全氟异丁烯急性吸入性肺损伤的病理机制研究
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摘要
作为重要的化工原料和副产物，全氟异丁烯（perfluoroisobutylene，PFIB）是一种常温下为气态的无色无味低分子量的高氟有机物，能穿透普通防毒面具，且无特效的防治措施，是常见的可引起急性肺损伤/急性呼吸窘迫综合征（acutelung injury，ALI/acute respiratory distress syndrome，ARDS）的有毒化学品。
目前，对PFIB所致ALI的发病机制研究认为直接氧化损伤可能是PFIB致病的重要机制之一；同时，多形核白细胞（polymorphonuclear leukocyte，PMN）介导的过度炎症反应可能是PFIB致病的另一重要机制。同时，肺泡巨噬细胞（alveolar macrophage，AM）参与PMN介导的PFIB急性吸入性肺损伤，并与PMN在肺内的扣押有关。PFIB急性吸入后可激活肺组织内多种细胞，尤其是间接激活AM内核因子κ B （nuclear factor-ha，NF-κ B），进而诱导AM表达多种致炎因子，这些致炎因子对PMN具有强烈的激活和趋化活性，但有关PFIB通过何种机制激活NF-κ B仍不清楚，PFIB急性吸入性肺损伤的机制研究仍有待完善。一、内源性血管紧张素II在PFIB急性吸入性肺损伤中的作用
近年研究发现，肾素-血管紧张素系统（renin-angiotensin system，RAS）在ALI/ARDS的发生、发展中发挥重要作用。血管紧张素II（angiotensin Ⅱ，AngⅡ）作为RAS的主要效应物质不仅是重要的体液调节因子参与调节血液循环，而且还是重要的前炎症因子，可上调NF-κB活性，增加白介素-8（Interleukin-8，IL-8）、白介素-1β（Interleukin-1beta，IL-1β）、肿瘤坏死因子-α（Tumor necrosisfactor-α，TNF-α）等炎症因子的表达释放，引发炎症反应。而血管紧张素转换酶（angiotensin convert enzyme，ACE）/血管紧张素转换酶2（angiotensin convertenzyme2，ACE2）分别作为AngⅡ的正/负性调节酶影响ALI的进程。减少AngⅡ的生成或阻断AngⅡ受体均可减轻多种原因所致的肺损伤。AngⅡ在PFIB急性吸入性肺损伤中的作用如何，它是否作为PFIB急性吸入性肺损伤的始动因素激活NF-κ B进而介导PFIB急性吸入性肺损伤，目前尚未见文献报道。本文的研究目的之一即是对上述问题进行初步探讨。
1、观察PFIB吸入染毒后，大鼠肺组织AngⅡ含量及其调节酶ACE的蛋白表达水平、酶活性与肺损伤程度的时间变化关系：大鼠PFIB急性吸入染毒后，肺系数、支气管肺泡灌洗液（bronchoalveolar lavage fluid，BALF）总蛋白含量和肺组织病理学检查等指标均显示肺损伤程度逐渐加重，至染毒后16h达峰值，染毒后24h明显缓解，因此确定本实验成功复制了大鼠PFIB急性吸入染毒病理模型。在此基础上，采用放射免疫分析方法检测PFIB染毒后大鼠的血液和肺组织匀浆中AngⅡ含量随时间的变化：血浆和肺组织中AngⅡ含量均呈现先升高后降低的变化趋势，其中肺组织AngⅡ含量在染毒后16h、24h较空白对照组显著降低。血浆AngⅡ含量与肺组织中AngⅡ变化趋势基本一致，但各时间点均无统计学差异；采用紫外线吸收法检测肺组织ACE酶活性随时间的变化：PFIB染毒后肺组织ACE活性在一定范围内波动，但各时间点无统计学差异。分析结果，肺组织AngⅡ含量和ACE酶活性的变化与肺损伤程度无明显的时间关联性，提示AngⅡ在大鼠PFIB急性吸入性肺损伤的发生、发展中可能不具有重要的病理学意义。
