Responses of pulmonary platelet-derived growth factor peptides and receptors to hyperoxia and nitric oxide in piglet lungs

The peptides platelet-derived growth factor-A (PDGF-A) and especially -B have important roles in lung development. The effect of hyperoxic exposure with and without inhaled nitric oxide (iNO) on lung expression of PDGF and its receptors is unknown. We hypothesized that hyperoxia exposure would suppress mRNA expression and protein production of these ligands and their receptors. The addition of iNO to hyperoxia may further aggravate the effects of hyperoxia. Thirteen-day-old piglets were randomized to breathe 1) room air (RA); 2) 0.96 fraction of inspired oxygen (O2), or 3) 0.96 fraction of inspired oxygen plus 50 ppm of NO (O2+NO), for 5 d. Lungs were preserved for mRNA, Western immunoblot, and immunohistochemical analyses for PDGF-A and -B and their receptors PDGFR-alpha and -beta. PDGF-B mRNA expression was greater than that of PDGF-A or PDGFR-alpha and -beta in RA piglet lungs (p<0.05). Hyperoxia with or without iNO reduced lung PDGF-B mRNA and protein expression relative to the RA group lungs (p<0.01). PDGF-B immunostain intensity was significantly increased in the alveolar macrophages, which were present in greater numbers in the hyperoxia-exposed piglet lungs, with or without NO (p<0.01). PDGFR-beta immunostaining was significantly increased in airway epithelial cells in O2- and O2+NO-exposed piglets. PDGF-A and PDGFR-alpha immunostain intensity and distribution pattern were unchanged relative to the RA group. Sublethal hyperoxia decreases PDGF-B mRNA and protein expression but not PDGF-A or their receptors in piglet lungs. iNO neither aggravates nor ameliorates this effect.     

High-dose inhaled nitric oxide and hyperoxia increases lung collagen accumulation in piglets

Nitric oxide (NO), a pro-oxidant gas, is used with hyperoxia (O(2)) to treat neonatal pulmonary hypertension and recently bronchopulmonary dysplasia, but great concerns remain regarding NO's potential toxicity. Based on reports that exposure to oxidant gases results in pulmonary extracellular matrix injury associated with elevated lavage fluid levels of extracellular matrix components, we hypothesized that inhaled NO with or without hyperoxia will have the same effect. We measured alveolar septal width, lung collagen content, lavage fluid hydroxyproline, hyaluronan and laminin levels in neonatal piglets after 5 days' exposure to room air (RA), RA + 50 ppm NO (RA + NO), O(2) (FiO(2) > 0.96) or O(2) + NO. Matrix metalloproteinase (MMP) activity and MMP-2 mRNA were also measured. In recovery experiments, we measured lung collagen content in piglets exposed to RA + NO or O(2) + NO and then allowed to recover for 3 days. The results show that lung collagen increased 4-fold in the RA + NO piglets, the O(2) and O(2) + NO groups had only a 2-fold elevation relative to RA controls. Unlike the RA + NO piglets, the O(2) and O(2) + NO groups had more than 20-fold elevation in lung lavage fluid hydroxyproline compared to the RA group. O(2) and O(2) + NO also had increased lung MMP activity, extravascular water, and lavage fluid proteins. MMP-2 mRNA levels were unchanged. After 3 days' recovery in room air, the RA + NO groups' lung collagen had declined from 4-fold to 2-fold above the RA group values. The O(2) + NO group did not decline. Alveolar septal width increased significantly only in the O(2) and O(2) + NO groups. We conclude that 5 days' exposure to NO does not result in pulmonary matrix degradation but instead significantly increases lung collagen content. This effect appears potentially reversible. In contrast, hyperoxia exposure with or without NO results in pulmonary matrix degradation and increased lung collagen content. The observation that NO increased lung collagen content represents a new finding and suggests NO could potentially induce pulmonary fibrosis.     

