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Objective To evaluate the role of endothelial nitric oxide synthase (eNOS) in inflammatory responses in mice with ventilator-induced lung injury using gene knockout.Methods Twenty-four male C578L/6J wild type mice and 24 male B6.129P2-Nos3tm1Unc/NJU (eNOS gene knockout) mice,aged 10-12 months,weighing 20-25 g,were randomly assigned into 2 groups (n=12 each) using a random number table:sham operation group (group S) and mechanical ventilation group (group MV).The animals were anesthetized with intraperitoneal 1％ pentobarbital sodium 70 mg/kg,and mechanically ventilated after tracheal intubation.The animals were mechanically ventilated for 4 h (oxygen flow rate 0.5 L/min,FiO2 50％,VT 15 ml/kg,RR 70 bpm,PEEP 2 cmH2O).After 4 h of ventilation,blood samples were obtained from the internal carotid artery for detection of PaO2.The animals were then sacrificed and the lungs were removed for determination of wet/dry lung weight (W/D) ratio,myeloperoxidase (MPO) activity,contents of malondialdehyde (MDA),interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α),and pulmonary microvascular permeability,and for microscopic examination of the pathological changes of lung (with electron microscope).Results Compared with group S of wild type mice,PaO2 was significantly decreased,while W/D ratio,MPO activity,contents of MDA,IL-6,TNF-oα and NO,and pulmonary microvascular permeability were increased in MV groups of wild type and gene knockout mice,and no significant change was found in the parameters mentioned above in group S of gene knockout mice.Compared with group MV of wild type mice,PaO2 was significantly increased,while W/D ratio,MPO activity,contents of MDA,IL-6,TNF-α and NO and pulmonary microvascular permeability were decreased in group MV of gene knockout mice.The pathological changes of lung were significantly attenuated in group MV of gene knockout mice as compared with group MV of wild type mice.Conclusion eNOS is involved in inflammatory responses in mice with ventilator-induced lung injury.
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Objective To investigate the efficiency of transduction of recombinant adenovirus-mediated human endothelial nitric oxide synthase (eNOS) into lung tissue by repeated intratracheal transfection in rats.Methods Sixty 3-4 month old male Wistar rats weighing 220-280 g were randomly divided into 2 groups:control group (group C,n =10) and eNOS gene transduction group (group T,n =50).The animals were anesthetized with intraperitoneal 10％ chloral hydrate 35 mg/kg,tracheally intubated and mechanically ventilated (VT 2.5 ml,RR 60 bpm,FiO2 1.0).Recombinant adenovirus carrying human eNOS gene was given as gift by Professor Gerard from Texas University,Southwest Medical Center.In group T 50 μl of the recombinant adenovirus in concentration of 5 × 109 PFU/ml was instilled into trachea every 5 minutes for 12 times,while in group C equal volume of vector conservation solution was instilled instead.Pulmonary arterial blood samples were obtained at 2,5,7,14 and 21 d after intratracheal transfection (n =10 at each time point) for determination of serum NO concentration.The animals were immediately sacrificed after blood sample collection for determination of expression of eNOS protein in the lung tissue and RNA.The eNOS expression in the trachea,bronchus,lung,liver,spleen and kidney was detected by immuno-histochemistry.Results The serum NO concentrations were significantly higher at all time points in group T than in group C.The eNOS expression was detected in the epithelial cells of trachea and bronchi,and endothelial cells of alveoli and pulmonary blood vessels in group T but not in group C.eNOS expression was not detected in liver,spleen and kidney at 7 d after intratracheal transfection in group T.Conclusion Human eNOS gene mediated by recombinant adenovirus was transducted into rat lung tissue with normal enzyme activity by repeated intratracheal administration without being detected in distant organs.
