【作者】 龚小慧；

【导师】 孙波；

【作者基本信息】 复旦大学 ， 儿科学， 2004， 博士

【摘要】 背景:急性肺损伤(ALI)是心源性以外的各种肺内外致病因素引起的弥漫性肺泡-毛细血管膜炎症性损伤,严重的ALI被定义为急性呼吸窘迫综合征(ARDS)。感染是ALI的主要诱因。其病理特征为严重的肺部炎症损伤,表现为肺泡换气功能受损、肺泡萎陷、继发于低氧性肺血管收缩的肺动脉高压、肺泡毛细血管膜通透性增加、肺表面活性物质功能及代谢受损及成分结构改变。吸入一氧化氮(iNO)作为选择性肺血管扩张剂,常规用于治疗新生儿持续低氧血症及肺动脉高压。其选择性舒张肺血管,减少肺动静脉分流的效果得到大量实验及临床研究证实。但是iNO改善成人ALI/ARDS病人氧合的作用不能持久,且未能降低ALI/ARDS的病死率,推测可能与iNO导致肺泡腔第一道防线-肺表面活性物质(PS)的蛋白A(SP-A)硝基化损伤有关。但过度炎症反应及高氧应激本身即可使SP-A产生硝基化。iNO对炎症损伤肺PS的主要活性成分-磷脂酰胆碱(PC)的影响少有报道。对PC代谢的具体环节,如合成、分泌、清除的影响,尚是空白。在成熟肺PS磷脂合成主要经再循环(recycling)和由原料(de novo)合成二条途径,前者约占90%,后者约占10%;而在胎肺de novo合成占主要地位。成熟肺在弥漫性损伤时,再循环比例下降,de novo合成加强。PC 的de novo 合成在ALI时机体抗损伤机制中有重要地位,是肺泡II型上皮细胞(AEC-II)抗损伤机制的一个主要方面。iNO治疗ALI/ARDS必定是和机械通气及高浓度氧气一起使用,长时间暴露会诱发肺损伤。有实验研究报道iNO可减轻动物肺高氧损伤并延长生存。但在肺部存在严重炎症损伤情况下,iNO加高氧对PS代谢的影响及其机制尚不清楚。iNO能减轻肺部的炎症损伤,可能成为预防感染性ALI的有效手段。炎症损伤与PS的代谢密切相关。炎症对PC的合成及分解均有影响,对PC合成的影响表现在对PC合成的关键酶,磷脂酰胆碱胞苷二酰基转移酶(CCT)功能的抑制。外源性及内源性NO对炎症损伤肺的免疫调节作用,构成肺抗损伤及修复机制的一部分。PC合成功能是反映肺泡Ⅱ型上皮细胞(AEC-II)在炎症损伤时抗损伤及修复功能的一个重要指标。iNO与高氧对炎症损伤肺PC合成产生的影响是否与此时肺<WP=6>部的炎症反应状态有关,尚未得到深入研究,值得探讨。目的:1. 从PC合成功能的角度,论证iNO在高氧条件下用于肺部炎症损伤的安全性。2. 从PC合成关键酶的功能和表达水平,研究iNO在高氧条件下对PC合成功能的影响机制。3. iNO及高氧对肺炎症反应的影响,及该影响与PC合成功能的关系。方法:清洁级成年雄性SD大鼠(180～200 g)先随机分组接受静脉注射内毒素2 mg/kg体重(简称LPS),和静脉注射等量生理盐水(作为对照组,简称C),24小时后LPS组及C组再分别随机给予吸空气(Air)、95%氧气(O)、20 ppm NO (NO)、20 ppm NO加95%氧气(ONO)4种干预。在开始气体干预的同时,大鼠尾静脉注射[3H]-氯化胆碱15 μCi,不同气体干预10分钟、及4、8、12和24小时(每组每时点n = 6~8 ),测各时点大鼠支气管肺泡灌洗液(BALF)及灌洗过的肺组织(LT)中总磷脂(TPL)和饱和磷脂酰胆碱(DSPC)放射活性,以反映合成及分泌TPL、DSPC的能力。并以生化方法检测BALF、LT磷脂代谢池中TPL、DSPC总量。并对BALF中白细胞计数(WCC)。另取大鼠,造模及分组干预同前,不注射示踪剂,不同气体干预4、24小时(每组每时点n = 6~8 )。以[14C]-磷酸胆碱为底物,测肺组织中CCT的活性。以RT-PCR、Western blot法测CCT mRNA与蛋白表达水平。以电泳迁移率法测肺组织核转录因子-κB(NF-κB)活性。以RT-PCR法测肿瘤坏死因子-α(TNF-α)、细胞因子诱导的中性粒细胞趋化因子-1(CINC-1)、白细胞介素-10(IL-10) mRNA表达水平;并检测肺组织湿干重比(W/D)、髓过氧化物酶(MPO)、丙二醛(MDA)水平。另取大鼠,造模及分组干预同前。不同气体干预4、24小时(每组每时点n = 5 )。全肺灌流固定用于组织形态学检查及损伤评分。结果:1. 肺PC合成代谢不同干预组PC合成高峰均在注射示踪剂后4小时。此时在Air干预下,全肺TPL放射活性约占示踪剂2.4 ‰,全肺DSPC放射活性约占示踪剂0.5 ‰ 。不同气体干预4小时后,LPS-NO组BALF与全肺(BALF+LT)中TPL(BALF 81±17.6 cpm/g,全肺931±158 cpm/g)、DSPC(BALF 21.2±4.3 cpm/g,全肺217±39.1 cpm/g)的放射活性均显著低于其它气体干预下的LPS大鼠(下降46～59%,P<0.01)。不同干预的LPS组之间,BALF与LT PC代谢池中TPL、DSPC总量,<WP=7>其差异无显著性。不同气体干预24小时后的LPS大鼠BALF与全肺中TPL、DSPC的放射活性均显著低于相同气体干预下的C组(下降24~47%)。LPS-O组BALF与全肺中TPL(BALF 68.8±17.5 cpm/g,全肺603±83.4 cpm/g)、DSPC(BALF 19.8±3.9 cpm/g,全肺102±13.6 cpm/g)的放射活性均显著低于其它气体干预下的LPS大鼠(下降42~53%,P<0.01)。LPS-NO及LPS-ONO组TPL、DSPC合成功能与LPS-Air组相比,其差异无显著性。LPS-O组BALF磷脂代谢池中TPL(4.1±0.8 mg/kg,下降35%)、DSPC总量(1.6±0.4 mg/kg,下降41%),及LT中DSPC(16.0±2.7 mg/kg)总量,显著低于LPS-Air组(TPL 6.3±1.6,DSPC 2.7±0.7,LT中DSPC 21.3±4.2 mg/kg)。LPS-NO、LPS-ONO组BALF及LT中TPL、DSPC总量与LPS-Air相比,其差异无显著性。更多还原