Inhaled nitric oxide decreases hyperoxia-induced surfactant abnormality in preterm rabbits

Inhaled nitric oxide (iNO) is a specific pulmonary vasodilator. By serving as a pro-oxidant or antioxidant, iNO may influence other pulmonary functions as well. This study was designed to test the hypothesis that iNO affects the alveolar lining after premature birth. Preterm rabbits (gestation 29 d, term 31 d) were nose-only exposed NO (14 ppm) and 98% O2, for 20 h. The others were exposed to either 98% O2 or air. In another experiment, premature rabbits were exposed to either NO in air or to air. After the exposure, bronchoalveolar lavage (BAL) was performed and the surfactant aggregates were isolated. The surfactant components and surface activity were analyzed. In total, 144 animals were studied. There were no significant differences in the number, distribution, or respiratory burst activity of cells recovered by BAL. Neither brief hyperoxia nor iNO increased plasma-derived proteins in BAL. Exposure to O2 decreased large surfactant aggregates, surface activity, and the content of surfactant protein B in BAL, whereas iNO prevented completely or partially these effects of acute hyperoxia on surfactant. Hyperoxia increased the content of malondialdehyde and decreased glutathione in epithelial lining fluid. iNO decreased malondialdehyde (p < 0.05) and tended to increase glutathione (p = 0.06) in animals breathing O2. Nitrotyrosine was not detectable in BAL, and NO2 was low in the breathing area. In room air, iNO had no significant effect on surfactant. According to the present results, a brief period of hyperoxia causes an oxidant stress and decreases the surface activity of alveolar surfactant in premature rabbits. In contrast, a low dosage of iNO decreased or prevented the O2-induced detrimental effects on alveolar surfactant and alleviated the oxidant stress.     

The effect of inhaled nitric oxide and oxygen on the hydroxylation of salicylate in rat lungs

Inhaled nitric oxide (iNO) is used as a selective pulmonary vasodilator, and often under conditions when a high fraction of inspired oxygen is indicated. However, little is known about the potential toxicity of iNO therapy with or without concomitant oxygen therapy. NO can combine with superoxide (O2-) to form peroxynitrite (ONOO-), which can in turn decompose to form hydroxyl radical (OH.). Both OH. and ONOO- are involved in various forms of lung injury. To begin evaluation of the effect of iNO under either normoxic or hyperoxic conditions on OH. and/or ONOO- formation, rats were exposed for 58 h to either 21% O2, 21% O2 + 10 parts per million (ppm) NO, 21% O2 + 100 ppm NO, 50% O2, 90% O2, 90% O2 + 10 ppm NO, or 90% O2 + 100 ppm NO. We used a salicylate hydroxylation assay to detect the effects of these exposures on lung OH. and/or ONOO- formation measured as the appearance of 2,3-dihydroxybenzoic acid (2,3-DHBA). Exposure to 90% O2 and 90% O2 + 100 ppm NO resulted in significantly (p < 0.05) greater lung wet weight (1.99 +/- 0.14 g and 3.14 +/- 0.30 g, respectively) compared with 21% O2 (1.23 +/- 0.01 g). Exposure to 21% O2 + 100 ppm NO led to 2.5 times the control (21% O2 alone) 2,3 DHBA formation (p < 0.05) and exposure to 90% O2 led to 2.4 times the control 2,3-DHBA formation (p < 0.05). However, with exposure to both 90% O2 and 100 ppm NO, the 2,3-DHBA formation was no greater than the control condition (21% O2). Thus, these results indicate that, individually, both the hyperoxia and the 100 ppm NO led to greater salicylate hydroxylation, but that the combination of hyperoxia and 100 ppm NO led to less salicylate hydroxylation than either did individually. The production of OH. and/or ONOO- in the lung during iNO therapy may depend on the ratio of NO to O2.     

Monocyte chemoattractant protein-1 and its receptor CCR-2 in piglet lungs exposed to inhaled nitric oxide and hyperoxia.

