甲泼尼龙预处理对兔复张性肺水肿肺通透性和肺泡表面活性物质的影响
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摘要
复张性肺水肿(reexpansion pulmanory edema ,RPE)是继发于任何原因所致的肺不张,在肺快速复张时或复张后发生的急性肺水肿,是一种罕见但可能致命的肺部并发症,主要表现为肺部微血管渗透性增加。尽管糖皮质激素因其具有强大的抗炎作用在各种肺损伤的治疗中一直受到关注,但将其用于对复张性肺水肿的预处理是否有效鲜有文献报道。本研究拟观察甲泼尼龙预处理对复张性肺水肿兔肺的肺通透性以及肺泡表面活性物质二棕榈酰磷脂酰胆碱(dipalmitoylphosphatidylcholine, DPPC)的影响。
RPE早期最基本的病理生理改变就是肺血管内皮细胞及肺泡上皮细胞屏障对液体、蛋白通透性的增加,进而导致肺泡内外液体转运失衡。已有研究发现在RPE水肿液中蛋白含量高,明显高于静水性肺水肿,而肺毛细血管嵌压(PCWP)正常。至于如何引起肺泡毛细血管通透性增加,血液成分渗漏,其机制尚未明了,近年来的研究包括这些方面：机械损伤；肺泡表面活性物质(PS)减少；肺泡的再灌注损伤；炎症介导反应。
RPE时,肺泡表面活性物质的减少已在临床检验中得到证实,PS是一种复合磷脂,起最主要作用的是饱和卵磷脂中的二棕榈酰磷脂酰胆碱(DPPC)。在RPE病理生理过程中,肺组织的缺血缺氧,直接导致肺泡表面活性物质生成与分泌减少,而复张后,PS的失活可由血浆蛋白,如白蛋白,纤维蛋白原,C反应蛋白,还有血红蛋白等与其结合而丧失；炎症还能促使磷脂酶A2(PLA2)释放,加速PS中的磷脂成分尤其是DPPC的分解,从而使PS含量减少。
本研究在建立RPE模型的基础上,进行肺泡灌洗,肺泡灌洗液(BALF)的总蛋白含量可在一定程度上反映血管内皮细胞和肺泡上皮细胞的屏障功能；BALF总蛋白与血浆总蛋白的比值为肺通透性指数,肺通透性指数反映肺组织对蛋白的通透性。有研究表明肺血管内皮细胞间完整性的破坏与血浆基质金属蛋白酶-9(MMP-9)的关系最为密切,MMP-9表达增多,活性升高,与肺组织损伤、肺泡毛细血管通透性升高密切相关。尚有研究表明使用甲泼尼龙可以减少中性粒细胞的活化从而抑制MMP-9的释放,因此可能降低复张性肺水肿的肺通透性。此外在BALF中,我们选择测定在肺泡表面活性物质中起最主要作用的二棕榈酰磷脂酰胆碱(DPPC)。有研究证实使用大剂量甲泼尼龙对PS活性的保持有良好的影响,甲泼尼龙能显著减轻肺损伤,并减少漏入肺泡腔的血浆蛋白对PS的灭活。综上所述,甲泼尼龙能降低复张性肺水肿兔肺的蛋白漏出,降低肺通透性指数,并能提高肺泡表面活性物质的活性成分DPPC的含量。
材料和方法：
1.实验动物准备与分组：
21只体重2.5-3.0kg的雄性新西兰大耳兔随机分为三组：C组为空白对照组,R组为复张性肺水肿组,R+M组为复张性肺水肿+甲泼尼龙预处理组。实验动物于模型制备前均禁食过夜。
R组：建立复张性肺水肿模型后饲养7天,于肺复张前20分钟,经耳缘静脉给予0.9%生理盐水2.0ml/kg。
R+M组：建立复张性肺水肿模型后饲养7天,于肺复张前20分钟,经耳缘静脉给予甲泼尼龙3mg/kg。
C组：经耳缘静脉给予0.9%生理盐水2.0ml/kg。
2.动物模型制作
经兔耳缘静脉缓慢推注3%的戊巴比妥钠,至角膜反射消失,固定于解剖台上按Sakao的方法改良复制复张性肺水肿模型：在右侧胸廓第四,五肋间切开皮肤,进入胸腔,置入无菌橡胶套囊,在套囊开口注入气体(10ml/kg),无菌丝线结扎套囊口,关闭胸腔。置于兔笼饲养7天后再次麻醉,行气管切开,右侧颈动脉置管测压,连接生理记录仪。在气囊取出肺复张前20分钟,分别经耳缘静脉给予R组0.9%生理盐水2.0ml/kg；R+M组甲泼尼龙3mg/kg。观察20分钟,生命体征稳定后,取出右侧胸腔内气囊,同时置入12F导尿管,固定于胸壁上。为促使肺复张完全,通过导管行负压抽吸,压力为-20cmH2O,排出胸腔内气体,同时经气管切开开口插管,并用简易呼吸器鼓肺5次,气道压力不超过20cmH2O。最后钳闭胸腔导管。在相应时间点完成血样采取后,行空气栓塞处死动物,打开胸腔,取右肺组织,用福尔马林固定,肺组织切片在光镜下证实具有肺间质及肺泡水肿,渗出明显,中性粒细胞及单核细胞浸润等一系列复张性肺水肿的病理表现。钳闭左侧支气管,取0.9%NaCl溶液20ml/kg,在气管切开开口处行支气管肺泡灌洗。
3.标本采集
复张后4h采集动脉血3ml,以备测定血清总蛋白。同期行支气管肺泡灌洗,用于测定灌洗液中的总蛋白和DPPC。
4.标本处理
(1)将3ml血液加入生化管中,置于4℃冰箱3小时,取上清液,将上清液于4℃离心机中离心(2500r/min,8 min),离心后再取其上清液置于-80℃冰箱保存。
(2)肺泡灌洗液制备成功后用透析带滤去杂质与痰液,置于4℃离心机中离心(3000 r/min,10 min),取上清液置于-80℃冰箱保存。
5.标本检测
血清与肺泡灌洗液中的总蛋白含量测定选用Bicinchoninic acid法(BCA法)。肺泡灌洗液中的二棕榈酰磷脂酰胆碱(DPPC)检测采用超高效液相色谱/串联质谱法(UPLC-MS/MS).