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ObjectiveTo investigate the effects of simvastatin preconditioning on the expression of inducible and endothelial nitric oxide synthase ( iNOS,eNOS) in thoracic aorta in a rat model of sepsis.Methods Eighty pathogen-free female Wistar rats aged 4 months weighing 200-250 g were randomly divided into 4 groups:group normal control (group Ⅰ,n =8) ; group sham operation (group Ⅱ,n =8) ; group sepsis (group Ⅲ,n =32) and group simvastatin preconditioning (group Ⅳ,n =32).Sepsis was induced by cecal ligation and puncture (CLP) in groups Ⅲ and Ⅳ.In group Ⅳ simvastatin 20 mg/kg was given via a gastric tube once a day for 2 weeksbefore CLP.The thoracic aorta specimens were taken at 3,6,24 and 48 h after CLP (n =8 at each time point)for detection of iNOS and eNOS protein expression by Western blot analysis.ResultsCLP significantly up-regulated iNOS expression and down-regulated eNOS expression in group Ⅲ as compared with groups Ⅰ and Ⅱ.Simvastatin pretreatment significantly attenuated CLP-induced increase in iNOS expression and decrease in eNOS expression in group Ⅳ as compared with group Ⅲ.ConclusionSimvastatin preconditioning can protect vascular endothelial cells from septic injury by down-regulating iNOS expression and up-regulating eNOS expression in vascular endothelial cells.
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Objective To investigate the role of PI3-kinase-Akt-endothelial nitric oxide synthase (PI3KAkt-eNOS) signaling pathway in the attenuation of myocardial ischemia-reperfusion (I/R) injury by sevoflurane postconditioning in rats.Methods Fifty healthy male Wistar rats weighing 250-280 g aged 2-3 months were randomly divided into 5 groups ( n =10 each):sham operation group (group S),I/R group,sevoflurane postconditioning group (group Spo),sevoflurane postconditioning + dimethyl sulfoxide (DMSO) group (group Spo + D),and sevoflurane postconditioning + LY294002 (a specific PI3K inhibitor) group (group Spo+ L).I/R was produced by occlusion of anterior descending branch of left coronary artery for 30 min followed by 120 min reperfusion in anesthetized rats.In group Spo,sevoflurane was inhaled for 5 min after the end-tidal concentration reached 2.5％-3.0％ at 1 min before reperfusion.In group Spo + L,LY294002 0.3 mg/kg in 0.02％ DMSO was injected intravenously at 5 min before reperfusion,and then sevoflurane postconditioning was performed.In group Spo + D,0.02％ DMSO equal to the volume of LY294002 was injected intravenously at 5 min before reperfusion,and then sevoflurane postconditioning was performed.Arterial blood samples were taken at 120 min of reperfusion for determination of the levels of creatine kinase isoenzyme MB (CK-MB),lactate dehydrogenase (LDH) and cardiac Troponin Ⅰ (cTnI).The myocardial infarct size (IS) and area at risk (AAR) were measured and IS/AAR ratio was calculated.The rats were sacrificed at 120 min of reperfusion and the myocardial tissues in the area at risk were taken for determination of the expression of Akt,phosphorylated Akt (p-Akt),eNOS and phosphorylated eNOS (peNOS) by Western blot.The ratios of p-Akt/Akt and p-eNOS/eNOS were calculated.Results Compared with group S,the levels of CK-MB,LDH and cTnI,IS/AAR ratio,p-Akt/Akt ratio and p-eNOS/eNOS ratio were significantly increased in the other groups ( P ＜ 0.05 or 0.01 ).Compared with group I/R,no significant change was found in the parameters mentioned above in group Spo+ L (P ＞ 0.05),and the levels of CK-MB,LDH and cTnI and IS/AAR ratio were significantly decreased,and the ratios of p-Akt/Akt and p-eNOS/eNOS were significantly increased in groups Spo and Spo + D ( P ＜ 0.05 or 0.01 ).There was no significant difference in the parameters mentioned above between group Spo and group Spo + D (P ＞ 0.05).Conclusion PI3K-Akt-eNOS signaling pathway mediates the attenuation of myocardial I/R injury by sevoflurane postconditioning in rats.
