猪肺表面活性物质对大鼠油酸型急性肺损伤的治疗作用
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摘要
急性呼吸窘迫综合征(acute respiratory distress syndrome,ARDS)是一种由多种原因,诸如严重感染、创伤、吸入有毒有害气体、严重缺氧以及溺水等,引起的以急性呼吸衰竭为表现的综合征,临床上以严重的呼吸困难、顽固性低氧血症和非心源性肺水肿为特征,其早期阶段为急性肺损伤(acute lunginjury,ALI)。近年来由于机械通气模式的优化及其他一些辅助疗法的合理应用,ARDS的救治率有所提高,但是迄今为止,其在我国的死亡率仍高达50%左右,而机械通气本身亦存在易并发感染以及造成呼吸机相关性肺损伤等问题。因此开展防治ALI/ARDS的研究,特别是增加有效药物的应用,对于进一步提高救治率具有重要意义。
本论文采用静脉注入油酸(oleic acid,OA)的方法复制成年SD大鼠ALI的模型。我们观察了经大鼠舌静脉注入不同剂量(0.05、0.08、0.10、0.15、0.20、0.25ml/Kg)油酸后大鼠肺损伤的情况,证实用0.20m1/Kg油酸舌静脉注入能成功复制大鼠ALI的模型。在此基础上,我们采用①大鼠存活率;②反映肺功能的指标:血气分析和呼吸频率(breathing rate,BR);③反映肺损伤程度和炎症反应的指标:肺系数(lung index,LI)、支气管肺泡灌洗液(bronchoalveolar lavage fluid,BALF)中总蛋白(total protein,TP)含量、血浆中TNF-a浓度、肺大体外观和组织病理学检查,评价了在大鼠自主呼吸的情况下本室研制的猪肺表面活性物质(porcine pulmonary surfactant,PPS)对ALI的治疗作用,并进一步探讨了不同剂量的PPS、不同时间点给予PPS以及氧气驱动雾化吸入PPS这一新的给药方式对ALI疗效的影响。
我们首先观察了静脉注入油酸30分钟后,大鼠气道滴入不同剂量的PPS混悬液(50、80、100、150、200mg/Kg)的治疗效果,结果发现,与气道滴入生理盐水的对照组相比,上述剂量的PPS均显示不同程度的治疗效果,其中50mg/Kg
The acute respiratory distress syndrome(ARDS) describes an overwhelming inflammatory reaction within the pulmonary parenchyma leading to life-threatening disturbances in pulmonary vasomotion, alveolar ventilation, and gas exchange. The early phase of ARDS is termed acute lung injury(ALI). Despite numerous efforts to develop causative or symptomatic treatment options, ARDS still has a high mortality rate of about 50%. Therefore there is a need for alternative therapeutic interventions.In this study, we used a rat model of acute lung injury(ALI) induced by oleic acid. We tried out different doses of oleic acid in order to choose the appropriate one. Through experiments, we found that the dose of 0.20ml/Kg would satisfy our requirements and meet the standard the ALL Then we evaluated the therapeutic effects of porcine pulmonary
surfactant(PPS) on the ALL We estimated survival studies and the respiratory function of lungs with breath rates and arterial blood gases, assessing the lung damage with appearance and pathological structure of lungs. The lung index(LI), the total protein(TP) in the bronchoalveoar lavage fluid(BALF) and TNF-a concentration in plasma were also measured as the sign of degree of lung injury.Firstly, we investigated the therapeutic effects and dose-effect relationship of intratracheal instillation of different doses of PPS (50、 80 、 100、 150、 200mg/Kg) in ALI rats. The results showed that PPS (50mg/Kg) group improved arterial blood gases during the first 2 hours and reduced breath rates. Other PPS-treatment groups not only improved arterial blood gases and reduced breath rates, but also significantly raised 4h-survival rates and decreased lung index, protein contents in BALF and TNF-a concentration in plasma, meliorated histological appearance compared with group given saline after OA. PPS(150, 200mg/Kg) groups provided more efficiently on lung injury than other groups.Secondly, we estimated the therapeutic effects of intratracheal instillating PPS (100, 150mg/Kg) at different time (30min、 2h) on ALI in rats. The experimental method and assessments were the same to mentioned above. The results showed that PPS ( ≥ 100mg/Kg ) administered at the early stage (30min) not only improved respiratory function, but also significantly decreased lung injury. More PPS (≥
150mg/Kg) needed at the late stage (2h) had the effects mentioned above. Comparatively the theraputic efficiency of 2h(PPS150mg/Kg) was worse than that of 30min(PPS150mg/Kg), indicated the importance of early administration.The usual way of administering PS is by instilling it as a bolus into the lungs through bronchoscope or endotracheal tube. In this study, we tried another method of inhaling PS driven by oxygen in ALI models. We used a laser aerosol particle counter to measure the size distribution of aerosolized PPS driven by oxygen under various flow rates. The outcome was