6.统计分析
统计分析采用SPSS13.0统计软件,计量资料以均数±标准差(x±s)表示,统计方法采用完全随机设计资料的方差分析,方差不齐时采用Welch校正,多重比较采用Bonferroni法,方差不齐时多重比较采用Dunnett T3(?)方法,P<0.05为差异有统计学意义。
结果
1.基本资料：三组新西兰雄性大耳兔的体重、肺泡灌洗液回收率的比较差异无统计学意义。
2.实验模型：本实验模型制作采用了在右侧胸腔置入气囊,饲养七天后复张的方法,置入气囊后第一天及第七天在X光线下可见：第一天右侧中下肺叶压缩达80%以上；第七天则压缩达到60%-70%。动物处死后,取肺组织制作病理切片,切片光镜下表现具有肺间质及肺泡水肿,渗出明显,中性粒细胞及单核细胞浸润等一系列复张性肺水肿病理表现
3.BALF总蛋白含量与肺通透性指数
BALF总蛋白含量R组最高,与C组及R+M组相比差异具有统计学意义。R+M组的BALF总蛋白含量为0.197±0.060,显著低于R组的0.443±0.098(p=0.000),C组与R+M比较,差异不存在统计学意义。在肺通透性指数的比较中,C组最低,与R组相比差异存在统计学意义(p=0.000),但与R+M组比较则差异无统计学意义(p=0.337)。R组及R+M组比较,R+M组肺通透性指数为0.007±0.002,显著低于R组的0.018±0.004(p=0.000；P<0.05)。
4.BALF中DPPC的含量
BALF中的DPPC含量C组最高,为198.065±21.525gg/ml,且与其余两组比较具有统计学意义；R+M组含量高于R组,R+M组为101.955±24.544μg/ml；R组为61.815±28.307μg/ml；两组比较具有显著差异(p=0.023)。
结论：
1.本实验采用了在兔右侧胸腔内置入气囊的方法制作RPE模型,动物死亡率低,操作便利。肺组织病理切片证实了动物RPE模型的成功。
2.甲泼尼龙能降低复张性肺水肿兔肺的蛋白漏出,降低肺通透性指数。
3.甲泼尼龙提高肺泡表面活性物质的活性成分DPPC的含量。
Reexpansion pulmonary edema(RPE) is an acute pulmonary edema which happens after any atelectasis when the lung is rapidly reexpansing or in after reexpansion. It is a rare but potentially fatal pulmonary complication, which mainly go with pulmonary capillary permeability increase. Although glucocorticoid has been drawing everybody's attention because of its powerful anti-inflammatory role in the treatment of various lung injuries, whether it is effective in its pre-treatment for acute lung injury is rarely reported in the literature. So, this study is to examine the effects of methylprednisolone pretreatment on reexpansion pulmonary edema in rabbit lung permeability and on lung surfactant dipalmitoylphosphatidylcholine(DPPC).
The most fundamental changes of early RPE is cause by the alveolar fluid transport and external imbalances right after the increase in liquid and protein permeability by the path physiology of pulmonary vascular endothelial cells and alveolar epithelial cell barrier. It has been found that protein percentage is significantly higher in the RPE edema fluid than in hydrostatic pulmonary edema when pulmonary capillary wedge pressure (PCWP) is normal. As for the mechanism, how to rise to increased alveolar capillary permeability and leakage of blood components is unknown. Recent studies including: mechanical damage, pulmonary surfactant Substances (PS) reduced, lung reperfusion injury and inflammatory-mediated response.
The reduction of pulmonary surfactant has been demonstrated in clinical tests when RPE happens:PS, a kind of complex phospholipids, containing DPPC which plays a main role when RPE. In the pathophysiological process of RPE, the ischemia and hypoxia of the lung tissue lead to a reduction of pulmonary surfactant's formation and secretion. While after reexpantion, PS inactivated by plasma proteins such as albumin, fibrinogen, C-reactive protein. And there would be a loss of its binding hemoglobin; inflammation can promote phospholipase A2 (PLA2) release, speed up the PS, especially DPPC decomposition, so that the PS content decreases.
RPE model was established in this study. Based on the model, we do bronchoalveolar by BALF. In some extent, the total protein proportion of BALF reflects that the endothelial cells and alveolar epithelial cell have barrier function. BALF's total protein and plasma's total protein ratio is the lung permeability index, and lung permeability index reflects the protein permeability of lung tissue. Research indicated that the damage of pulmonary vascular endothelial cell and plasma matrix metalloproteinase -9 (MMP-9) are most closely related. The increase of MMP-9 expression and activity are closed related to lung tissue damage and alveolar capillary permeability increase. Also, other studies shows that the using of methylprednisolone reduce the activation of neutrophils and inhibit the release of MMP-9 and may therefore reduce the reexpansion pulmonary edema in lung permeability. In addition, in BALF, we chose the determination of DPPC. Research has shown the large doses of methylprednisolone on the PS activity in maintaining a good influence. Methylprednisolone can significantly reduce lung injury, and reduce the leakage of plasma protein into the alveolar space of the inactivation of PS. In conclusion, methylprednisolone can reduce the reexpansion pulmonary edema in rabbit lung protein leakage and reduce the lung permeability index, and can improve the pulmonary surfactant DPPC content of the active ingredients.