Monocyte chemoattractant protein-1 (MCP-1), acting through its C-C chemokine receptor 2 (CCR-2), has important roles in inflammation, angiogenesis, and wound repair. The individual and combined effects of inhaled nitric oxide (NO) and hyperoxia on lung MCP-1 and CCR-2 in relation to lung leukocyte dynamics are unknown. Because MCP-1 gene is up-regulated by oxidants, we hypothesized that inhaled NO with hyperoxia will increase MCP-1 production and CCR-2 expression more than either gas alone. We randomly assigned young piglets to breathe room air (RA), RA+50 ppm NO (RA+NO), O(2), or O(2)+NO for 1 or 5 d before sacrifice. Lungs were lavaged and tissues preserved for hybridization studies, Western blotting, histology, and immunohistochemistry. The results show that lung MCP-1 production and alveolar macrophage count were significantly elevated in the 5-d O(2) and O(2)+NO groups relative to the RA group (p < or = 0.05). In contrast, lung CCR-2 abundance was diminished in the O(2) group (p 相似文献   

新生大鼠高氧肺损伤血管内皮生长因子蛋白及其mRNA表达变化研究

目的：血管内皮生长因子（VEGF）参与肺的发育和损伤修复,VEGF具有促进肺泡和肺血管增殖以及预防新生儿支气管肺发育不良（BPD）的作用。该研究的目的是探讨新生大鼠高氧肺损伤后肺组织VEGF蛋白及VEGF mRNA表达的变化。方法：新生Sprague-Dawley大鼠48只随机分为高氧实验组和空气对照组,高氧实验组吸入95%以上高氧建立高氧肺损伤模型。分别采用免疫组化法和逆转录多聚酶链式反应（RT-PCR）检测新生大鼠3 d,7 d,14 d肺组织VEGF蛋白及VEGF mRNA表达变化。结果：空气对照组新生大鼠生后随着肺发育,肺组织中VEGF蛋白及VEGF mRNA表达逐渐增加。高氧实验组新生鼠吸入高氧3 d,肺VEGF蛋白表达出现降低,在7 d,14 d明显降低与对照组比较差异有显著性（VEGF 蛋白:12.67±3.82 vs 7.79±5.23; 15.10±8.91 vs 5.85±3.37, 均P<0.01）;高氧实验组VEGF mRNA表达在3 d,7 d,14 d均明显低于对照组（VEGF mRNA: 1.19±0.63 vs 0.78±0.22, 1.52±0.47 vs 0.53±0.18, 1.89±0.81 vs 0.48±0.12, 均P<0.01）。结论：VEGF能促进新生大鼠肺发育,VEGF蛋白及VEGF mRNA表达与新生大鼠肺发育有密切关系。高氧可抑制VEGF蛋白及VEGF mRNA在新生大鼠肺内的表达,VEGF在新生鼠肺发育和高氧肺损伤发病机制中起重要作用。     

一氧化氮吸入对新生鼠高氧肺损伤时表面活性蛋白A和肺甘露糖结合力的影响

目的：通过观察吸入一氧化氮(iNO)对新生大鼠高氧肺损伤时表面活性蛋白A(SP-A)和肺组织甘露糖结合力(MBA)的影响,探讨iNO对高氧肺损伤保护作用的可能机制。方法：新生大鼠随机分为对照组(空气);高氧组(>95%O2,6d);NO组(空气+10ppmNO,24h);高氧+NO组(>95%O2,6d+10ppmNO,24h)。观察暴露后2d和6d肺组织病理变化,肺SP-AmRNA基因表达、蛋白含量和MBA的变化。结果：高氧组病理损伤明显,暴露后2d时SP-A的mRNA含量(0.81±0.04vs1.53±0.25)和蛋白表达(59.45±18.37vs89.77±16.41)比对照组减少,6d时分别比对照组增加(0.81±0.02vs0.63±0.03),(93.57±13.71vs47.73±21.69),(P<0.05)。高氧+NO组暴露后2d时病理损伤比高氧组明显减轻,SP-AmRNA(0.55±0.91)比对照组和高氧组降低,SP-A蛋白表达(55.12±17.53)比对照组降低(P<0.01);6d时SP-A蛋白表达(67.33±18.59)比高氧组降低(P<0.05)。甘露糖结合力在暴露后2d时NO组比对照组增加(0.821±0.133vs0.580±0.158)、高氧+NO组比高氧组增加(0.430±0.175vs0.738±0.141)(P<0.05)。结论：小剂量NO吸入可降低高氧肺组织SP-A蛋白表达的升高,增加肺组织的MBA,减轻肺组织的病理损伤。     

