Lipopolysaccharide-induced diaphragmatic contractile dysfunction and sarcolemmal injury in mice lacking the neuronal nitric oxide synthase

In this study we evaluated the role of the neuronal nitric oxide synthase (nNOS) in lipopolysaccharide (LPS)-induced diaphragmatic contractile dysfunction and sarcolemmal injury. Wild-type (WT) mice or mice deficient in the nNOS gene (nNOS(-/-)) were injected with either saline (control) or Escherichia coli LPS (LPS groups) and sacrificed 12 h later. The diaphragm was then examined for NOS expression, NOS activity, and in-vitro contractility. We also assessed sarcolemmal injury in isolated muscle strips under resting condition and after 3 min of artificial stimulations. In WT mice, LPS injection reduced maximum force to about 75% of that of control animals and raised total NOS activity significantly due to the induction of the iNOS isoform. Although muscle fiber injury was minimal under resting condition, the percentage of injured fibers in control and LPS-injected mice approached 27% and 40% of total fibers, respectively, in response to artificial stimulation. By comparison, LPS injection in nNOS(-/-) mice elicited a worsening of muscle contractility (maximum force < 60% of control animals) but elicited degrees of sarcolemmal injury similar to those observed in the WT animals. In addition, muscle NOS activity and iNOS protein level in nNOS(-/-) mice injected with LPS reached about 10% and 60% of that of WT animals, respectively (p < 0.05 compared with WT animals). Protein level of endothelial NOS isoform in the diaphragm was not altered by LPS injection in either WT or nNOS(-/-) animals. We conclude that nNOS plays a protective role in attenuating the negative influence of sepsis on diaphragmatic contractility but is not involved in the pathogenesis of sepsis-induced sarcolemmal injury.     

iNOS gene expression modulates microvascular responsiveness in endotoxin-challenged mice

Septic shock is characterized by vasodilation and decreased responsiveness to vasoconstrictors. Recent studies suggest this results from nitric oxide (NO) overproduction after expression of the calcium-independent isoform of NO synthase (iNOS) in smooth muscle cells. However, direct evidence linking iNOS (NOS2) expression and decreased microvascular responsiveness after septic stimuli is lacking. In the present study, we determined the effect of bacterial lipopolysaccharide (LPS, 20 mg/kg, IP) on smooth muscle contraction and endothelial relaxation in mesenteric resistance arteries from wild-type and iNOS knockout mice. Four hours after challenge with LPS or saline in vivo, concentration-dependent responses to norepinephrine (NE) and acetylcholine (NE+ACh) were measured in cannulated, pressurized vessels ex vivo. In vessels from wild-type mice, NE-induced contraction was markedly impaired after LPS, and pretreatment with the iNOS inhibitor aminoguanidine (AG) partly restored the NE contraction. In contrast, NE contraction in microvessels from iNOS knockout mice was unaffected by LPS. ACh-induced relaxation was unaffected by LPS in vessels from either genotype. These data provide direct evidence that iNOS gene expression mediates the LPS-induced decrease in microvascular responsiveness to vasoconstrictors. Moreover, the observation that AG did not fully restore NE contraction after LPS, whereas iNOS gene deficiency did, suggests that iNOS expression plays a central role in the development of the NO-independent effect of LPS on microvascular responsiveness. Finally, our data indicate that LPS or iNOS expression has little effect on endothelium-dependent relaxation, and eNOS activity does not appear to play a role in the decreased smooth muscle responsiveness after LPS in this model. The full text of this article is available at http://www.circresaha.org.     

Lack of inducible nitric oxide synthase does not prevent aging-associated insulin resistance