Materials and methods:
1. Experimental animal preparation and grouping:
21 New Zealand male white rabbits which weighted 2.5-3.0kg were randomly divided into three groups:group C, group R and group R+M. Group C was the control group, group R was the reexpansion pulmonary edema group, group R + M was the reexpansion pulmonary edema + methylprednisolone pretreatment group. The experimental animals in the model are fasted the night before.
Group R: with 7 days'feeding after the establishment of reexpansion pulmonary edema, inject the rabbits'ear vein with 2.0ml/kg 0.9% saline in the first 20 minutes of its lung reexpansion.
Group R + M:with 7 days' feeding after the establishment of reexpansion pulmonary edema, inject the rabbits'ear vein with 3mg/kg methylprednisolone in the first 20 minutes of lung reexpansion.
Group C:the ear vein was inject with 2.0ml/kg 0.9% saline.
2. Animal model
Slowly inject the Rabbit's ear vein with 3% sodium pentobarbital until corneal reflex disappears. Then fix the rabbit on the autopsy table. Modify the reexpansion pulmonary edema model by Sakao modeling: In the right side of the thorax, incision the fourth and fifth intercostal of the skin, place a sterile rubber cuff into the chest, inject gas (10ml/kg), and sterile silk ligature cuff mouth, then closed the chest. Place the rabbit in the cage for 7 days, then anesthetized again, tracheotomy. After that, measure the pressure of the right carotid artery catheter and connect the polygraph.20 minutes before removing the air bag, inject the rabbit's ear vein by 2.0ml/kg 0.9% group R and 3mg/kg of methylprednisolone of group R + M. Observe for 20 minutes, if vital signs are stable, remove the right side of the chest air bags. In the mean while insertl2F catheter and fix it on the chest wall. In order to completely promote lung recruitment, do vacuum suction through the catheter with a pressure of 20cmH2O, exhaust the chest's air. In the mean while open the tracheotomy tube and ventilator with a simple drum, pulmonary the airway 5 times with a pressure that does not exceed 20 cmH2O. Finally, clamp the chest tube. Take the blood samples correspond. Kill the rabbit by air embolism, open the chest, take the right lung tissue and fix with formalin. It is proved in the light microscopy that the lung tissue has lung biopsy pulmonary interstitial and alveolar edema. The exudate evidently happens, and the neutrophils and monocytes and a series of reexpansion pulmonary edema pathology appears. Finally, clamp the left bronchus, take 20ml/kg 0.9% NaCl solution, do bronchoalveolar lavage in the opening of the airway.
3. Specimen collection
After 4 hours of reexpansion, in preparing for serum total protein, collect 3ml arterial blood. Bronchial lavage over the same period for the determination of total protein and fluid in the DPPC
4. Specimen handling
(1) The blood were saved in refrigerator at 4℃for 3 hours, and then the supernatant was centrifuged at 4℃centrifuge (2500r/min,8 min), supernatant after centrifugation home Refrigerator at-80℃.
(2) Prepare bronchoalveolar lavage fluid after the success with filtered of impurities by dialysis and sputum, centrifugate centrifuge at 4℃(3000 r/min,10 min), was then place supernatant in-80℃refrigerator.
5. Sample testing
The study use Bicinchoninic acid(BCA). And serum and BALF total protein content determination method BALF dppc detected by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS).
6. Statistics Analysis
SPSS 13.0 statistical software is used for statistical analysis, measurement data as mean±standard deviation (x±s), P<0.05 was considered statistically significant
Results
1. The differential of the weight among the three groups of male New Zealand white rabbits, and of the recovery lavage fluid showed no statistical significant.
2. This experimental model produced by a balloon inserted in the right side of the chest, keeping seven days before the reexpansion. When placing in the first day and seventh day under the X light it can be seen: in the first day,80% compression in the right middle and lower lobe, the seventh day 60% -70%. After the animals were sacrificed, the lung biopsy tissue was produced. Under the light microscope, there was pulmonary interstitial and alveolar edema, exudate clear neutrophils and monocytes and a series of reexpansion pulmonary edema pathology
3. The lung permeability index in group R+M (0.007±0.002) was significantly lower than that in group R (0.177±0.004) (p <0.05) with respective DPPC concentration of 101.955±24.544μg/ml and 61.815±28.307μg/ml (p<0.05).
Conclusion Methylprednisolone pretreatment may increase the density of pulmonary surfactant and improve lung permeability in rabbit model of reexpansion pulmonary edema.