Independent and combined effects of prolonged inhaled nitric oxide and oxygen on lung inflammation in newborn piglets

Clinical use of nitric oxide (NO) is usually in conjunction with high oxygen concentrations, the effects of which may include lung neutrophil accumulation, apoptosis and upregulation of antioxidant enzyme activity. To define the effects of NO on neutrophils from young piglets and its relationship to lung neutrophil dynamics during hyperoxia we exposed thirty piglets to room air (RA), RA+NO (50 ppm NO), O2 (FiO2> or =0.96) or O2+NO for 5 days. Ten additional animals breathed RA+NO or O2+NO, then recovered in RA for 3 days before sacrifice. Neutrophil CD18 and intracellular oxidant production were measured by flow cytometry. Lung apoptosis were assessed by TUNEL assay. Lung myeloperoxidase, SOD and catalase were measured biochemically. When compared to RA group, there was significant reduction in neutrophil CD18 and intracellular oxidant production in the RA+NO group, but lung MPO was unchanged. The O2 and O2+NO groups did not differ in CD18 expression or in intracellular oxidant production, but had significant increase in lung myeloperoxidase compared to the RA group. Apoptosis increased significantly only in the O2+NO group. The O2 group showed significantly increased lung SOD and catalase activity compared to the RA group, whereas the RA+NO and O2+NO groups did not. We conclude that inhaled NO at 50 ppm decreases neutrophil CD18 expression as well as intracellular oxidant production. However, this effect does not impact lung neutrophil accumulation during concurrent hyperoxia. The combination of NO and O2 exposure produces an increase in lung apoptosis. Finally, NO may prevent upregulation of SOD and catalase activity during hyperoxia, potentially increasing injury.     

持续高氧暴露降低新生大鼠肺组织血管内皮生长因子的表达

目的：高氧肺损伤是导致支气管肺发育不良(BPD)的最常见原因。血管内皮生长因子(VEGF)在新生肺中具有促进内皮细胞增殖、分化、诱导血管发生的作用。本研究动态观察高氧暴露对新生鼠肺组织VEGF表达的影响,探讨BPD的发生机制。方法：新生Wistar大鼠出生后随机分为空气组和高氧组(均n=30)。高氧组在生后12h内开始,予以持续吸入95%氧气。分别于生后1、2、3、7、14、21d各处死5只大鼠,留取肺组织标本。苏木精-伊红染色观察病理改变,免疫组化检测VEGF蛋白表达,原位杂交方法检测VEGFmRNA表达。结果以切片视野灰度值表示,值越高说明蛋白或mRNA表达越少。结果：新生鼠高氧暴露7d后肺泡间隔减少,肺微血管发育异常,间质纤维化,且病变随高氧暴露时间延长而加重。高氧组大鼠第3,7天时肺组织VEGF蛋白表达明显低于相应空气对照组(81.9±0.8vs80.8±1.0,82.8±1.2vs79.2±1.6,均P<0.01)。VEGFmRNA表达亦显著减少(89.5±1.1vs88.0±1.0,91.1±1.5vs87.7±1.7,均P<0.001)。随着暴露时间的延长,VEGF蛋白和mRNA进行性降低,在高氧暴露14、21d时几乎检测不到VEGF蛋白和mRNA的表达。结论：高氧暴露抑制新生鼠肺VEGF的表达,这可能与高氧诱导BPD的发生有关。     