Inducible nitric oxide synthase (iNOS) is involved in obesity-induced insulin resistance. Since aging is accompanied by increased iNOS expression, the effect of iNOS gene deletion on aging-associated insulin resistance was investigated in 7-month-old (adult) and 22-month-old (old) iNOS knockout and wild-type mice using the hyperinsulinemic–euglycemic clamp. While body weight and fat mass were increased, muscle mass was reduced with aging in wild-type mice. However, body composition was not changed with aging in iNOS knockout mice due to increased locomotor activity. NO metabolites in plasma, and protein levels of iNOS and nitrotyrosine in skeletal muscle increased with aging in wild-type mice. Deletion of iNOS gene attenuated NO metabolites and nitrotyrosine with aging in iNOS knockout mice. Glucose uptake in whole body and skeletal muscle was reduced with aging in both wild-type and iNOS knockout mice and there was no difference between two groups. Plasma level of tumor necrosis factor-α and gene expression of proinflammatory cytokines in peripheral tissues were increased with aging in both groups, and that was more heightened in iNOS knockout mice. These results suggest that lack of iNOS does not prevent aging-associated insulin resistance in mice and heightened production of proinflammatory cytokines may be involved.     

The therapeutic effect of natriuretic peptides in heart failure; differential regulation of endothelial and inducible nitric oxide synthases

The abnormal regulation of nitric oxide synthase activity represents an underlying feature of heart failure. Increased peripheral vascular resistance, and decreased renal function may be in part related to impaired endothelium-dependent nitric oxide (NO) synthesis. Paradoxically, the chronic production of NO by inducible nitric oxide synthase (iNOS) in heart failure exerts deleterious effects on ventricular contractility, and circulatory function. Consequently, pharmacologically improving endothelium-dependent NO synthesis and the concomitant inhibition of iNOS activity would be therapeutically advantageous. Interestingly, natriuretic peptides have been shown to differentially regulate endothelial NOS (eNOS) and iNOS activity. Moreover, in both patients and animal models of heart failure, pharmacologically increasing plasma natriuretic peptide levels ameliorated vascular tone, renal function, and ventricular contractility. Based on these observations, the following review will explore whether the therapeutic benefit of the natriuretic peptide system in heart failure may occur in part via the amelioration of endothelium-dependent NO synthesis, and the concomitant inhibition of cytokine-mediated iNOS expression.     

Melatonin counteracts inducible mitochondrial nitric oxide synthase-dependent mitochondrial dysfunction in skeletal muscle of septic mice

Mitochondrial nitric oxide synthase (mtNOS) produces nitric oxide (NO) to modulate mitochondrial respiration. Besides a constitutive mtNOS isoform it was recently suggested that mitochondria express an inducible isoform of the enzyme during sepsis. Thus, the mitochondrial respiratory inhibition and energy failure underlying skeletal muscle contractility failure observed in sepsis may reflect the high levels of NO produced by inducible mtNOS. The fact that mtNOS is induced during sepsis suggests its relation to inducible nitric oxide synthase (iNOS). Thus, we examined the changes in mtNOS activity and mitochondrial function in skeletal muscle of wild-type (iNOS(+/+)) and iNOS knockout (iNOS(-/-)) mice after sepsis. We also studied the effects of melatonin administration on mitochondrial damage in this experimental paradigm. After sepsis, iNOS(+/+) but no iNOS(-/-) mice showed an increase in mtNOS activity and NO production and a reduction in electron transport chain activity. These changes were accompanied by a pronounced oxidative stress reflected in changes in lipid peroxidation levels, oxidized glutathione/reduced glutathione ratio, and glutathione peroxidase and reductase activities. Melatonin treatment counteracted both the changes in mtNOS activity and rises in oxidative stress; the indole also restored mitochondrial respiratory chain in septic iNOS(+/+) mice. Mitochondria from iNOS(-/-) mice were unaffected by either sepsis or melatonin treatment. The data suggest that inducible mtNOS, which is coded by the same gene as that for iNOS, is responsible for mitochondrial dysfunction during sepsis. The results also suggest the use of melatonin for the protection against mtNOS-mediated mitochondrial failure.     