that if the flow rate was over 2.0 L/min, the particle size distribution of PPS was almost ranging from 0.3um to 2.0um. With raising the flow rate, more PPS particle size was smaller than 1 .Oum. This size of PPS could arrive at the pulmonary alveolus to perform function. Combined with the clinical fraction of inspired oxygen, we decided on the flow rate of 4.5L/min to drive the bolus of PPS into aerosol.Then we evaluated the new method of administering PPS in ALI rats and compared the efficiency of intilling PPS with inhaling PPS. This test showed that two inhaling groups not only improved arterial blood gases and reduced breath rates, but also significantly raised 4h-survival rates and decreased lung index > protein contents in BALF and TNF-a level in plasma, meliorated histological appearance compared with the group given OA. PPS-inhaling group had better effects than saline-inhaling
group on lung index> protein contents in BALF> TNF-a level in plasma and histological appearance. Inhaling PPS (160mg/Kg) had the same effects with instilling PPS (lOOmg/Kg).From what we studied, we had proved that PPS provided therapeutic effects on animal models of ALI induced by oleic acid. PPS=^80mg/Kg administered at early stage might alleviate the lung injury, however more PPS^150mg/Kg was needed at late stage. Aerosolized PPS (160mg/Kg) also had the therapeutic effects, but the effect was inferior to that obtained with bolus PPS. As this method of inhaling PPS is more convenient and safer, more investigation about the implication of aerosolized PPS is needed.Our findings provide the proofs of efficiency of PPS and help to make up the clinical medication of PS. We also set up a new method of aerosolized PS.
本论文采用静脉注入油酸(oleic acid,OA)的方法复制成年SD大鼠ALI的模型。我们观察了经大鼠舌静脉注入不同剂量(0.05、0.08、0.10、0.15、0.20、0.25ml/Kg)油酸后大鼠肺损伤的情况,证实用0.20m1/Kg油酸舌静脉注入能成功复制大鼠ALI的模型。在此基础上,我们采用①大鼠存活率;②反映肺功能的指标:血气分析和呼吸频率(breathing rate,BR);③反映肺损伤程度和炎症反应的指标:肺系数(lung index,LI)、支气管肺泡灌洗液(bronchoalveolar lavage fluid,BALF)中总蛋白(total protein,TP)含量、血浆中TNF-a浓度、肺大体外观和组织病理学检查,评价了在大鼠自主呼吸的情况下本室研制的猪肺表面活性物质(porcine pulmonary surfactant,PPS)对ALI的治疗作用,并进一步探讨了不同剂量的PPS、不同时间点给予PPS以及氧气驱动雾化吸入PPS这一新的给药方式对ALI疗效的影响。
我们首先观察了静脉注入油酸30分钟后,大鼠气道滴入不同剂量的PPS混悬液(50、80、100、150、200mg/Kg)的治疗效果,结果发现,与气道滴入生理盐水的对照组相比,上述剂量的PPS均显示不同程度的治疗效果,其中50mg/Kg
The acute respiratory distress syndrome(ARDS) describes an overwhelming inflammatory reaction within the pulmonary parenchyma leading to life-threatening disturbances in pulmonary vasomotion, alveolar ventilation, and gas exchange. The early phase of ARDS is termed acute lung injury(ALI). Despite numerous efforts to develop causative or symptomatic treatment options, ARDS still has a high mortality rate of about 50%. Therefore there is a need for alternative therapeutic interventions.In this study, we used a rat model of acute lung injury(ALI) induced by oleic acid. We tried out different doses of oleic acid in order to choose the appropriate one. Through experiments, we found that the dose of 0.20ml/Kg would satisfy our requirements and meet the standard the ALL Then we evaluated the therapeutic effects of porcine pulmonary
surfactant(PPS) on the ALL We estimated survival studies and the respiratory function of lungs with breath rates and arterial blood gases, assessing the lung damage with appearance and pathological structure of lungs. The lung index(LI), the total protein(TP) in the bronchoalveoar lavage fluid(BALF) and TNF-a concentration in plasma were also measured as the sign of degree of lung injury.Firstly, we investigated the therapeutic effects and dose-effect relationship of intratracheal instillation of different doses of PPS (50、 80 、 100、 150、 200mg/Kg) in ALI rats. The results showed that PPS (50mg/Kg) group improved arterial blood gases during the first 2 hours and reduced breath rates. Other PPS-treatment groups not only improved arterial