【Abstract】 BackgroundSeptic acute respiratory distress syndrome (ARDS) is often encountered as a complication of various diseases, such as pneumonia, pancreatitis, trauma, burn injury, cardiovascular and gastrointestinal operations, or as a part of multiple system organ failure. Pathogenesis of acute lung injury (ALI) and ARDS in septic patients is related to intrapulmonary neutrophil accumulation and inflammatory damage of lungs, especially in alveoli. Such changes lead to impairment of lung mechanics and gas exchange, surfactant dysfunction and deficiency, pulmonary hypertension secondary to hypoxic intrapulmonary vasoconstriction, and increased vascular-to-alveolar permeability.Inhaled nitric oxide (iNO) was introduced primarily as a selective pulmonary vasodilator to alter ventilation-perfusion mismatching in ALI/ARDS. It may also act to prevent development of ARDS from pulmonary infection and septic lung injury, Although in patients with ARDS, iNO significantly reduces the pulmonary hypertension and the intrapulmonary shunt, it has not yet proved to be effective in reducing mortality.It has been speculated that iNO may cause secondary surfactant damage by formation of nitrotyrosine in its protein moiety due to production of peroxynitrite under hyperoxic condition. iNO is usually used in combination with high oxygen supply in critical conditions. Whether iNO alone or iNO together with hyperoxia would have any adverse effects on surfactant phosphatidylcholine (PC) de novo synthesis in inflammatory injury in the lungs remains an unanswered question, and of clinical importance in terms of new indication of iNO.Recent studies showed that iNO attenuates lung inflammatory injury. Whether iNO alone or iNO plus hyperoxia would affect the course of lung inflammation is not clear. In ARDS patients, elevated levels of TNF-α may be primarily responsible for inhibition of surfactant phospholipid synthesis. In sepsis, bacterial lipopolysaccharide (LPS) triggers the release of TNF-α from alveolar macrophages and alveolar epithelial cells <WP=10>and subsequently initiates an inflammatory cascade leading to diffusive lung injury. TNF-α inhibits PC synthesis via decreasing activity of cytidine triphosphorylate: phosphocholine cytidylyltransferase (CCT), a key rate-limiting enzyme in synthesis of PC. Our aim is to find out the mechanisms by which iNO alone or iNO plus hyperoxia affect PC de novo synthesis and its relation to the lung inflammatory injury.Objectives1. To explore safety and efficacy of iNO and/or hyperoxia in mature lungs with inflammatory injury in association with de novo synthesis and secretion of phosphatidylcholine (PC).2. To determine function and expression of cytidine triphosphorylate: phosphocholine cytidylyltransferase (CCT), a key rate-limiting enzyme in synthesis of PC, and influence of iNO and hyperoxia on CCT.3. Interaction between iNO and/or hyperoxia and lung inflammation in association with synthesis of PC.MethodsAdult male SD rats (clean conventional rats, 180~200 g) were used to induce lung inflammatory injury with bolus LPS (2 mg/kg, i.v) 24 hours prior to experiment. A saline control group was used with identical manner for comparison. LPS-treated (LPS) and control (C) rats were randomly allocated to subgroups exposed to: air, 95% oxygen (O), 20 ppm iNO (NO), 95% oxygen and 20 ppm iNO (ONO).PC de novo synthesis and secretion was determined by measurement of incorporated [3H]-choline chloride into PC. Each rat was injected intravenously with 15 μCi [3H]-choline chloride followed by exposure to various gases. They were sacrificed at 10 min, 4, 8, 12 and 24 hours after the exposure (n = 6~8). The radioactivity of 3H incorporated into total phospholipid (TPL) and disaturated phosphatidylcholine (DSPC) was measured in bronchoalveolar lavage fluid (BALF) and lung tissue (LT, after lavage) Pool size of PC was also measured in both BALF and LT.In a separate cohort study, rats were exposed for 4 and 24 hours in the same protocol of gas exposure. Eight or six rats in each subgroup we更多还原