Altered response to inflammatory cytokines in hepatic energy metabolism in inducible nitric oxide synthase knockout mice

BACKGROUND/AIMS: Production of nitric oxide (NO) in the liver is believed to be a critical factor for carbohydrate and energy metabolism in endotoxin shock. The present study focuses on the involvement of NO produced by inducible nitric oxide synthase (iNOS) in glycogen synthesis and energy metabolism stimulated by insulin.METHODS: Primary hepatocytes prepared from wild-type and iNOS knockout (iNOS(-/-)) mice were employed.RESULTS: Incubation of wild-type hepatocytes with a combination of cytokines (interleukin-1beta, tumor necrosis factor-alpha and interferon-gamma) and lipopolysaccharide (cytokines/LPS) inhibited insulin-stimulated glycogen synthesis and adenosine triphosphate (ATP) increase, and decreased the ketone body ratio (KBR) at 8-12 h, concomitant with expression of iNOS protein and NO production. While the glycogen synthesis was suppressed by cytokines/LPS, reduction of the ATP increase and a decrease in KBR by cytokines/LPS were not observed in iNOS(-/-) hepatocytes. Further, N(G)-monomethyl-L-arginine, a NOS inhibitor, reversed the inhibition of ATP increase and decrease in KBR by cytokines/LPS, but not the inhibition of glycogen synthesis. Conversely, addition of S-nitroso-N-acetylpenicillamine, a NO donor, inhibited the insulin-stimulated ATP increase synthesis in iNOS(-/-) hepatocytes, but not the insulin-stimulated glycogen synthesis.CONCLUSIONS: These results demonstrate that NO mediates the suppression of insulin-stimulated energy metabolism, but not glycogen synthesis, in cytokines/LPS-treated hepatocytes.     

Disruption of inducible nitric oxide synthase improves beta-adrenergic inotropic responsiveness but not the survival of mice with cytokine-induced cardiomyopathy

Transgenic (TG) mice with cardiac-specific overexpression of tumor necrosis factor-alpha develop congestive heart failure. We have previously reported that short-term inhibition of inducible nitric oxide synthase (iNOS) ameliorates beta-adrenergic hyporesponsiveness in TG mice. To examine whether long-term inhibition of iNOS may rescue TG mice from developing congestive heart failure, we disrupted iNOS gene by crossing TG mice with iNOS knockout mice. Myocardial levels of iNOS protein were significantly increased in TG mice compared with age- and sex-matched wild-type (WT) mice. No iNOS protein was detected in TG mice with the disruption of iNOS. Myocardial levels of endothelial NOS were not different among these mice. To examine the effects of iNOS disruption on myocardial contractility, left ventricular pressure was measured. In TG mice, +dP/dt(max) was significantly suppressed, and its beta-adrenergic responsiveness was blunted. As in the case with short-term inhibition of iNOS, the disruption of iNOS gene improved beta-adrenergic inotropic responsiveness in TG mice but not in WT mice. However, the iNOS disruption did not alter myocardial inflammation, ventricular hypertrophy, or the survival of these mice. These results indicate that although myocardial expression of iNOS plays a key role in the attenuation of beta-adrenergic inotropic responsiveness, NO-independent mechanisms might be more important in the development of congestive heart failure.     

Gastric nitric oxide synthase expression during endotoxemia: implications in mucosal defense in rats

BACKGROUND & AIMS: This study was performed to examine expression of gastric nitric oxide synthase (NOS) isoforms during endotoxemia in rats and to assess their role(s) in gastric injury from bile and ethanol. METHODS: Lipopolysaccharide (LPS) enhanced the expression and activity of inducible nitric oxide synthase in gastric mucosa in a dose- and time-dependent manner. RESULTS: Endothelial nitric oxide synthase and neural nitric oxide synthase expression did not significantly change, but constitutive nitric oxide synthase activity decreased over time. LPS alone caused injury to the gastric mucosa and disrupted F-actin filaments in the same cells with enhanced immunostaining for inducible nitric oxide synthase. LPS also exacerbated gastric injury from the mild irritants 5 mmol/L acidified taurocholate and 20% ethanol as did local intra-arterial infusion of the nitric oxide donor S-nitroso-N-acetyl-penicillamine. The selective inducible nitric oxide synthase inhibitor aminoguanidine negated LPS-induced exacerbation of gastric injury from these irritants. The nonselective NOS inhibitor N(G)-nitro-L-arginine methyl ester augmented the deleterious effects of LPS, an effect reversed by L-arginine but not D-arginine. Aminoguanidine, but not N(G)-nitro-L-arginine methyl ester, negated LPS-induced accumulation of gastric luminal nitrates. CONCLUSIONS: These data suggest that increased inducible NOS activity and decreased constitutive nitric oxide synthase activity are primarily responsible for exacerbating gastric injury from luminal irritants during endotoxemia. Moreover, septic patients may be more susceptible to gastric injury from bile during gastrointestinal ileus.     