2、针对RAS发挥病理生理作用的最关键成分AngⅡ，选择AngⅡ的AngⅡ1型受体（angiotensionⅡ type1receptor，AT1-R）阻断剂氯沙坦，从时间-效应和剂量-效应两个方面考察AngⅡ在大鼠PFIB急性肺损伤中的作用：①时间-效应关系，PFIB染毒对照组大鼠的各项肺系数和BALF总蛋白含量较溶剂对照组显著升高，说明染毒模型建立成功。但不同时间给予氯沙坦（分别在PFIB染毒前1h、染毒前0.5h、染毒后0.5h、染毒后1h、染毒后2h、染毒后4h和染毒后8h腹腔注射氯沙坦10mg/kg）阻断AngⅡ受体进行预防或治疗，各组大鼠肺系数和BALF总蛋白含量与染毒对照组比较没有显著性差异。②剂量-效应关系，PFIB染毒对照组大鼠的各项肺系数和BALF总蛋白含量显著高于溶剂对照组，说明染毒模型建立成功。但给予不同剂量氯沙坦（分别在PFIB染毒前1h腹腔注射氯沙坦1.25、2.5、5、10和15mg/kg）阻断AngⅡ受体进行预防，各组大鼠肺系数和BALF总蛋白含量与染毒对照组比较没有显著性差异。以上结果表明，不同给药时间和不同给药剂量给予氯沙坦阻断AT1-R对PFIB急性肺损伤的影响微弱，进一步提示AngⅡ在PFIB急性吸入性肺损伤中可能不具有重要意义。
3、比较两种来源不同的AngⅡ受体阻断剂对PFIB急性吸入性肺损伤的防治效果：采用大样本量的小鼠为实验对象，观察国产和进口两种不同来源的氯沙坦对小鼠全身暴露吸入PFIB染毒后24h肺系数和72h死亡率的影响，实验结果表明染毒对照组和各个时间给药组（染毒前0.5h、染毒后0.5h和染毒后1h给药40mg/kg）比较均无显著性差异。这说明国产和进口来源的氯沙坦对小鼠PFIB急性吸入性肺损伤均无明显的防治作用。结果提示，AngⅡ在PFIB急性吸入性肺损伤中病理学意义有限。
综合分析，大鼠PFIB急性吸入性肺损伤过程中肺组织AngⅡ及其调节酶ACE与肺损伤程度无明显的时间相关性。时间-效应和剂量-效应关系研究揭示AT1-R阻断剂氯沙坦对大鼠PFIB急性肺损伤无明显影响。选择多个时间点腹腔注射两种不同来源的氯沙坦对小鼠PFIB急性吸入性肺损伤均无明显的改善作用。结果表明，AngⅡ在PFIB急性吸入性肺损伤中的病理作用微弱，RAS在PFIB急性肺损伤的发生、发展过程中可能不发挥主要作用，PFIB通过AngⅡ激活NF-κB启动炎症级联的通路可能并不存在。以上结果可能与不同ALI模型对RAS的影响不同有关，也可能与RAS复杂的生物学作用有关，其具体机理有待进一步研究。
二、PFIB对PMVEC功能的影响
肺气血屏障（blood-gas barrier）的结构和功能受损在各种原因所致ALI中居中心地位。其中，肺毛细血管内皮细胞（pulmonary microvascular endothelialcell，PMVEC）作为各种致病因素作用的靶细胞首先受到攻击，也是PMN扣押和游出血管时首先接触的细胞，所以PMVEC在ALI时的结构和功能变化受到越来越多的学者关注。
研究表明，肺损伤时气血屏障的细胞连接破坏、细胞骨架重排是炎性细胞渗出的关键环节，结果导致液体、蛋白、炎性介质等进入肺泡，这些构成了ALI的主要病理基础。PFIB染毒除了引起肺气血屏障主要细胞的结构发生病理改变，还影响细胞的分泌功能。一般情况下，血管内皮细胞受到炎症刺激时其胞内转录因子NF-κB被激活，促使多种炎性介质mRNA表达，进而诱发合成并释放肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α），同时诱导细胞间粘附分子-1（intercellular adhension molecular-1，ICAM-1）、血管细胞粘附分子-1（vascular celladhesion molecule-1，VCAM-1）、E-选择素等粘附分子的表达，促进PMN趋化和聚集，分泌基质金属蛋白酶（matrix metalloproteinases，MMPs）如基质金属蛋白酶-2（matrix metalloproteinase-2，MMP-2）、基质金属蛋白酶-9（matrixmetalloproteinase-9，MMP-9）等降解基底膜、破坏正常肺泡结构，并引发更多的炎性细胞聚集和活化。
