix (a mixture of TNF-alpha, IL-1beta, and IFN-gamma) or TNF-alpha alone, but not with IL-1beta or IFN-gamma alone, caused significant caspase-3 activation (P < 0.01). TP-induced caspase-3 activation was virtually abolished by an anti-TNF-alpha antibody, and TP isolated from TNF-alpha(-/-) mice failed to induce caspase-3 activation. Moreover, incubation of cardiomyocytes with TP upregulated inducible nitric oxide (NO) synthase (iNOS)/NADPH oxidase expression, increased NO/superoxide production, and increased cardiomyocyte protein nitration (measured by nitrotyrosine content). These oxidative/nitrative stresses and the resultant cardiomyocyte caspase-3 activation can be blocked by neutralization of TNF-alpha (anti-TNF-alpha antibody), inhibition of iNOS (1400W), or NADPH oxidase (apocynin) and scavenging of peroxynitrite (FP15) (P < 0.01). Taken together, our study demonstrated that there exists a TNF-alpha-initiated, cardiomyocyte iNOS/NADPH oxidase-dependent, peroxynitrite-mediated signaling pathway that contributes to posttraumatic myocardial apoptosis. Therapeutic interventions that block this signaling cascade may attenuate posttraumatic cardiac injury and reduce the incidence of secondary organ dysfunction after trauma.     

Aldose reductase mediates endotoxin-induced production of nitric oxide and cytotoxicity in murine macrophages

Aldose reductase (AR) is a ubiquitously expressed protein with pleiotrophic roles as an efficient catalyst for the reduction of toxic lipid aldehydes and mediator of hyperglycemia, cytokine, and growth factor-induced redox-sensitive signals that cause secondary diabetic complications. Although AR inhibition has been shown to be protective against oxidative stress signals, the role of AR in regulating nitric oxide (NO) synthesis and NO-mediated apoptosis has not been elucidated to date. We therefore investigated the role of AR in regulating lipopolysaccharide (LPS)-induced NO synthesis and apoptosis in RAW 264.7 macrophages. Inhibition or RNA interference ablation of AR suppressed LPS-stimulated production of NO and overexpression of iNOS mRNA. Inhibition or ablation of AR also prevented the LPS-induced apoptosis, cell cycle arrest, activation of caspase-3, p38-MAPK, JNK, NF-kappaB, and AP1. In addition, AR inhibition prevented the LPS-induced down-regulation of Bcl-xl and up-regulation of Bax and Bak in macrophages. L-Arginine increased and L-NAME decreased the severity of cell death caused by LPS and AR inhibitors prevented it. Furthermore, inhibition of AR prevents cell death caused by HNE and GS-HNE, but not GS-DHN. Our findings for the first time suggest that AR-catalyzed lipid aldehyde-glutathione conjugates regulate the LPS-induced production of inflammatory marker NO and cytotoxicity in RAW 264.7 cells. Inhibition or ablation of AR activity may be a potential therapeutic target in endotoximia and other inflammatory diseases.     

Inducible nitric oxide synthase modulates lipolysis in adipocytes

The role of inducible nitric oxide synthase (iNOS) in the modulation of adipocyte lipolysis was investigated. Treatment of white and brown adipose cell lines and mouse adipose explants with a mixture of tumor necrosis factor-alpha, interferon-gamma, and lipopolysaccharide (LPS) doubled the lipolytic rate, and this was associated with marked induction of iNOS expression and nitric oxide (NO) production. iNOS inhibition by 1400W, aminoguanidine, or L-NIL pretreatment further increased the cytokine/LPS-mediated lipolysis by 30% (P < 0.05) in cultured adipocytes and in adipose explants. However, this potentiating effect of iNOS inhibition was abolished in adipose explants isolated from iNOS knockout mice. Pharmacological inhibitors of adenylyl cyclase or protein kinase A reduced cytokine/LPS-induced lipolysis and also blunted the potentiating effect of iNOS inhibition on the lipolytic rate. Furthermore, addition of the antioxidants l-cystine and l-glutathione to cytokine/LPS-stimulated adipocytes mimicked the lipolytic effect of iNOS inhibition. In conclusion, inhibition of iNOS activity in adipocytes potentiates cytokine/LPS-induced lipolysis. This effect was fully reversed by adenylyl cyclase and protein kinase A inhibitors but was mimicked by cellular antioxidants. These data suggest that iNOS-mediated NO production counteracts cytokine/LPS-mediated lipolysis in adipocytes and that this feedback mechanism involves an oxidative process upstream of cAMP production in the signaling pathway.     