RPE早期最基本的病理生理改变就是肺血管内皮细胞及肺泡上皮细胞屏障对液体、蛋白通透性的增加,进而导致肺泡内外液体转运失衡。已有研究发现在RPE水肿液中蛋白含量高,明显高于静水性肺水肿,而肺毛细血管嵌压(PCWP)正常。至于如何引起肺泡毛细血管通透性增加,血液成分渗漏,其机制尚未明了,近年来的研究包括这些方面：机械损伤；肺泡表面活性物质(PS)减少；肺泡的再灌注损伤；炎症介导反应。
RPE时,肺泡表面活性物质的减少已在临床检验中得到证实,PS是一种复合磷脂,起最主要作用的是饱和卵磷脂中的二棕榈酰磷脂酰胆碱(DPPC)。在RPE病理生理过程中,肺组织的缺血缺氧,直接导致肺泡表面活性物质生成与分泌减少,而复张后,PS的失活可由血浆蛋白,如白蛋白,纤维蛋白原,C反应蛋白,还有血红蛋白等与其结合而丧失；炎症还能促使磷脂酶A2(PLA2)释放,加速PS中的磷脂成分尤其是DPPC的分解,从而使PS含量减少。
本研究在建立RPE模型的基础上,进行肺泡灌洗,肺泡灌洗液(BALF)的总蛋白含量可在一定程度上反映血管内皮细胞和肺泡上皮细胞的屏障功能；BALF总蛋白与血浆总蛋白的比值为肺通透性指数,肺通透性指数反映肺组织对蛋白的通透性。有研究表明肺血管内皮细胞间完整性的破坏与血浆基质金属蛋白酶-9(MMP-9)的关系最为密切,MMP-9表达增多,活性升高,与肺组织损伤、肺泡毛细血管通透性升高密切相关。尚有研究表明使用甲泼尼龙可以减少中性粒细胞的活化从而抑制MMP-9的释放,因此可能降低复张性肺水肿的肺通透性。此外在BALF中,我们选择测定在肺泡表面活性物质中起最主要作用的二棕榈酰磷脂酰胆碱(DPPC)。有研究证实使用大剂量甲泼尼龙对PS活性的保持有良好的影响,甲泼尼龙能显著减轻肺损伤,并减少漏入肺泡腔的血浆蛋白对PS的灭活。综上所述,甲泼尼龙能降低复张性肺水肿兔肺的蛋白漏出,降低肺通透性指数,并能提高肺泡表面活性物质的活性成分DPPC的含量。
材料和方法：
1.实验动物准备与分组：
21只体重2.5-3.0kg的雄性新西兰大耳兔随机分为三组：C组为空白对照组,R组为复张性肺水肿组,R+M组为复张性肺水肿+甲泼尼龙预处理组。实验动物于模型制备前均禁食过夜。
R组：建立复张性肺水肿模型后饲养7天,于肺复张前20分钟,经耳缘静脉给予0.9%生理盐水2.0ml/kg。
R+M组：建立复张性肺水肿模型后饲养7天,于肺复张前20分钟,经耳缘静脉给予甲泼尼龙3mg/kg。
C组：经耳缘静脉给予0.9%生理盐水2.0ml/kg。
2.动物模型制作
经兔耳缘静脉缓慢推注3%的戊巴比妥钠,至角膜反射消失,固定于解剖台上按Sakao的方法改良复制复张性肺水肿模型：在右侧胸廓第四,五肋间切开皮肤,进入胸腔,置入无菌橡胶套囊,在套囊开口注入气体(10ml/kg),无菌丝线结扎套囊口,关闭胸腔。置于兔笼饲养7天后再次麻醉,行气管切开,右侧颈动脉置管测压,连接生理记录仪。在气囊取出肺复张前20分钟,分别经耳缘静脉给予R组0.9%生理盐水2.0ml/kg；R+M组甲泼尼龙3mg/kg。观察20分钟,生命体征稳定后,取出右侧胸腔内气囊,同时置入12F导尿管,固定于胸壁上。为促使肺复张完全,通过导管行负压抽吸,压力为-20cmH2O,排出胸腔内气体,同时经气管切开开口插管,并用简易呼吸器鼓肺5次,气道压力不超过20cmH2O。最后钳闭胸腔导管。在相应时间点完成血样采取后,行空气栓塞处死动物,打开胸腔,取右肺组织,用福尔马林固定,肺组织切片在光镜下证实具有肺间质及肺泡水肿,渗出明显,中性粒细胞及单核细胞浸润等一系列复张性肺水肿的病理表现。钳闭左侧支气管,取0.9%NaCl溶液20ml/kg,在气管切开开口处行支气管肺泡灌洗。
3.标本采集
复张后4h采集动脉血3ml,以备测定血清总蛋白。同期行支气管肺泡灌洗,用于测定灌洗液中的总蛋白和DPPC。
4.标本处理
(1)将3ml血液加入生化管中,置于4℃冰箱3小时,取上清液,将上清液于4℃离心机中离心(2500r/min,8 min),离心后再取其上清液置于-80℃冰箱保存。
(2)肺泡灌洗液制备成功后用透析带滤去杂质与痰液,置于4℃离心机中离心(3000 r/min,10 min),取上清液置于-80℃冰箱保存。
5.标本检测
血清与肺泡灌洗液中的总蛋白含量测定选用Bicinchoninic acid法(BCA法)。肺泡灌洗液中的二棕榈酰磷脂酰胆碱(DPPC)检测采用超高效液相色谱/串联质谱法(UPLC-MS/MS).