本课题的研究目的是，拟在前期PFIB染毒对肺气血屏障形态学研究的基础上，观察体外培养的PMVEC经PFIB染毒后分泌功能的变化特点，重点观察细胞因子（TNF-α、IL-1β）、粘附分子（ICAM-1）和基质金属蛋白酶（MMP-2、MMP-9）的表达情况，探讨PMVEC在PFIB急性吸入性肺损伤过程中形态结构改变与功能变化的关联性，以丰富对PFIB染毒后气血屏障结构和功能的变化规律及PFIB急性吸入性肺损伤发病机制的认识，为更有效和针对性地防治PFIB急性吸入性肺损伤提供重要依据。
实验结果如下：①PFIB刺激PMVEC染毒后0.5h即迅速合成大量TNF-α，至2h达峰值。PMVEC合成大量TNF-α的同时缓慢释放到细胞外，至染毒后4h培养上清液中TNF-α含量才达峰值。②PFIB染毒后2h内刺激PMVEC合成大量的IL-1β，至2h达峰值，2h后伴随着PMVEC细胞凋亡数的增多合成的IL-1β也慢慢减少，但合成的IL-1β始终未释放到细胞外。③PFIB染毒后迅速促进PMVEC大量合成ICAM-1，但始终未有ICAM-1大量释放到细胞外。④染毒后PMVEC的培养上清液中MMP-2含量渐升高，至染毒后2h显著性增高，然后渐降低。PFIB染毒刺激PMVEC培养上清液中MMP-2迅速表达生物学活性。⑤PFIB染毒并未刺激PMVEC生成和释放大量MMP-9，提示PFIB急性吸入性肺损伤时PMVEC可能不是MMP-9的主要来源。
综合前期研究结果分析，一方面PFIB染毒促进PMVEC大量凋亡，使肺气血屏障的完整性遭到破坏；另一方面刺激存活的PMVEC合成并释放大量TNF-α，表达ICAM-1。在上述因子与其它因素的综合作用下，PMN激活并粘附于PMVEC，释放活性氧自由基（reactive oxygen species，ROS）与蛋白酶类，对PMVEC及其基底膜造成进一步损伤。此外，PFIB染毒刺激PMVEC迅速释放大量MMP-2，后者可加速肺泡微血管内皮细胞连接的破坏、细胞骨架的重排，并破坏细胞外基质。以上因素综合作用的结果是肺气血屏障完整性受到破坏，失去对大分子的屏障作用，血浆蛋白漏出形成渗透梯度，血浆水分也随之进入血管外造成水肿，炎症介质等进入肺泡内，这些也是ALI主要的病理基础。
As an important chemical raw materials or toxic byproducts in fluoroplastic industry,perfluoroisobutylene (PFIB) is a colorless and odorless fluoric gas at roomtemperature with low molecular weight. It can penetrate ordinary gas masks and notherapeutic measures are available up to now. It is one of the common toxic chemicalswhich can induce acute lung injury/acute respiratory distress syndrome(ALI/ARDS).