Lipopolysaccharide (LPS) Stimulates the Production of Tumor Necrosis Factor (TNF)-α and Expression of Inducible Nitric Oxide Synthase (iNOS) by Osteoclasts (OCL) in Murine Bone Marrow Cell Culture

Osteoclasts (OCL) resorb bone. They are essential for the development of normal bones and the repair of impaired bones. The function of OCL is presumed to be supported by cytokines and other biological mediators, including tumor necrosis factor (TNF)-α and nitric oxide (NO). Bacterial lipopolysaccharide (LPS) is a potent inducer of TNF-α and inducible nitric oxide synthase (iNOS), which is the specific enzyme for synthesizing NO from L-arginine. To obtain direct evidence on LPS-induced TNF-α production and iNOS expression by OCL, OCL-enriched cultures were prepared by 7-day cocultures of bone marrow cells of adult BALB/c mice and osteoblastic cells (OBs) derived from calvaria of newborn BALB/c mice, and the generation of TNF-α and iNOS in OCL stimulated with LPS was examined immunocytochemically. When the cultured cells were stimulated with 100 ng/ml of LPS, OCL clearly showed TNF-α and iNOS expression. Without LPS-stimulation, no expression was observed. TNF activity in the culture supernatants of the OCL-enriched cultures in the presence of LPS was also detected by cytotoxic assay that used TNF-sensitive L929 cells. The dentin resorption activity of OCL was estimated by area and number of pits formed on dentin slices, which were covered by the OCL fraction and cultured in the presence or absence of LPS, sodium nitroprusside (SNP; a NO generating compound), N^G-monomethyl L-arginine acetate (L-NMMA; a competitive inhibitor of NO synthase (NOS)), or LPS plus L-NMMA. Pit formation was obviously inhibited in the presence of SNP and slightly inhibited in the presence of L-NMMA, but it was not affected in the presence of LPS or LPS plus L-NMMA. These findings indicate that OCL produces TNF and expresses iNOS in response to LPS, but the LPS-activation of OCL scarcely affects pit formation by them.     

Minocycline inhibits apoptotic cell death via attenuation of TNF-alpha expression following iNOS/NO induction by lipopolysaccharide in neuron/glia co-cultures

We attempted to ascertain the neuroprotective effects and mechanisms of minocycline in inflammatory-mediated neurotoxicity using primary neuron/glia co-cultures treated with lipopolysaccharide (LPS). Neuronal cell death was induced by treatment with LPS for 48 h, and the cell damage was assessed using lactate dehydrogenase (LDH) assays and by counting microtubule-associated protein-2 (MAP-2) positive cells. Through terminal transferase deoxyuridine triphosphate-biotin nick end labeling (TUNEL)-staining and by measuring caspase-3 activity, we found that LPS-induced neuronal cell death was mediated by apoptosis. We determined that pre-treatment with minocycline significantly inhibited LPS-induced neuronal cell death. In addition, LPS induced inducible nitric oxide synthase (iNOS) expression significantly, resulting in nitric oxide (NO) production within glial cells, but not in neurons. Both nitric oxide synthase (NOS) inhibitors (N(G)-monomethyl-L-arginine monoacetate (L-NMMA) and S-methylisothiourea sulfate (SMT)) and minocycline inhibited iNOS expression and NO release, and increased neuronal survival in neuron/glia co-cultures. Pre-treatment with minocycline significantly inhibited the rapid and extensive production of tumor necrosis factor-alpha (TNF-alpha) mediated by LPS in glial cells. We also determined that the signaling cascade of LPS-mediated iNOS induction and NO production was mediated by TNF-alpha by using neutralizing antibodies to TNF-alpha. Consequently, our results show that the neuroprotective effect of minocycline is associated with inhibition of iNOS induction and NO production in glial cells, which is mediated by the LPS-induced production of TNF-alpha.     