Effect of lipopolysaccharide on diarrhea and gastrointestinal transit in mice： Roles of nitric oxide and prostaglandin E2

AIM: To investigate the effect of lipopolysaccharide (LPS) on the diarrheogenic activity, gastrointestinal transit (GIT), and intestinal fluid content and the possible role of nitric oxide (NO) and prostaglandin E2 (PGE2) in gastrointestinal functions of endotoxin-treated mice. METHODS: Diarrheogic activity, GIT, and intestinal fluid content as well as nitric oxide and PGE2 products were measured after intraperitoneal administration of LPS in mice. RESULTS: LPS dose-dependently accumulated abundant fluid into the small intestine, induced diarrhea, but decreased the GIT. Both nitric oxide and PGE2 were found to increase in LPS-treated mice. Western blot analysis indicated that LPS significantly induced the protein expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 in mice intestines. Pretreatment with NG-nitro-L-arginine-methyl ester (L-NAME, a non-selective NOS inhibitor) or indomethacin (an inhibitor of prostaglandin synthesis) significantly attenuated the effects of LPS on the diarrheogenic activity and intestine content, but reversed the GIT. CONCLUSION: The present study suggests that the pathogenesis of LPS treatment may mediate the stimulatory effect of LPS on nitric oxide and PGE2 production and NO/ prostaglandin pathway may play an important role on gastrointestinal function.     

Interaction between enteric epithelial cells and Peyer''s patch lymphocytes in response to lipopolysaccharide: Effect on nitric oxide and IL-6 release

AIM: To investigate the effect of interaction between enteric epithelial cells and lymphocytes of Peyer's patch on the release of nitric oxide (NO) and IL-6 in response to Shigella lipopolysaccharide (LPS).METHODS: Human colonic epithelial cells (Caco-2)were mixed cocultured with lymphocytes of Peyer's patch from wild-type (C57 mice) and inducible NO synthase knockout mice, and challenged with Shigella F2a-12LPS. Release of NO and mIL-6 was measured by Griess colorimetric assay and enzyme-linked immunosorbent assay (ELISA), respectively.RESULTS: In the absence of LPS challenge, NO was detected in the culture medium of Caco-2 epithelial cells but not in lymphocytes of Peyer's patch, and the NO release was further up-regulated in both cocultures with lymphocytes from either the wild-type or iNOS knockout mice, with a significantly higher level observed in the coculture with iNOS knockout lymphocytes. After Shigella F2a-12 LPS challenge for 24-h, NO production was significantly increased in both Caco-2 alone and the coculture with lymphocytes of Peyer's patch from the wild-type mice but not from iNOS knockout mice.LPS was found to stimulate the release of mIL-6 from lymphocytes, which was suppressed by coculture with Caco-2 epithelial cells. The LPS-induced mIL-6production in lymphocytes from iNOS knockout mice was significantly greater than that from the wild-type mice.CONCLUSION: Lymphocytes of Peyer's patch maintain a constitutive basal level of NO production from the enteric epithelial cell Caco-2. LPS-induced mIL-6 release from lymphocytes of Peyer's patch is suppressed by the cocultured epithelial cells. While no changes are detectable in NO production in lymphocytes from both wild-type and iNOS knockout mice before and after LPS challenge, NO from lymphocytes appears to play an inhibitory role in epithelial NO release and their own mIL-6 release in response to LPS.     