Modulation of pulmonary cytochrome P4501A1 expression by hyperoxia and inhaled nitric oxide in the newborn rat: implications for lung injury

Inhaled nitric oxide (iNO), with supplemental oxygen, is used in the treatment of hypoxic respiratory failure of the newborn. In this study, we tested the hypothesis that exposure of newborn rats to iNO, hyperoxia, or iNO + hyperoxia would modulate the expression of pulmonary cytochrome P450 (CYP)1A1 in relation to acute lung injury. Newborn Fischer 344 rats were maintained in room air, or exposed to iNO, hyperoxia (>95%), or iNO (20 or 40 ppm) + hyperoxia for up to 168 h, and lung injury parameters and CYP1A1 expression were studied. Animals given iNO (40 ppm) + hyperoxia were more susceptible to lung injury than those exposed to hyperoxia or iNO alone. On the other hand, animals exposed to iNO (20 ppm) + hyperoxia did not elicit lung damage. Pulmonary CYP1A1 protein and mRNA expression were induced by hyperoxia, iNO (20 or 40 ppm), or iNO (20 ppm) + hyperoxia for up to 168 h, compared with air-breathing controls. In animals given iNO (40 ppm) + hyperoxia, pulmonary CYP1A1 was enhanced at 48 h, followed by down-regulation at later time points. Immunohistochemistry experiments showed localization of CYP1A1 in the pulmonary epithelial and endothelial cells. In conclusion, because previous studies have shown beneficial effects of CYP1A1 induction in hyperoxic lung injury, our current observations showing maintenance of pulmonary CYP1A1 induction by iNO (20 ppm) + hyperoxia through the 168-h period support the hypothesis that this phenomenon may contribute to the protective effects of iNO against hyperoxic injury.     

早产鼠高氧暴露后肺组织血管内皮生长因子和一氧化氮合酶的动态变化及相互关系

目的：最近研究表明,血管内皮生长因子（VEGF）及内皮型一氧化氮合酶(eNOS)功能的缺失,在支气管肺发育不良（BPD）发病机制中发挥了重要的作用。该文通过动态观察持续中浓度高氧暴露（60％O2）对早产大鼠肺内VGEF蛋白及mRNA和eNOS蛋白及mRNA表达的影响,探讨BPD的发病机制。方法：将21 d 孕早产鼠随机分为高氧暴露组(简称高氧组) 和空气对照组(简称空气组) ,分别置于常压高氧仓中（60％O2）和正常空气中暴露。分别于生后1,4,7,11,14 d每组各处死6只大鼠,留取肺组织标本。苏木精-伊红染色观察病理改变,免疫组化检测VEGF和eNOS蛋白表达,逆转录-聚合酶链反应方法检测VEGF和eNOS mRNA表达。结果：早产鼠高氧暴露4 d后出现肺泡间隔减少,微血管发育异常,间质纤维化,且病变随着高氧暴露时间的延长而加重。高氧组大鼠第4,7天时肺组织VEGF蛋白表达明显低于相应空气对照组(P< 0. 05), VEGF mRNA表达亦显著减少(P< 0. 05)。随着暴露时间的延长,VEGF蛋白和mRNA进行性降低。高氧组大鼠在高氧暴露过程中肺组织eNOS蛋白和mRNA表达亦随着暴露时间的延长而降低。结论：高氧暴露导致早产鼠肺组织VEGF和eNOS表达持续性减少,微血管发育异常和肺泡化受阻。这些由高氧暴露诱导产生的BPD样损害,可能与VEGF和eNOS的表达下调有关,且二者之间存在着密切的联系。［中国当代儿科杂志,2007,9（5）：473-478］     

Inhaled nitric oxide enhances distal lung growth after exposure to hyperoxia in neonatal rats