Late preconditioning induced by NO donors, adenosine A1 receptor agonists, and delta1-opioid receptor agonists is mediated by iNOS

Although ischemia-induced late preconditioning (PC) is known to be mediated by inducible nitric oxide (NO) synthase (iNOS), the role of this enzyme in pharmacologically induced late PC remains unclear. We tested whether targeted disruption of the iNOS gene abrogates late PC elicited by three structurally different NO donors [diethylenetriamine/NO (DETA/NO), nitroglycerin (NTG), and S-nitroso-N-acetyl-penicillamine (SNAP)], an adenosine A1 receptor agonist [2-chloro-N6-cyclopentyladenosine (CCPA)], and a delta1-opioid receptor agonist (TAN-670). The mice were subjected to a 30-min coronary occlusion followed by 24 h of reperfusion. In iNOS knockout (iNOS-/-) mice, infarct size was similar to wild-type (WT) controls, indicating that iNOS does not modulate infarct size in the absence of PC. Pretreatment of WT mice with DETA/NO, NTG, SNAP, TAN-670, or CCPA 24 h before coronary occlusion markedly reduced infarct size. In iNOS-/- mice, however, the late PC effect elicited by DETA/NO, NTG, SNAP, TAN-670, and CCPA was completely abrogated. Furthermore, in WT mice pretreated with TAN-670 or CCPA, the selective iNOS inhibitor 1400W also abolished the delayed PC properties of these drugs; 1400W had no effect in WT mice. These data demonstrate that iNOS plays an obligatory role in NO donor-induced, adenosine A1 receptor agonist-induced, and delta1-opioid receptor agonist-induced late PC, underscoring the critical role of this enzyme as a common mediator of cardiac adaptations to stress.     

Platelet-activating factor mediates Helicobacter pylori lipopolysaccharide interference with gastric mucin synthesis

Platelet-activating factor (PAF) is now recognized as the most proximal mediator of cellular events triggered by bacterial infection. In this study, we report that a specific PAF antagonist, BN52020, impedes the reduction in mucin synthesis evoked in gastric mucosal cells by H. pylori LPS. The impedance by BN52020 of the LPS inhibitory effect on mucin synthesis was blocked by wortmannin, an inhibitor of phosphatidylinositol 3-kinase (P13K), which also obviated the inhibitory effect of BN52020 on the LPS-induced upregulation in apoptosis, TNF-alpha, and NO generation. A reduction in the impedance by BN52020 of the LPS detrimental effect on mucin synthesis was also attained with cNOS inhibitor, L-NNA, whereas NOS-2 inhibitor, 1400W caused a potentiation in the impedance effect of BN52020. However, while 1400W and BN52020 countered the potentiating effect of wortmannin on the LPS-induced decrease in mucin synthesis, a further exacerbation of the potentiating effect of wortmannin was attained in the presence of cNOS inhibitor, L-NNA. Our findings suggest that PAF, through the interference with PI3K-dependent cNOS activation, plays a critical role in influencing the extent of pathological consequences of H. pylori infection on the synthesis of gastric mucin.     

Nitric oxide augments oridonin-induced efferocytosis by human histocytic lymphoma U937 cells via autophagy and the NF-κB-COX-2-IL-1β pathway