糖尿病大鼠肾脏一氧化氮系统基因表达的研究

目的 研究糖尿病大鼠肾脏一氧化氮系统基因的表达。方法 观察了１２周ＳＴＺ－糖尿病大鼠肾脏皮质一氧化氮（ＮＯ）含量及一氧化氮合成酶（ＮＯＳ）活力,并应用ＲＴ－ＰＣＲ技术检测了肾脏皮质诱生型一氧化氮合成酶ｍＲＮＡ水平的改变。结果 糖尿病大鼠肾脏皮质ＮＯＳ活性及ｉＮＯＳ ｍＲＮＡ水平明显高于正常对照组（Ｐ〈０．０５）,然而ＮＯ含量却反而下降（Ｐ〈０．０５）。结论 糖尿病大鼠肾脏一氧化氮合成障碍,灭活加速     

Interaction between enteric epithelial cells and Peyer＇s patch lymphocytes in response to Shigella lipopolysaccharide： Effect on nitric oxide and IL-6 release

AIM: To investigate the effect of interaction between enteric epithelial cells and lymphocytes of Peyer's patch on the release of nitric oxide (NO) and IL-6 in response to Shigella lipopolysaccharide (LPS). METHODS: Human colonic epithelial cells (Caco-2) were mixed cocultured with lymphocytes of Peyer's patch from wild-type (C57 mice) and inducible NO synthase knockout mice, and challenged with Shigella F2a-12 LPS. Release of NO and mIL-6 was measured by Griess colorimetric assay and enzyme-linked immunosorbent assay (ELISA), respectively. RESULTS: In the absence of LPS challenge, NO was detected in the culture medium of Caco-2 epithelial cells but not in lymphocytes of Peyer's patch, and the NO release was further up-regulated in both cocultures with lymphocytes from either the wild-type or iNOS knockout mice, with a significantly higher level observed in the coculture with iNOS knockout lymphocytes. After Shigella F2a-12 LPS challenge for 24-h, NO production was significantly increased in both Caco-2 alone and the coculture with lymphocytes of Peyer's patch from the wild-type mice but not from iNOS knockout mice. LPS was found to stimulate the release of mIL-6 from lymphocytes, which was suppressed by coculture with Caco-2 epithelial cells. The LPS-induced mIL-6 production in lymphocytes from iNOS knockout mice was significantly greater than that from the wild-type mice. CONCLUSION: Lymphocytes of Peyer's patch maintain a constitutive basal level of NO production from the enteric epithelial cell Caco-2. LPS-induced mIL-6 release from lymphocytes of Peyer's patch is suppressed by the cocultured epithelial cells. While no changes are detectable in NO production in lymphocytes from both wild-type and iNOS knockout mice before and after LPS challenge, NO from lymphocytes appears to play an inhibitory role in epithelial NO release and their own mIL-6 release in response to LPS.     

动脉平滑肌细胞一氧化氮合酶与高血压发病的关系

目的 探讨高血压病患者血管平滑肌细胞（ＶＳＭＣ）一氧化氮合酶（ＮＯＳ）活性和ＮＯＳ基因表达的特点及其与高血压发病的关系。方法 采用复合胶原酶法分离培养高血压病患者和血压正常者动脉ＶＳＭＣ，并以血红蛋白分光光度法、原位反转录聚合酶链反应检测ＶＳＭＣ的ＮＯＳ活性和诱生型ＮＯＳ（ｉＮＯＳ）ｍＲＮＡ表达量。结果 （１）高血压组ＶＳＭＣ的ＮＯＳ活性和ｉＮＯＳ ｍＲＮＡ表达量均显著低于正常组（Ｐ〈０．０１）；     

Calcineurin regulates NFAT-dependent iNOS expression and protection of cardiomyocytes: co-operation with Src tyrosine kinase

OBJECTIVE: To determine the role of calcineurin and Src tyrosine kinase in the regulation of inducible nitric oxide synthase (iNOS) expression and protection in cardiomyocytes. METHODS: iNOS expression was studied in isolated neonatal rat ventricular myocyte cultures in response to bacterial lipopolysaccharide (LPS) or following transfection with constitutively active calcineurin or Src and in hearts isolated from wild-type or calcineruin Abeta knockout mice. Cell injury in response to simulated ischemia-reperfusion was studied following overexpression of active calcineurin. Regulation of the iNOS gene promoter by calcineurin was studied using promoter-luciferase reporter and chromatin immunoprecipitation assays. RESULTS: Overexpression of constitutively active Src co-operated with [Ca2+]c elevation to induce iNOS expression, and LPS-induced iNOS expression was abrogated by pharmacological inhibition of calcineurin or tyrosine kinase. LPS also induced tyrosine kinase-dependent but calcineurin-independent phosphorylation of Src Tyr418. LPS induced myocardial iNOS expression in wild-type but not calcineurin Abeta knockout mice. Overexpression of constitutively active calcinuerin in isolated cardiomyocytes caused dephosphorylation and nuclear accumulation of the c1 isoform of nuclear factor of activated T-cells (NFATc1), induced strong iNOS expression, and induced NOS-dependent protection against simulated ischemia-reperfusion prior to cardiomyocyte hypertrophy. Co-transfection of a mouse iNOS promoter-luciferase reporter in combination with active calcineurin and wild-type or dominant negative Src confirmed that constitutive activation of calcineurin was sufficient for transactivation. Chromatin immunoprecipitation confirmed calcineurin-dependent in vivo binding of NFATc1 to consensus sites within the iNOS promoter. CONCLUSIONS: These results support a cardioprotective role for calcineurin mediated by NFAT-dependent induction of iNOS expression and co-operativity between calcineurin and Src.     