6.统计分析
统计分析采用SPSS13.0统计软件,计量资料以均数±标准差(x±s)表示,统计方法采用完全随机设计资料的方差分析,方差不齐时采用Welch校正,多重比较采用Bonferroni法,方差不齐时多重比较采用Dunnett T3(?)方法,P<0.05为差异有统计学意义。
结果
1.基本资料：三组新西兰雄性大耳兔的体重、肺泡灌洗液回收率的比较差异无统计学意义。
2.实验模型：本实验模型制作采用了在右侧胸腔置入气囊,饲养七天后复张的方法,置入气囊后第一天及第七天在X光线下可见：第一天右侧中下肺叶压缩达80%以上；第七天则压缩达到60%-70%。动物处死后,取肺组织制作病理切片,切片光镜下表现具有肺间质及肺泡水肿,渗出明显,中性粒细胞及单核细胞浸润等一系列复张性肺水肿病理表现
3.BALF总蛋白含量与肺通透性指数
BALF总蛋白含量R组最高,与C组及R+M组相比差异具有统计学意义。R+M组的BALF总蛋白含量为0.197±0.060,显著低于R组的0.443±0.098(p=0.000),C组与R+M比较,差异不存在统计学意义。在肺通透性指数的比较中,C组最低,与R组相比差异存在统计学意义(p=0.000),但与R+M组比较则差异无统计学意义(p=0.337)。R组及R+M组比较,R+M组肺通透性指数为0.007±0.002,显著低于R组的0.018±0.004(p=0.000；P<0.05)。
4.BALF中DPPC的含量
BALF中的DPPC含量C组最高,为198.065±21.525gg/ml,且与其余两组比较具有统计学意义；R+M组含量高于R组,R+M组为101.955±24.544μg/ml；R组为61.815±28.307μg/ml；两组比较具有显著差异(p=0.023)。
结论：
1.本实验采用了在兔右侧胸腔内置入气囊的方法制作RPE模型,动物死亡率低,操作便利。肺组织病理切片证实了动物RPE模型的成功。
2.甲泼尼龙能降低复张性肺水肿兔肺的蛋白漏出,降低肺通透性指数。
3.甲泼尼龙提高肺泡表面活性物质的活性成分DPPC的含量。
Reexpansion pulmonary edema(RPE) is an acute pulmonary edema which happens after any atelectasis when the lung is rapidly reexpansing or in after reexpansion. It is a rare but potentially fatal pulmonary complication, which mainly go with pulmonary capillary permeability increase. Although glucocorticoid has been drawing everybody's attention because of its powerful anti-inflammatory role in the treatment of various lung injuries, whether it is effective in its pre-treatment for acute lung injury is rarely reported in the literature. So, this study is to examine the effects of methylprednisolone pretreatment on reexpansion pulmonary edema in rabbit lung permeability and on lung surfactant dipalmitoylphosphatidylcholine(DPPC).
The most fundamental changes of early RPE is cause by the alveolar fluid transport and external imbalances right after the increase in liquid and protein permeability by the path physiology of pulmonary vascular endothelial cells and alveolar epithelial cell barrier. It has been found that protein percentage is significantly higher in the RPE edema fluid than in hydrostatic pulmonary edema when pulmonary capillary wedge pressure (PCWP) is normal. As for the mechanism, how to rise to increased alveolar capillary permeability and leakage of blood components is unknown. Recent studies including: mechanical damage, pulmonary surfactant Substances (PS) reduced, lung reperfusion injury and inflammatory-mediated response.
The reduction of pulmonary surfactant has been demonstrated in clinical tests when RPE happens:PS, a kind of complex phospholipids, containing DPPC which plays a main role when RPE. In the pathophysiological process of RPE, the ischemia and hypoxia of the lung tissue lead to a reduction of pulmonary surfactant's formation and secretion. While after reexpantion, PS inactivated by plasma proteins such as albumin, fibrinogen, C-reactive protein. And there would be a loss of its binding hemoglobin; inflammation can promote phospholipase A2 (PLA2) release, speed up the PS, especially DPPC decomposition, so that the PS content decreases.
RPE model was established in this study. Based on the model, we do bronchoalveolar by BALF. In some extent, the total protein proportion of BALF reflects that the endothelial cells and alveolar epithelial cell have barrier function. BALF's total protein and plasma's total protein ratio is the lung permeability index, and lung permeability index reflects the protein permeability of lung tissue. Research indicated that the damage of pulmonary vascular endothelial cell and plasma matrix metalloproteinase -9 (MMP-9) are most closely related. The increase of MMP-9 expression and activity are closed related to lung tissue damage and alveolar capillary permeability increase. Also, other studies shows that the using of methylprednisolone reduce the activation of neutrophils and inhibit the release of MMP-9 and may therefore reduce the reexpansion pulmonary edema in lung permeability. In addition, in BALF, we chose the determination of DPPC. Research has shown the large doses of methylprednisolone on the PS activity in maintaining a good influence. Methylprednisolone can significantly reduce lung injury, and reduce the leakage of plasma protein into the alveolar space of the inactivation of PS. In conclusion, methylprednisolone can reduce the reexpansion pulmonary edema in rabbit lung protein leakage and reduce the lung permeability index, and can improve the pulmonary surfactant DPPC content of the active ingredients.
Materials and methods:
1. Experimental animal preparation and grouping:
21 New Zealand male white rabbits which weighted 2.5-3.0kg were randomly divided into three groups:group C, group R and group R+M. Group C was the control group, group R was the reexpansion pulmonary edema group, group R + M was the reexpansion pulmonary edema + methylprednisolone pretreatment group. The experimental animals in the model are fasted the night before.
Group R: with 7 days'feeding after the establishment of reexpansion pulmonary edema, inject the rabbits'ear vein with 2.0ml/kg 0.9% saline in the first 20 minutes of its lung reexpansion.
Group R + M:with 7 days' feeding after the establishment of reexpansion pulmonary edema, inject the rabbits'ear vein with 3mg/kg methylprednisolone in the first 20 minutes of lung reexpansion.
Group C:the ear vein was inject with 2.0ml/kg 0.9% saline.