Abstract We previously demonstrated that oridonin-induced autophagy enhanced efferocytosis (phagocytosis of apoptotic cells) by macrophage-like U937 cells through activation of the inflammatory pathways. In this study, exposure of U937 cells to 2.5 μM oridonin caused up-regulation of inducible nitric oxide synthase (iNOS) expression and continuous endogenous generation of nitric oxide (NO), which was reversed by pre-treatment with the inhibitors of nitric oxide synthase 1400 W (dihydrochloride) or L-NAME (hydrochloride). NO donor sodium nitroprusside (SNP) and efferocytosis irritant lipopolysaccharide (LPS) could also exert NO generation and iNOS expression. Moreover, oridonin-induced stimulation of efferocytosis was significantly suppressed by 1400 W or L-NAME. In addition, 1400 W or L-NAME impaired oridonin-induced autophagy. Inhibition of autophagy with 3-methyladenine (3MA) or Beclin-1 siRNA attenuated the uptake of apoptotic cells with a slight increase in the production of NO. The pro-inflammatory cytokine interleukin-1β (IL-1β) has been reported to be involved in oridonin-induced efferocytosis in U937 cells and interact with NO to contribute to inflammatory responses. 1400 W or L-NAME blocked the secretion of IL-1β and the activation of NF-κB and COX-2. Provision of SNP or LPS in place of oridonin resulted in the similar enhancement of efferocytosis, autophagy, the release of IL-1β and the expression of signal protein. NO augmented the oridonin-induced efferocytosis by mediating autophagy and activating the NF-κB-COX-2-IL-1β pathway. Inhibition of NF-κB or COX-2 in turn decreased the production of NO and the expression of iNOS. There exists a positive feedback loop between NO generation and NF-κB-COX-2-IL-1β pathway.     

iNOS/NO signaling regulates apoptosis induced by glycochenodeoxycholate in hepatocytes

Inducible nitric oxide synthase (iNOS) and nitric oxide (NO) can ameliorate apoptosis induced by toxic glycochenodeoxycholate (GCDC) in hepatocytes. However, the underlying molecular mechanisms are not yet understood in detail. This study is to clarify the function of iNOS/NO and its mechanisms during the apoptotic process. The apoptosis was brought about by GCDC in rat primary hepatocytes. iNOS/NO signaling was then investigated. iNOS inhibitor 1400 W enhanced the GCDC-induced apoptosis as reflected by caspase-3 activity and TUNEL assay. Exogenous NO regulated the apoptosis subsequent to NO donor S-nitroso-N-acetyl-penicillamine (SNAP) or sodium nitroprusside (SNP). The GCDC-induced apoptosis was decreased with 0.1 mM SNAP or 0.15 mM SNP, while it was increased with 0.8 mM SNAP or 1.2 mM SNP. The endogenous iNOS inhibited apoptosis, but the exogenous NO played a dual role during the GCDC-induced apoptosis. There was a potential iNOS/Akt/survivin axis that inhibited the hepatocyte apoptosis in low doses of NO donors. In contrast, high doses of NO donors activated CHOP through p38MAP-kinase (p38MAPK), upregulated TRAIL receptor DR5, and suppressed survivin. Consequently the high doses of NO donors promoted the apoptosis in hepatocytes. Our data suggest that the iNOS/NO signaling can modulate Akt/survivin and p38MAPK/CHOP pathways to mediate the GCDC-induced the apoptosis in hepatocytes. These signaling pathways may serve as targets for therapeutic intervention in cholestatic liver disease.     

Endothelial E- and P-selectin expression in iNOS- deficient mice exposed to polymicrobial sepsis

In vitro, nitric oxide (NO) decreases leukocyte adhesion to endothelium by attenuating endothelial adhesion molecule expression. In vivo, lipopolysaccharide-induced leukocyte rolling and adhesion was greater in inducible NO synthase (iNOS)-/- mice than in wild-type mice. The objective of this study was to assess E- and P-selectin expression in the microvasculature of iNOS-/- and wild-type mice subjected to acute peritonitis by cecal ligation and perforation (CLP). E- and P-selectin expression were increased in various organs within the peritoneum of wild-type animals after CLP. This CLP-induced upregulation of E- and P-selectin was substantially reduced in iNOS-/- mice. Tissue myeloperoxidase (MPO) activity was increased to a greater extent in the gut of wild-type than in iNOS-/- mice subjected to CLP. In the lung, the reduced expression of E-selectin in iNOS-/- mice was not associated with a decrease in MPO. Our findings indicate that NO derived from iNOS plays an important role in sepsis-induced increase in selectin expression in the systemic and pulmonary circulation. However, in iNOS-/- mice, sepsis-induced leukocyte accumulation is affected in the gut but not in the lungs.