Exposure of newborn rats to hyperoxia impairs alveolarization and vessel growth, causing abnormal lung structure that persists during infancy. Recent studies have shown that impaired angiogenesis due to inhibition of vascular endothelial growth factor (VEGF) signaling decreases alveolar and vessel growth in the developing lung, and that nitric oxide (NO) mediates VEGF-dependent angiogenesis. The purpose of this study was to determine whether hyperoxia causes sustained reduction of lung VEGF, VEGF receptor, or endothelial NO synthase (eNOS) expression during recovery, and whether inhaled NO improves lung structure in infant rats after neonatal exposure to hyperoxia. Newborn rat pups were randomized to hyperoxia [fraction of inspired oxygen (Fio(2)), 1.00] or room air exposure for 6 d, and then placed in room air with or without inhaled NO (10 ppm) for 2 wk. Rats were then killed for studies, which included measurements of body weight, lung weight, right ventricular hypertrophy (RVH), morphometric analysis of alveolarization (by mean linear intercept (MLI), radial alveolar counts (RAC), and vascular volume (Vv), and immunostaining and Western blot analysis. In comparison with controls, neonatal hyperoxia reduced body weight, increased MLI, and reduced RAC in infant rats. Lung VEGF, VEGFR-2, and eNOS protein expression were reduced after hyperoxia. Inhaled NO treatment after hyperoxia increased body weight and improved distal lung growth, as demonstrated by increased RAC and Vv and decreased MLI. We conclude that neonatal hyperoxia reduced lung VEGF expression, which persisted during recovery in room air, and that inhaled NO restored distal lung growth in infant rats after neonatal hyperoxia.     

高氧对胎鼠肺成纤维细胞p53和增殖细胞核抗原表达的影响

目的 探讨高氧对胎鼠肺成纤维细胞（LFs）p53 和增殖细胞核抗原（PCNA）表达的影响。方法 原代培养胎鼠肺 LFs,待生长至亚汇合状态时,随机分为：空气组和高氧组（95% O2/5% CO2）。于培养 12 h 和 24 h 时,采用噻唑蓝（MTT）实验测定细胞增殖状况,半定量 RT-PCR 方法检测 p53 mRNA 表达,Western blot 技术检测 p53 和 PCNA 蛋白的表达。结果（1）与空气组比较,高氧组 12 h 和 24 h 的 LFs 生长抑制率分别为 8% 和 23%;（2）高氧组在 12 h 和 24 h 时 p53 mRNA 表达明显高于空气组（PPP结论 高氧暴露抑制 PCNA 表达、促进 p53 表达,从而抑制 LFs 增殖和 DNA 复制,是导致肺发育异常的重要因素。     

Hyperoxia decreases matrix metalloproteinase-9 and increases tissue inhibitor of matrix metalloproteinase-1 protein in the newborn rat lung: association with arrested alveolarization

Matrix metalloproteinases (MMP) are likely effectors of normal lung development, especially branching morphogenesis, angiogenesis, and extracellular matrix degradation. Because hyperoxia exposure (>95% O(2)) from d 4 to 14 in newborn rat pups leads to arrest of alveolarization and mimics newborn chronic lung disease, we tested whether hyperoxia altered MMP-2 and -9 mRNA, protein, and enzymatic activity, and the mRNA and protein expression of the endogenous tissue inhibitor of MMP, TIMP-1. No changes due to hyperoxia exposure were observed in MMP-2 mRNA or pro-enzyme (72 kD) protein levels between d 6 and 14, although the overall protein mass and zymographic activity of the active (68 kD) enzyme were diminished (p < 0.05, ANOVA). However, hyperoxia significantly decreased levels of MMP-9 mRNA and pro-MMP-9 protein and diminished overall MMP-9 pro-enzyme activity. TIMP-1 mRNA was not elevated by hyperoxia until d 14, but protein levels were significantly (p < 0.001) elevated by hyperoxia from d 9 to 14. To estimate the potential of MMP inhibition to arrest alveolarization, administration of doxycycline (20 mg/kg, twice daily by gavage), a pan-MMP proteolysis inhibitor, arrested lung alveolarization. We conclude that hyperoxia decreases MMP-9 mRNA, protein, and activity and elevates TIMP-1 protein, and these changes have the potential to contribute to the arrest of normal lung development.     