blood gases and reduced breath rates, but also significantly raised 4h-survival rates and decreased lung index, protein contents in BALF and TNF-a concentration in plasma, meliorated histological appearance compared with group given saline after OA. PPS(150, 200mg/Kg) groups provided more efficiently on lung injury than other groups.Secondly, we estimated the therapeutic effects of intratracheal instillating PPS (100, 150mg/Kg) at different time (30min、 2h) on ALI in rats. The experimental method and assessments were the same to mentioned above. The results showed that PPS ( ≥ 100mg/Kg ) administered at the early stage (30min) not only improved respiratory function, but also significantly decreased lung injury. More PPS (≥
150mg/Kg) needed at the late stage (2h) had the effects mentioned above. Comparatively the theraputic efficiency of 2h(PPS150mg/Kg) was worse than that of 30min(PPS150mg/Kg), indicated the importance of early administration.The usual way of administering PS is by instilling it as a bolus into the lungs through bronchoscope or endotracheal tube. In this study, we tried another method of inhaling PS driven by oxygen in ALI models. We used a laser aerosol particle counter to measure the size distribution of aerosolized PPS driven by oxygen under various flow rates. The outcome was that if the flow rate was over 2.0 L/min, the particle size distribution of PPS was almost ranging from 0.3um to 2.0um. With raising the flow rate, more PPS particle size was smaller than 1 .Oum. This size of PPS could arrive at the pulmonary alveolus to perform function. Combined with the clinical fraction of inspired oxygen, we decided on the flow rate of 4.5L/min to drive the bolus of PPS into aerosol.Then we evaluated the new method of administering PPS in ALI rats and compared the efficiency of intilling PPS with inhaling PPS. This test showed that two inhaling groups not only improved arterial blood gases and reduced breath rates, but also significantly raised 4h-survival rates and decreased lung index > protein contents in BALF and TNF-a level in plasma, meliorated histological appearance compared with the group given OA. PPS-inhaling group had better effects than saline-inhaling
group on lung index> protein contents in BALF> TNF-a level in plasma and histological appearance. Inhaling PPS (160mg/Kg) had the same effects with instilling PPS (lOOmg/Kg).From what we studied, we had proved that PPS provided therapeutic effects on animal models of ALI induced by oleic acid. PPS=^80mg/Kg administered at early stage might alleviate the lung injury, however more PPS^150mg/Kg was needed at late stage. Aerosolized PPS (160mg/Kg) also had the therapeutic effects, but the effect was inferior to that obtained with bolus PPS. As this method of inhaling PPS is more convenient and safer, more investigation about the implication of aerosolized PPS is needed.Our findings provide the proofs of efficiency of PPS and help to make up the clinical medication of PS. We also set up a new method of aerosolized PS.
引文
[1] Wilson DF, Zaritsky A, Bauman LA, Dockery K, James RL, Conrad D, Croft H, Novotny WE, Egan EA, Dalton H: Instillation of calf lung surfactant extract (calfactant) is beneficial in pediatric acute hypoxemic respiratory failure. Members of the Mid-Atlantic Pediatric Critical Care Network. Crit Care Med 1999,27:188-195.
[2] Walmrath D, Gunther A, Ghofrani HA, Schermuly R, Schneider T, Grimminger F, Seeger W: Bronchoscopie surfactant administration in patients with severe adult respiratory distress syndrome and sepsis. Am J Respir Crit Care Med 1996,154:57-62.
[3] Spragg RG, Lewis JF, Walmrath D, Johannigman J, Bellingan G, Laterre PF, Witte MC, Richards GA, Rippin G, Rathgeb F, et al: Effect of recombinant surfactant protein C-based surfactant on the acute respiratory distress syndrome. N Engl J Med 2004,351:884-892.