At present, the study on the pathogenesis of PFIB-induce ALI suggests that directoxidative damage to the lung tissue may be one of the important mechanisms. Besides,polymorphonuclear leukocyte (PMN) also plays a pivotal role, in which alveolarmacrophage (AM) is involved as an initiator of PMN sequestration in the lung. PFIBcan indirectly activate nuclear factor-κB (NF-κB) in AM which induces theexpression of many kinds of proinflammatory cytokines and chemokines. Theseproinflammatory cytokines and chemokines have strong activation and chemotaxiseffect on PMN. But the mechanism by which PFIB activates NF-κB remains unclearand the pathogenesis of PFIB-induced ALI remains to be fully elucidated.
The role of angiotensin Ⅱin PFIB-induce ALI
The renin-angiotensin system (RAS) plays an important role in the onset anddevelopment of ALI/ARDS. As the main effector of RAS, Angiotensin II (Ang II) notonly regulates the blood circulation, but also acts as a proinflammatory factor whichcan initiate inflammatory response in the lung by activating NF–κB and thereforeup-regulating the expression and excretion of IL-8, IL-1and TNF-. Angiotensinconverting enzyme (ACE) and angiotensin converting enzyme2(ACE2) acts aspositive and negative regulatory enzyme, respectively, to influence the process of ALI.Reducing the production of Ang Ⅱ or blocking AngⅡ receptor can alleviate ALI. Upto now it has not been documented in the literature that whether Ang Ⅱ is involved inPFIB inhalation-induce ALI by activating NF–κB and initiating the inflammatorycascade. One of the purposes of the present study is to preliminarily investigate theabove questions.
1. Correlation between the time-course of lung injury induced by PFIB inhalation and the level of AngⅡ and its regulatory enzyme ACE in the lung tissue: as indicated bylung coefficients (including wet lung-to-body weight ratio, dry lung-to-body weightratio, water content in the lung, and lung wet-to-dry weight ratio), total proteincontent in bronchoalveolar lavage fluid (BALF) and lung histopathology examination,the severity of lung injury worsen gradually after PFIB exposure, which peaked at16h and relieved at24h post exposure, demonstrating that the modeling of PFIBinhalation-induced ALI was successful. The content of Ang Ⅱ in lung tissueincreased firstly and then decreased, with significant decrease at16h and24h postexposure compared to the control group. The time-course of AngⅡin Plasma afterPFIB exposure showed similar changing trend as that in lung tissue with nosignificant difference compared to the control group. The activity of ACE in the lungtissue fluctuated after PFIB exposure. These results revealed that there were noobvious time-course correlation between the content of AngⅡ and the activity ofACE in the lung tissue and the degree of lung injury, suggesting AngⅡ may not havesignificant pathological significance in PFIB inhalation-induced ALI.
2. The role of AngⅡ in PFIB inhalation-induced ALI was determined by usinglosartan, an antagonist of AngⅡ type1receptor (AT1R) as the tool drug. The lungcoefficients and total protein content in BALF in the rats exposed to PFIB weresignificantly higher than those in the control rats, indicating the model ofPFIB-induced ALI was successfully established. However, injection of the rats withdifferent doses (1.25,2.5,5,10and15mg/kg injected1h before PFIB inhalation) oflosartan at different time points (1h,0.5h before PFIB and1h,2h,4h,8h afterPFIB inhalation) did not change the lung coefficients and total protein content inBALF. These results indicated that antagonizing of AT1-R does not have significanteffect on PFIB-induced ALI. These results further suggest that AngⅡ does not playimportant role in PFIB-induced ALI.