Age-dependent lipopolysaccharide-induced iNOS expression and multiorgan failure in rats: effects of melatonin treatment

Senescence amplifies the sensitivity to endotoxemia, which correlates with increased nitric oxide (NO) levels and mortality. Melatonin displays antioxidant and anti-inflammatory effects, but its levels decrease with age. Lipopolysaccharide (LPS) (10 mg/kg) was injected to 3- and 18-month-old rats 6 h before they were killed, and melatonin (60 mg/kg) was injected before and/or after LPS. Inducible nitric oxide synthase (iNOS) expression and activity, nitrite content, lipoperoxidation (LPO) levels, and serum markers of liver, renal, and metabolic dysfunction, were measured in liver and lung of these animals. An age-dependent increase in iNOS activity, NO content, and LPO levels was observed, and these changes were augmented further by LPS. Melatonin decreased the expression and activity of iNOS, reducing NO and LPO levels to basal values in both septic LPS-treated groups. Liver, kidney, and metabolic dysfunctions were also significantly higher in aged that in young rats and further increased by LPS. Melatonin treatment counteracted these alterations in young and aged septic rats. Melatonin reduced LPS-dependent iNOS expression and multiorgan failure in a similar extent in young and aged rats. Because aged rats showed higher organ and metabolic impairment than young animals in response to LPS, the results also suggest an increased efficacy of the anti-septic properties of melatonin in the aged animals.     

Deletion of neuronal NOS prevents impaired vasodilation in septic mouse skeletal muscle

OBJECTIVE: Sepsis-stimulated nitric oxide (NO) production impairs arteriolar responsiveness in skeletal muscle. Using wild type (WT), eNOS(-/-), iNOS(-/-) and nNOS(-/-) mice, we aimed to determine the key nitric oxide synthase (NOS) isoenzyme(s) responsible for the arteriolar hyporesponsiveness to acetylcholine (ACh) in septic skeletal muscle. METHODS: Sepsis was induced by the cecal ligation and perforation procedure (24 h model). We measured the post-ACh increase in red blood cell velocity (V(RBC)) in a capillary fed by the stimulated arteriole as an index of vasodilation. NOS activity and protein expression in the muscle were measured by standard procedures. RESULTS: In all non-septic mice, ACh increased V(RBC) by approximately 150% from baseline. Sepsis impaired this response in WT, eNOS(-/-) and iNOS(-/-) mice, but not in nNOS(-/-) mice. Accordingly, pharmacological inhibition of nNOS with 7-nitroindazole reversed this impairment in WT mice. cNOS (eNOS+nNOS) activity was elevated in septic WT mice; Western blots indicated that this occurred through a post-translational mechanism. iNOS protein activity/expression was negligible. ACh caused dilation via endothelial-derived relaxing factor (EDRF) in WT mice and via endothelial-derived hyperpolarizing factor (EDHF) in eNOS(-/-) mice. Although exogenous NO reduced EDHF-mediated dilation in eNOS(-/-) mice, NOS inhibition did not reverse the sepsis-impaired dilation in these mice. CONCLUSIONS: In our 24-h mouse model of sepsis, NO in skeletal muscle is primarily derived from nNOS. Sepsis impairs both EDRF- and EDHF-mediated dilation in response to ACh. Both genetic deletion and inhibition of nNOS protect against this impairment when the dilation occurs via the EDRF but not EDHF pathway.     