[4] Gregory T J, Steinberg KP, Spragg R, Gadek JE, Hyers TM, Longmore WJ, Moxley MA, Cal GZ, Hire RD, Smith RM, Hudson LD, Crim C, Newton P, Mitchell BR, Gold AJ: Bovine surfactant therapy for patients with acute respiratory distress syndrome. Am J Respir Crit Care Med 1997,155:1309-1315.
[5] 陆月明:外源性肺表面活性物质在ARDS中的治疗作用。中国处方药,2004,2(23):11-16。
[6] 刘辉,王国军,丁正年:外源性肺泡表面活性物质对急性肺损伤治疗效果及量效关系的研究。Acta Universitatis Medicinalis Nanjing (Natural Science),2005,25(3): 176-179.
[7] Joan C Meidter, Venkatareman Balaraman, Tercia KU, et al: Lavage administration of diluted recombinant surfactant in ALl in piglets. Pediatric Research, 2000,47(2):240-245.
[8] Sun B, Herting E, Curstedt T, et al: Exogenous surfaetant improves lung compliance and oxygenation in adult rats with meconium adpiration. J Appl Physiol, 1994,77(4): 1961-1971.
[9] 朱光发,张国清,郭海英,张旻,周新:肺表面活性物质对急性呼吸窘迫综合征肺内炎症反应的影响。中国呼吸与危重监护杂志,2002,1(4):206-209。
[10] Zeenat Safdar, Maimiti Yiming, Gabriele Grunig, Jahar Bhattacharya: Inhibition of Acid-induced Lung Injury by Hyperosmolar Sucrose in Rats[J]. American Journal of Respiratory and Critical Care Medicine, 2005,172( 8): 1002-1007.
[11] Karen, Lu Bengt Robertson H, William Taeusch: Dextran or polyethylene glycol added to curosurf for treatment of meconium Lung Injury in Rats[J]. Biol Neonate, 2005,88:46-53.
[2] Walmrath D, Gunther A, Ghofrani HA, Schermuly R, Schneider T, Grimminger F, Seeger W: Bronchoscopie surfactant administration in patients with severe adult respiratory distress syndrome and sepsis. Am J Respir Crit Care Med 1996,154:57-62.
[3] Spragg RG, Lewis JF, Walmrath D, Johannigman J, Bellingan G, Laterre PF, Witte MC, Richards GA, Rippin G, Rathgeb F, et al: Effect of recombinant surfactant protein C-based surfactant on the acute respiratory distress syndrome. N Engl J Med 2004,351:884-892.
[4] Gregory T J, Steinberg KP, Spragg R, Gadek JE, Hyers TM, Longmore WJ, Moxley MA, Cal GZ, Hire RD, Smith RM, Hudson LD, Crim C, Newton P, Mitchell BR, Gold AJ: Bovine surfactant therapy for patients with acute respiratory distress syndrome. Am J Respir Crit Care Med 1997,155:1309-1315.
[5] 陆月明:外源性肺表面活性物质在ARDS中的治疗作用。中国处方药,2004,2(23):11-16。
[6] 刘辉,王国军,丁正年:外源性肺泡表面活性物质对急性肺损伤治疗效果及量效关系的研究。Acta Universitatis Medicinalis Nanjing (Natural Science),2005,25(3): 176-179.
[7] Joan C Meidter, Venkatareman Balaraman, Tercia KU, et al: Lavage administration of diluted recombinant surfactant in ALl in piglets. Pediatric Research, 2000,47(2):240-245.
[8] Sun B, Herting E, Curstedt T, et al: Exogenous surfaetant improves lung compliance and oxygenation in adult rats with meconium adpiration. J Appl Physiol, 1994,77(4): 1961-1971.
[9] 朱光发,张国清,郭海英,张旻,周新:肺表面活性物质对急性呼吸窘迫综合征肺内炎症反应的影响。中国呼吸与危重监护杂志,2002,1(4):206-209。
[10] Zeenat Safdar, Maimiti Yiming, Gabriele Grunig, Jahar Bhattacharya: Inhibition of Acid-induced Lung Injury by Hyperosmolar Sucrose in Rats[J]. American Journal of Respiratory and Critical Care Medicine, 2005,172( 8): 1002-1007.
[11] Karen, Lu Bengt Robertson H, William Taeusch: Dextran or polyethylene glycol added to curosurf for treatment of meconium Lung Injury in Rats[J]. Biol Neonate, 2005,88:46-53.