2. Animal model
Slowly inject the Rabbit's ear vein with 3% sodium pentobarbital until corneal reflex disappears. Then fix the rabbit on the autopsy table. Modify the reexpansion pulmonary edema model by Sakao modeling: In the right side of the thorax, incision the fourth and fifth intercostal of the skin, place a sterile rubber cuff into the chest, inject gas (10ml/kg), and sterile silk ligature cuff mouth, then closed the chest. Place the rabbit in the cage for 7 days, then anesthetized again, tracheotomy. After that, measure the pressure of the right carotid artery catheter and connect the polygraph.20 minutes before removing the air bag, inject the rabbit's ear vein by 2.0ml/kg 0.9% group R and 3mg/kg of methylprednisolone of group R + M. Observe for 20 minutes, if vital signs are stable, remove the right side of the chest air bags. In the mean while insertl2F catheter and fix it on the chest wall. In order to completely promote lung recruitment, do vacuum suction through the catheter with a pressure of 20cmH2O, exhaust the chest's air. In the mean while open the tracheotomy tube and ventilator with a simple drum, pulmonary the airway 5 times with a pressure that does not exceed 20 cmH2O. Finally, clamp the chest tube. Take the blood samples correspond. Kill the rabbit by air embolism, open the chest, take the right lung tissue and fix with formalin. It is proved in the light microscopy that the lung tissue has lung biopsy pulmonary interstitial and alveolar edema. The exudate evidently happens, and the neutrophils and monocytes and a series of reexpansion pulmonary edema pathology appears. Finally, clamp the left bronchus, take 20ml/kg 0.9% NaCl solution, do bronchoalveolar lavage in the opening of the airway.
3. Specimen collection
After 4 hours of reexpansion, in preparing for serum total protein, collect 3ml arterial blood. Bronchial lavage over the same period for the determination of total protein and fluid in the DPPC
4. Specimen handling
(1) The blood were saved in refrigerator at 4℃for 3 hours, and then the supernatant was centrifuged at 4℃centrifuge (2500r/min,8 min), supernatant after centrifugation home Refrigerator at-80℃.
(2) Prepare bronchoalveolar lavage fluid after the success with filtered of impurities by dialysis and sputum, centrifugate centrifuge at 4℃(3000 r/min,10 min), was then place supernatant in-80℃refrigerator.
5. Sample testing
The study use Bicinchoninic acid(BCA). And serum and BALF total protein content determination method BALF dppc detected by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS).
6. Statistics Analysis
SPSS 13.0 statistical software is used for statistical analysis, measurement data as mean±standard deviation (x±s), P<0.05 was considered statistically significant
Results
1. The differential of the weight among the three groups of male New Zealand white rabbits, and of the recovery lavage fluid showed no statistical significant.
2. This experimental model produced by a balloon inserted in the right side of the chest, keeping seven days before the reexpansion. When placing in the first day and seventh day under the X light it can be seen: in the first day,80% compression in the right middle and lower lobe, the seventh day 60% -70%. After the animals were sacrificed, the lung biopsy tissue was produced. Under the light microscope, there was pulmonary interstitial and alveolar edema, exudate clear neutrophils and monocytes and a series of reexpansion pulmonary edema pathology
3. The lung permeability index in group R+M (0.007±0.002) was significantly lower than that in group R (0.177±0.004) (p <0.05) with respective DPPC concentration of 101.955±24.544μg/ml and 61.815±28.307μg/ml (p<0.05).
Conclusion Methylprednisolone pretreatment may increase the density of pulmonary surfactant and improve lung permeability in rabbit model of reexpansion pulmonary edema.
引文
1.Riesman D. Albuminous expectoration following thoracocentesis.Am J Med Sci 1902; 123:620-30
2. Trapnell DH, Thuraton JG. Unilateral pulmonary oedema after pleural aspiration, Lancet 1970; 1:1367-9.
3. Johnstone W. Can you diagnose this ? Reexpansion pulmonary edema. Va Med 1980; 107:791-2.
4. Sprung CL, Loewenherz. JW, Baier H, Hauser MJ. Evidence for increased permeability in reexpansion. Am j Med 1981; 71:497-500.
5. Carlson RI, Classen KL, Gollan F, et al. Pulmonary edema following the rapid reexpansion of a totally collapsed lung due to a pneumothorax:a clinical and experimental study. Surg Forum 1959;9:367-71.
6. Fukuda T, Okutani R, Kono K, et al. A case of reexpansion pulmonary edema during fenestration of a giant hepatic cyst. Masui 1989; 38:1509-13.(in Jpse. With Engl. Abstr.)
7. Matsumiya N, Dohi S, Kimura T, Naito H. Reexpansion pulmonary edema after mediastinal tumor removal. Anesth Analg 1991;73:646-8.
8. Matsuura Y, Nomimura T, Murakami H.Clinical analysis of reexpansion pulmonary edema. Chest,1991,100:1562-6
9.土应果,王福,复张性肺水肿的救治,急诊医学,1997,6(3)：167-168.
10. Yim AP,Liu HP.Complications and failures of viedo-assisted thoracic surgery: Experience from two centers in Asia. Ann Thorac Surg,1996,61(2):538-41.
11.朱恩良,单根法,张辅贤,胸腔镜外科并发复张性肺水肿,上海第二医科大学学报,1998,18(3)：238-40.
12.李剑峰,刘桐林,王俊,等,胸腔镜术后复张性肺水肿的预防和治疗,中国内镜杂志,1998,4(1)：60-1.
13. Ragozzino MW,Greene R.Bilateral reexpansion pulmonary edema following unilateral pleurocentesis. Chest,1991,99(2):506-8.
14. Matsumiya N, Dohi S, Kimura T. Reexpansion pulmonary edema after mediastinal tumor removal. Anesht Analg,1991,73(5):648-8.
15. Pavlin DJ. Reexpansion hypotension--A complication of rapid evacuation of prolonged pneumothoxax. Chest,1986,89:70-4.
16. Suzuki S,Tanita T, Koike K. Evidence of acute inflammatory response reexpansion pulmonary edema. Chest,1992,101 (1):275-6.
17. Jackson RM,Ressell WJ,Veal CF,et al. Endogenous and exogenous catalase in reoxygenation lung injury.J Appl Physiol,1992,72:858-64.