Decreased neointimal thickening after arterial wall injury in inducible nitric oxide synthase knockout mice

Mechanical injury in vivo results in the expression of the inducible form of nitric oxide synthase (iNOS) in vascular smooth muscle cells. However, the role of iNOS in modulating neointima formation after arterial wall injury is not clear. To determine whether the induction of iNOS gene expression promotes or attenuates the neointimal response to injury, we used a murine model of perivascular injury induced by placing a periadventitial collar around the carotid arteries in both wild-type and iNOS knockout mice (iNOS-KO mice). Periadventitial injury induced iNOS expression in the wild-type but not the iNOS-KO mice. Neointimal area and the intima/media ratio were significantly less in the iNOS-KO mice compared with the wild-type mice at 21 days. Injury-induced proliferation of medial cells and vascular cell adhesion molecule-1 expression were also attenuated in iNOS-KO mice compared with wild-type mice. The induction of iNOS and the activation of the nuclear factor-kappaB-mediated pathway were also demonstrated in an in vitro injury model. We conclude that mechanical injury in vivo and in vitro induces iNOS expression and that lack of iNOS expression attenuates neointima formation after perivascular arterial injury. Taken together, these findings suggest that iNOS expression after vascular injury may promote neointima formation.     

慢阻肺患者内源性NO合酶表达、膈肌组织改变对膈肌功能变化的影响

目的研究分析轻中度慢性阻塞性肺疾病(慢阻肺)患者膈肌组织学结构变化的特点及内源性一氧化氮合酶(NOS)表达情况,探讨一氧化氮(NO)在慢阻肺患者膈肌功能变化中发挥的作用。方法选取实施开胸手术的22例患者作为研究对象,其中12例为轻中度慢阻肺患者(慢阻肺组),10例为肺功能正常的患者(对照组),采用免疫组化及HE染色观察膈肌组织学改变,采用Westernblot检测两组患者的NOS表达变化,并分析两组患者的肺功能差异。结果慢阻肺组患者的FEV_1、FEV1%、FEV_1/FVC检测值均显著低于对照组且差异均具有统计学意义(P0.05)。慢阻肺组患者的膈肌肌球蛋白重链(MHC)亚型阳性(MHCslow、MHCfast)细胞所占比例与对照组比较差异均不具有统计学意义(P0.05)。慢阻肺组患者的胶原沉着面积(22.84±1.86)%、毛细血管数量(12.87±3.82)个/视野、NOS(0.321±0.126)OD值均显著高于对照组(P0.05),两组膈肌纤维横截面积差异不具有统计学意义(P0.05)。结论慢阻肺患者的膈肌纤维超微结构较非慢阻肺患者发生显著的改变,同时NOS表达率升高,这对慢阻肺患者的膈肌收缩力、耐力均有不利的影响。     

Role of neuronal nitric oxide synthase and inducible nitric oxide synthase in intestinal injury in neonatal rats

AIM: To investigate the dynamic change and role of neuronal nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS) in neonatal rat with intestinal injury and to define whether necrotizing enterocolitis (NEC) is associated with the levels of nitric oxide synthase (NOS) in the mucosa of the affected intestine tissue. METHODS: Wistar rats less than 24 h in age received an intraperitoneal injection with 5 mg/kg lipopolysaccharide (IPS). Ileum tissues were collected at 1, 3, 6, 12 and 24 h following LPS challenge for histological evaluation of NEC and for measurements of nNOS and iNOS. The correlation between the degree of intestinal injury and levels of NOS was determined. RESULTS: The LPS-injected pups showed a significant increase in injury scores versus the control. The expression of nNOS protein and mRNA was diminished after LPS injection. There was a negative significant correlation between the nNOS protein and the grade of median intestinal injury within 24 h. The expression of iNOS protein and mRNA was significantly increased in the peak of intestinal injury. CONCLUSION: nNOS and iNOS play different roles in LPS-induced intestinal injury. Caution should be exerted concerning potential therapeutic uses of NOS inhibitors in NEC.