Inhaled nitric oxide attenuates hyperoxic lung injury in lambs

Cytochrome P450 (CYP) inhibition with cimetidine reduces hyperoxic lung injury in young lambs. Nitric oxide (NO), also a CYP inhibitor, has been shown to either aggravate or protect against oxidant stress depending on experimental context. The objective of this study was to determine whether NO, like cimetidine, would protect young lambs against hyperoxic lung injury, and whether its effect was associated with CYP inhibition. Three groups of lambs were studied: 1) room air exposure, 2) >95% O2, and 3) >95% O2 plus inhaled NO. After 72 h, hyperoxia alone resulted in a significant increase in arterial P(CO2) and number of polymorphonuclear leukocytes in bronchoalveolar lavage (BAL), and a significant decrease in arterial/alveolar O2 tension (a/A). The addition of inhaled NO significantly decreased the hypercarbia and BAL polymorphonuclear cellular response associated with hyperoxia but had no beneficial effect on a/A ratio. There were no significant differences in F2-isoprostanes or isofurans (markers of lipid peroxidation) measured in BAL or lung tissue among study groups. No intergroup differences were detected in BAL epoxyeicosatrienoic acid levels (index of CYP activity). The results of this study indicate that hypercarbia and inflammation accompanying hyperoxic lung injury in young lambs can be attenuated by inhaled NO. However, this study provides no direct evidence that NO is inhibiting CYP-mediated oxidant lung injury.     

高氧和脂多糖诱导人胚肺成纤维细胞NF-κB的动态表达

目的近年研究表明,支气管肺发育不良的发生与宫内炎性因子暴露和出生后机械通气、高氧暴露有着密切的联系。该研究选取人胚肺成纤维细胞,模拟炎性暴露和高氧暴露环境,探讨高氧和脂多糖（lipopolysaccharide,LPS）对人胚肺成纤维细胞核转录因子NF κB表达的影响。方法60%高氧,LPS（100 ng/mL）及两者同时刺激人胚肺成纤维细胞0.5,1,2及4 h,用免疫细胞化学观察其亚基p50及p65的核转位,RT PCR方法检测NF κB p50和p65 mRNA。结果LPS的直接刺激迅速诱导p50及p65的核转位,0.5 h即可致NF κB迅速活化,1 h 达到高峰,后逐渐下降;高氧刺激诱导p50 及p65核转位,1 h达高峰,之后迅速下降;高氧和LPS联合刺激p50及p65亚基核转位,在2 h达高峰,然后缓慢下降。但4 h时活化效应明显高于LPS组和高氧组。结论同时暴露于高氧和LPS组人胚肺成纤维细胞中NF κB的活化比单独暴露因素下活化更为显著,活化持续时间更长,提示暴露于宫内炎性环境中的患儿生后又高氧/辅助通气更容易导致肺部疾病。     

VEGF attenuates hyperoxic injury through decreased apoptosis in explanted rat embryonic lung