18. Nakamura H ,Ishizaka A,Sawafuji M,et al. Elevated levels of interleukin-8 and leukotrieneB4 in pulmonary edema fluid of a patient with reexpansion pulmonary edema.Am J Respir Crit Care Med,1994,149:1037-40.
19. Matsuura Y, Nomimura T,Murakami H.Clinical analysis of reexpansion pulmonary edema. Chest,1991,100:1562-6.
20. Hao H, Wendt CH,Sandhu G, et al.Dexamethasone stimulates transcription of the Na+-K+ATP ase betal gene in adult rat lung epithelial cells.Am J Physiol Lung Cell Mol Physiol,2003,285(3):L593-L601
21. Wissink S, van Heerde EC,Vander Burg B,et al.A dual mechanism mediates repression of NFxB activity by glucocorticoids. Mol Endocrinol, 1998,12:355-63.
22. Lanchou J,Coroel M,Tanguy M,etal.Imbalance between matrix metalloproteinases (MMP-9 and MMP-2) and tissueinhibitors of metalloproteinases (TIM P-l and TTMP-2) in acute respiratory distress syndrome patients [J]. Crit Care Med,2003,31(2):53642
23. Joffs C, Gunasinghe H R, Multani M M, et al, Cardiopulmonary bypass induces the synthesis and release of matrix metalloproteinases[J]. Ann T horac S urg, 2001,71(5):1518-23
24. Keck T, Balcom J H, Femandez del Castillo C, et al. Matrix metalloproteinase 9 promotes neutrophil migration and alveolar capillary leakage in pancreatitis-associated lung injury in the rat[J]. Gastroenterology,2002,122(1):188-201
25.郭克芳,曾真,姜桢,甲泼尼龙对犬低温体外循环后肺血管通透性的影及机制,复旦学报(医学版)2006 Jul.,33(4)：470-4
26. Lema G,, Enhorning G., Surface properties after a simulated PLA2 hydrolysis of pulmonary surfactants main component, DPPC. Biochim Biophys Acta,1997; 1345:86-92.
27.岑坚正,张镜方,庄建,大剂量甲泼尼龙对幼猪体外循环肺表面活性物质的影响,中山大学学报(医学科学版),Vol.27 No.1 Jan.2006：42-6
28. Yukinori Sakao, Osamu Kajikawa, Thomas R. Martin, et al. Association of IL-8 and MCP-1 with the development of reexpansion pulmonary edema in rabbits[J]. Ann Thorac Surg.2001,71(6):1825-32.
29.占向鸿,顾泗荣,复张性肺水肿实验模型,江苏大学学报(医学版),August2002,12(4)：339-41
30.李百花,董殿军,张秋香,林晓明,高效液相色谱-蒸发光散射检测器测定大鼠支气管肺泡灌洗液中肺泡表面活性物质,分析实验室,Vol.26.NO.6.2007-6：6-9.
31.徐伟东,聂亮,高效液相色谱法-蒸发光散射检测器测定中/长链脂肪乳注射液中磷脂酞胆碱和磷脂酞乙醇胺含量,药物分析杂志,Anal2006,26(5)：661-3.
32.王凤君,赵云,谢尔凡,尤忠义,高效液相色谱法测定生物样品中磷脂含量,第三军医大学学报,Vol.18.No.1.Feb.1996：67-8.
33. Mora R, Arold S, Marzan Y, et al. Determinants of surfactant function in acute lung injury and early recovery[J]. Am J Physiol Lung Cell Mol Physiol,2000, 279(2):L342-349.
34. Williason PD,Jennifex L,Pavies SW. Changes in pulmonary microvascular permeability accompanying reexpansion pulmonary edema: evidence from dual isotopic scintigraphy. Thorax,1990,45:456-9
35. OLSON N, GREUL AK, HRISTOVA M, et al. Nitric oxide and airway epithelial barrier function:Regulation of tight junction proteins and epithelial permeability [J]. Arch Biochem Biophys,2009,484(2):205-13.
36. Alexander JS, Elrod JW. Extracellular matrix Junctional integrity and matrixmetallop roteinase interactions in endothelial permeability regulation. J Anat,2002,200(6):561-74
37.花少栋,杜江,唐雯,安胜利,杨丽华,封志纯,新生兔机械通气肺损伤MMP-9与白细胞相关性的研究,中国妇幼保健,2007年第22卷：4916-4920.
2. Trapnell DH, Thuraton JG. Unilateral pulmonary oedema after pleural aspiration, Lancet 1970; 1:1367-9.
3. Johnstone W. Can you diagnose this ? Reexpansion pulmonary edema. Va Med 1980; 107:791-2.
4. Sprung CL, Loewenherz. JW, Baier H, Hauser MJ. Evidence for increased permeability in reexpansion. Am j Med 1981; 71:497-500.
5. Carlson RI, Classen KL, Gollan F, et al. Pulmonary edema following the rapid reexpansion of a totally collapsed lung due to a pneumothorax:a clinical and experimental study. Surg Forum 1959;9:367-71.
6. Fukuda T, Okutani R, Kono K, et al. A case of reexpansion pulmonary edema during fenestration of a giant hepatic cyst. Masui 1989; 38:1509-13.(in Jpse. With Engl. Abstr.)
7. Matsumiya N, Dohi S, Kimura T, Naito H. Reexpansion pulmonary edema after mediastinal tumor removal. Anesth Analg 1991;73:646-8.
8. Matsuura Y, Nomimura T, Murakami H.Clinical analysis of reexpansion pulmonary edema. Chest,1991,100:1562-6
9.土应果,王福,复张性肺水肿的救治,急诊医学,1997,6(3)：167-168.