3. We also determined the effect of two kinds of losartan from different sources(domestic and imported) on the prevention and treatment of PFIB-induced ALI. Ourresults showed that treatment of the rats with either type of losartan (0.5h before and0.5h or1h after PFIB inhalation at the dose of40mg/kg) did not have significanteffect on24h-post-inhalation lung coefficients and72h-post-inhalation mortality.These results indicated that domestic or imported losartan does not have significant preventive or therapeutic effect on PFIB-induced ALI and AngⅡ is not involved inthe pathogenesis of PFIB-induced ALI.
Taking together, Ang Ⅱ and its regulatory enzyme ACE do not have significantcorrelation with the degree of PFIB-induced ALI. Time-and dose-dependencestudies showed that PFIB-induced ALI was not affected by AT1-R antagonist losartan.Intraperitoneal injection of two different kinds of losartan at multiple time points hadno significant improvement to ALI, indicating that Ang Ⅱ and RAS play a minorrole in the pathogenesis of PFIB-induced ALI. These results also suggest that Ang Ⅱ-activated NF-κB inflammatory cascade may not exist in PFIB-induced ALI. Theseresults may be related to different models of ALI and their effect on RAS. It may alsobe related to complex biological role of RAS. The role of RAS on ALI needs to befurther investigated.
The effect of PFIB on the function of PMVEC
Impairment of the structure and function of blood-gas barrier play a central role inALI caused by different factors. Pulmonary microvascular endothelial cell (PMVEC)is the first target attacked by the pathogenic factors. PMVEC is also the first cellcontacted by PMN released from vessel. Therefore, structural and functionalalteration of PMVEC in ALI has attracted more and more attentions. Studies haveshown that blood-gas barrier damage and cytoskeletal rearrangement are the key stepto the exudation of inflammatory cells, resulting in the entering of fluid, protein, andother inflammatory mediators into the alveoli. These constitute the main pathogenicmechanism of ALI. PFIB exposure not only causes main pathological changes ofblood-gas barrier, but also affects cell secretion function. Generally, when endothelialcells are stimulated with inflammatory factors, transcriptional factor NF-κB isactivated, leading to the expression of multiple inflammatory mediators includingTNF-α, ICAM-1, VCAM-1and E-selectin, which consequently promotes PMNchemotaxis and aggregation, secretion of matrix metalloproteinases (MMPs), e.g.,MMP-2and MMP-9. MMPs degrade the basement membrane and disrupt normalalveolar structure, resulting in more inflammatory cell recruitment and activation. Based on the morphology of blood-gas barrier of rat exposed to PFIB, the secondobjective of this study was to characterize the secretion function of in vitro culturedPMVEV exposed to PFIB with a focus on cytokines (TNF-α, IL-1β), adhesionmolecules (ICAM-1) and matrix metalloproteinase (MMP-2and MMP-9). Thecorrelation between the structural alteration and functional changes in PFIB-exposedPMVEC was explored in order to understand the mechanisms of structural alterationof blood-gas barrier and provide fundamental bases for the effective treatment ofPFIB inhalation-induced ALI.
Our results showed that TNF-α was rapidly expressed by PMVEC at0.5h post PFIBstimulation and the maximum value was achieved at2h post PFIB stimulation. Thenewly synthesized TNF-α was slowly released to outside of the cells. The maximumTNF-α in the supernatant was achieved at4h post stimulation. Within2h ofstimulation, PMVEC synthesizes large amount of IL-1β and peaks at2h.After2h ofstimulation, the synthesized IL-1β was decreased with the apoptosis of PMVEC.However, IL-1β was never released to the extracellular milieu. Large amount ofICAM-1was rapidly synthesized by PMVEC after PFIB stimulation, but was notreleased. After stimulation with PFIB, MMP-2in the supernatant of PMVEC culturewas gradually increased, peaked at2h and then decreased subsequently. Thebiological activity of MMP-2in the supernatant was also enhanced after PFIBstimulation. PFIB did not stimulate synthesis and secretion of MMP-9, indicating thatPMVEC is not the main source of MMP-9during PFIB inhalation-induced ALI.