Ambient oxygen concentration and vascular endothelial growth factor (VEGF)-A are vital in lung development. Since hypoxia stimulates VEGF-A production and hyperoxia reduces it, we hypothesized that VEGF-A down-regulation by exposure of airways to hyperoxia may result in abnormal lung development. An established model of in vitro rat lung development was used to examine the effects of hyperoxia on embryonic lung morphogenesis and VEGF-A expression. Under physiologic conditions, lung explant growth and branching is similar to that seen in vivo. However, in hyperoxia (50% O2) the number of terminal buds and branch length was significantly reduced after 4 d of culture. This effect correlated with a significant increase in cellular apoptosis and decrease in proliferation compared with culture under physiologic conditions. mRNA for Vegf164 and Vegf188 was reduced during hyperoxia and addition of VEGF165, but not VEGF121, to explants grown in 50% O2 resulted in partial reversal of the decrease in lung branching, correlating with a decrease in cell apoptosis. Thus, hyperoxia suppresses VEGF-A expression and inhibits airway growth and branching. The ability of exogenous VEGF165 to partially reverse apoptotic effects suggests this may be a potential approach for the prevention of hyperoxic injury.     

Effects of pulmonary oxygen injury on airway content of surfactant-associated protein A

The use of therapeutic hyperoxia has greatly improved the survival of infants born prematurely. However, high concentrations of oxygen cause pulmonary injury, leading to decreased pulmonary compliance and decreased oxygen diffusion. This injury can result in chronic pulmonary insufficiency. It has been hypothesized that the adverse effects of hyperoxia are mediated, in part, through changes in the pulmonary surfactant system. We investigated the effects of hyperoxia on surfactant-associated protein A (SP-A), the abundant surfactant-specific glycoprotein. Adult male rats were exposed to 85% oxygen for 72 h. Total lung volume and pulmonary compliance were measured, and alveolar surfactant material recovered by lavage. Hyperoxia decreased total lung capacity, and altered inflation and deflation hysteresis patterns. Disaturated phosphatidylcholine and SP-A content were significantly increased in alveolar surfactant material isolated from oxygen-treated rats. SP-A content was also significantly increased in lung tissue from oxygen-treated rats. The SP-A in the lavage of oxygen-treated rats appeared to be intact protein as no proteolytic fragments were detected and the SP-A migrated identically to that recovered from room air animals when analyzed by two-dimensional isoelectric focusing. We conclude that the decreased pulmonary compliance associated with pulmonary oxygen injury is not due to quantitative decreases in two major surfactant components, disaturated phosphatidylcholine and SP-A.     

高氧致慢性肺疾病早产鼠肺组织CDK4 p21的表达及意义

目的：早产儿慢性肺疾病（CLD）的发病机制目前研究还不十分清楚,但CLD的最终病理变化与肺细胞增殖有关。该文采用高氧诱导早产鼠CLD模型为对象,探讨CDK4和p21基因动态表达与肺细胞增殖调控的关系。方法：高浓度氧致早产鼠CLD模型（实验组）和正常对照组各40例为研究对象,每组分别于实验后的1,3,7,14和21 d随机选取8只大鼠处死, 取出肺组织,常规制成5 μm切片。检测观察：①肺组织形态学;②肺组织纤维化评分;③采用免疫组化检测肺组织内PCNA表达;④采用原位杂交检测肺组织CDK4 mRNA和p21 mRNA的表达。结果：两组肺组织细胞PCNA指数：与对照组比较,实验组1 d,3 d PCNA表达均减弱（P﹤0.05）,7 d开始表达增强（P＜0.01）, 14 d和21 d明显高于对照组（P＜0.01）。两组肺组织细胞CDK4 mRNA表达强度： 从7 d开始实验组高于对照组（P﹤0.05）, 14 d,21 d明显高于对照组（P﹤0.01）。两组肺组织细胞p21 mRNA表达强度：实验组1 d,3 d表达明显高于对照组（P﹤0.01）, 7 d 后持续下降, 但也高于对照组（P﹤0.05）。7~21 d肺组织细胞CDK4 mRNA,p21 mRNA 表达分别与PCNA呈显著正、负相关（r分别为0.83和-0.81,P﹤0.05）。结论：高氧可诱导早产鼠肺细胞增殖。肺组织细胞CDK4基因的过度表达、p21基因的表达下降,可能是高氧诱导肺细胞增殖的机制之一。［中国当代儿科杂志,2007,9（6）：595-600］