10. Yim AP,Liu HP.Complications and failures of viedo-assisted thoracic surgery: Experience from two centers in Asia. Ann Thorac Surg,1996,61(2):538-41.
11.朱恩良,单根法,张辅贤,胸腔镜外科并发复张性肺水肿,上海第二医科大学学报,1998,18(3)：238-40.
12.李剑峰,刘桐林,王俊,等,胸腔镜术后复张性肺水肿的预防和治疗,中国内镜杂志,1998,4(1)：60-1.
13. Ragozzino MW,Greene R.Bilateral reexpansion pulmonary edema following unilateral pleurocentesis. Chest,1991,99(2):506-8.
14. Matsumiya N, Dohi S, Kimura T. Reexpansion pulmonary edema after mediastinal tumor removal. Anesht Analg,1991,73(5):648-8.
15. Pavlin DJ. Reexpansion hypotension--A complication of rapid evacuation of prolonged pneumothoxax. Chest,1986,89:70-4.
16. Suzuki S,Tanita T, Koike K. Evidence of acute inflammatory response reexpansion pulmonary edema. Chest,1992,101 (1):275-6.
17. Jackson RM,Ressell WJ,Veal CF,et al. Endogenous and exogenous catalase in reoxygenation lung injury.J Appl Physiol,1992,72:858-64.
18. Nakamura H ,Ishizaka A,Sawafuji M,et al. Elevated levels of interleukin-8 and leukotrieneB4 in pulmonary edema fluid of a patient with reexpansion pulmonary edema.Am J Respir Crit Care Med,1994,149:1037-40.
19. Matsuura Y, Nomimura T,Murakami H.Clinical analysis of reexpansion pulmonary edema. Chest,1991,100:1562-6.
20. Hao H, Wendt CH,Sandhu G, et al.Dexamethasone stimulates transcription of the Na+-K+ATP ase betal gene in adult rat lung epithelial cells.Am J Physiol Lung Cell Mol Physiol,2003,285(3):L593-L601
21. Wissink S, van Heerde EC,Vander Burg B,et al.A dual mechanism mediates repression of NFxB activity by glucocorticoids. Mol Endocrinol, 1998,12:355-63.
22. Lanchou J,Coroel M,Tanguy M,etal.Imbalance between matrix metalloproteinases (MMP-9 and MMP-2) and tissueinhibitors of metalloproteinases (TIM P-l and TTMP-2) in acute respiratory distress syndrome patients [J]. Crit Care Med,2003,31(2):53642
23. Joffs C, Gunasinghe H R, Multani M M, et al, Cardiopulmonary bypass induces the synthesis and release of matrix metalloproteinases[J]. Ann T horac S urg, 2001,71(5):1518-23
24. Keck T, Balcom J H, Femandez del Castillo C, et al. Matrix metalloproteinase 9 promotes neutrophil migration and alveolar capillary leakage in pancreatitis-associated lung injury in the rat[J]. Gastroenterology,2002,122(1):188-201
25.郭克芳,曾真,姜桢,甲泼尼龙对犬低温体外循环后肺血管通透性的影及机制,复旦学报(医学版)2006 Jul.,33(4)：470-4
26. Lema G,, Enhorning G., Surface properties after a simulated PLA2 hydrolysis of pulmonary surfactants main component, DPPC. Biochim Biophys Acta,1997; 1345:86-92.
27.岑坚正,张镜方,庄建,大剂量甲泼尼龙对幼猪体外循环肺表面活性物质的影响,中山大学学报(医学科学版),Vol.27 No.1 Jan.2006：42-6
28. Yukinori Sakao, Osamu Kajikawa, Thomas R. Martin, et al. Association of IL-8 and MCP-1 with the development of reexpansion pulmonary edema in rabbits[J]. Ann Thorac Surg.2001,71(6):1825-32.
29.占向鸿,顾泗荣,复张性肺水肿实验模型,江苏大学学报(医学版),August2002,12(4)：339-41
30.李百花,董殿军,张秋香,林晓明,高效液相色谱-蒸发光散射检测器测定大鼠支气管肺泡灌洗液中肺泡表面活性物质,分析实验室,Vol.26.NO.6.2007-6：6-9.
31.徐伟东,聂亮,高效液相色谱法-蒸发光散射检测器测定中/长链脂肪乳注射液中磷脂酞胆碱和磷脂酞乙醇胺含量,药物分析杂志,Anal2006,26(5)：661-3.
32.王凤君,赵云,谢尔凡,尤忠义,高效液相色谱法测定生物样品中磷脂含量,第三军医大学学报,Vol.18.No.1.Feb.1996：67-8.
33. Mora R, Arold S, Marzan Y, et al. Determinants of surfactant function in acute lung injury and early recovery[J]. Am J Physiol Lung Cell Mol Physiol,2000, 279(2):L342-349.
34. Williason PD,Jennifex L,Pavies SW. Changes in pulmonary microvascular permeability accompanying reexpansion pulmonary edema: evidence from dual isotopic scintigraphy. Thorax,1990,45:456-9
35. OLSON N, GREUL AK, HRISTOVA M, et al. Nitric oxide and airway epithelial barrier function:Regulation of tight junction proteins and epithelial permeability [J]. Arch Biochem Biophys,2009,484(2):205-13.
36. Alexander JS, Elrod JW. Extracellular matrix Junctional integrity and matrixmetallop roteinase interactions in endothelial permeability regulation. J Anat,2002,200(6):561-74
37.花少栋,杜江,唐雯,安胜利,杨丽华,封志纯,新生兔机械通气肺损伤MMP-9与白细胞相关性的研究,中国妇幼保健,2007年第22卷：4916-4920.