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ObjectiveTo investigate the effect of propofol on endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) expression in thoracic aorta of hypertensive rats.MethodsHealthy SD rats of both sexes weighing 240-280 g were used in this study.Hypertension was induced by subcutaneous deoxycorticosterone 25 mg/kg twice a week for consecutive 7 weeks.Sixty-four hypertensive rats were randomly divided into four groups (n ＝ 16 each):hypertension group (group H),low,medium and high dose propofol group ( groups P1,P2,P3 ).Groups P1,P2 and P3 received infusion of propofol at a rate of 20,30 and 40 mg* kg- 1 · h- 1 for 3 h respectively,while group H received equal volume normal saline instead of propofol.Mean arterial pressure (MAP) was monitored and recorded before,1 h and 3 h after the start of propofol or normal saline infusion.All animals were sacrificed at 3 h of intravenous administration.Blood samples were collected by taking out the eyeballs for determination of serum NO concentrations by nitrate reductase method.The expression of eNOS mRNA,iNOS mBNA was determined by reverse transcription-polymerase chain reaction.The expression of eNOS and iNOS protein was determined by Western blot.ResultsCompared to group H,MAP was decreased significantly,the serum NO concentrations were increased significantly,the expression of eNOS mRNA and protein in thoracic aorta was up-regulated,and the expression of iNOS mRNA and protein in thoracic aorta was down-regulated in a dose-dependent manner in groups P1,P2 and P3 ( P < 0.05 or 0.01 ).ConclusionPropofol can down-regulate iNOS expression and up-regudate eNOS expression in endothelial cells of thoracic aorta and promote NO release in hypertensive rats,Which is the mechanism of propofol decreasing pressure.
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Objective To explore the expression of nitric oxide synthases including neuronal nitric oxide synthases(nNOS), inducible nitric oxide synthases(iNOS), endothelial nitric oxide synthases (eNOS) in neurogenic bladder tissues, and analyze it's producing feature and significance. Methods There were 30 cases with neurogenic bladder(18 males, 12 females). The average age was 6.3±3.1 years. All patients appeared with myelodysplasia, urinary and fecal incontinence in different degree. Twenty-six cases were manifested with hyperreflexia bladders, and all patients were treated with surgical procedures. During operation, collected bladder tissue samples including tissues of apex vesicae and tissues of bladder neck, and all tissues were enveloped with mineral wax. All tissues were detected for nNOS, iNOS, and eNOS respectively in tissues of apex vesicae and tissues of bladder neck,and with normal bladder tissues as control group (bladder tissues of hypospadia, 10 cases), and according to clinical features, to explore the expression of NOS, and to analyze the relationship among them. Results In normal apex vesicae tissues, all cases stained with nNOS, and distributed among bundles of smooth muscles, and surface of smooth muscles and interstitial tissue, histochemica;score (HS) 2.8-4.0 and 1.2-2.7. There were no stained cells in bladder tissues of iNOS, and HS was very low, HS:0-0. 4 and 0-0.1 ;eNOS mainly distributed in interstitial tissues in rarefaction manners, and mainly in vascular endothelial cell (VEC), and smooth muscles had no stainings the most expression among them was nNOS, and mainly distributed in bladder neck tissues. In neurogenic bladder tissues, the main expression of NOS type was iNOS, and nNOS decreased significantly. eNOS mainly expressed in VEC among interstitial tissues, and had no staining in smooth muscle cells and collagenoblast and rarefaction of microvessel in bladder tissues, and microvessel density decreased significantly than normal bladder tissues. Microvessal density(MVD) in bladder tisssus (6. 8± 3.2/100per square) was less than that in normal tissues (16.7±6.3/100 per square). Conclusions In normal bladder tissues, nNOS mainly distributes in bladder neck and urethra, and nitric oxide mainly derives from nNOS. Much more matrix fibers, fewer nitrogenergic nerves, and less nNOS expression are seen in neurogenic bladder interstitial tissue. There are more iNOS expressions in bladder tissues,and NO is mainly derived from iNOS, and it may play an important role in pathological bladder tissues, especially in fibrosis of bladder wall. eNOS may be considered as angiopoietic labeling, and may evaluate the blood supply of bladder.