Based on these results, we concluded that on one hand PFIB promotes apoptosis ofPMVEC and disrupts the blood-gas barrier. On the other hand, PFIB stimulates thesurviving PMVEC to synthesize large amount of TNF-α. Subsequently, PMN isactivated and adheres to PMVEC, leading to the release of ROS and proteases andfurther damages to the basement membrane. In addition, large amount of MMP-2secreted by PFIB-stimulated PMVEC can accelerate the destruction of alveolarcapillary endothelial cell connections, cytoskeleton rearrangement and destruction ofthe extracellular matrix. Consequently, the integrity of the blood-gas barrier isdamaged, leading to the loss of barrier function to the macromolecules, plasmaprotein leakage, osmotic gradient formation, plasma water entering to theextravascular milieu, formation of edema and entering of inflammatory mediators into the alveoli. These are also the main pathological basis of ALI.
目前，对PFIB所致ALI的发病机制研究认为直接氧化损伤可能是PFIB致病的重要机制之一；同时，多形核白细胞（polymorphonuclear leukocyte，PMN）介导的过度炎症反应可能是PFIB致病的另一重要机制。同时，肺泡巨噬细胞（alveolar macrophage，AM）参与PMN介导的PFIB急性吸入性肺损伤，并与PMN在肺内的扣押有关。PFIB急性吸入后可激活肺组织内多种细胞，尤其是间接激活AM内核因子κ B （nuclear factor-ha，NF-κ B），进而诱导AM表达多种致炎因子，这些致炎因子对PMN具有强烈的激活和趋化活性，但有关PFIB通过何种机制激活NF-κ B仍不清楚，PFIB急性吸入性肺损伤的机制研究仍有待完善。一、内源性血管紧张素II在PFIB急性吸入性肺损伤中的作用
近年研究发现，肾素-血管紧张素系统（renin-angiotensin system，RAS）在ALI/ARDS的发生、发展中发挥重要作用。血管紧张素II（angiotensin Ⅱ，AngⅡ）作为RAS的主要效应物质不仅是重要的体液调节因子参与调节血液循环，而且还是重要的前炎症因子，可上调NF-κB活性，增加白介素-8（Interleukin-8，IL-8）、白介素-1β（Interleukin-1beta，IL-1β）、肿瘤坏死因子-α（Tumor necrosisfactor-α，TNF-α）等炎症因子的表达释放，引发炎症反应。而血管紧张素转换酶（angiotensin convert enzyme，ACE）/血管紧张素转换酶2（angiotensin convertenzyme2，ACE2）分别作为AngⅡ的正/负性调节酶影响ALI的进程。减少AngⅡ的生成或阻断AngⅡ受体均可减轻多种原因所致的肺损伤。AngⅡ在PFIB急性吸入性肺损伤中的作用如何，它是否作为PFIB急性吸入性肺损伤的始动因素激活NF-κ B进而介导PFIB急性吸入性肺损伤，目前尚未见文献报道。本文的研究目的之一即是对上述问题进行初步探讨。
1、观察PFIB吸入染毒后，大鼠肺组织AngⅡ含量及其调节酶ACE的蛋白表达水平、酶活性与肺损伤程度的时间变化关系：大鼠PFIB急性吸入染毒后，肺系数、支气管肺泡灌洗液（bronchoalveolar lavage fluid，BALF）总蛋白含量和肺组织病理学检查等指标均显示肺损伤程度逐渐加重，至染毒后16h达峰值，染毒后24h明显缓解，因此确定本实验成功复制了大鼠PFIB急性吸入染毒病理模型。在此基础上，采用放射免疫分析方法检测PFIB染毒后大鼠的血液和肺组织匀浆中AngⅡ含量随时间的变化：血浆和肺组织中AngⅡ含量均呈现先升高后降低的变化趋势，其中肺组织AngⅡ含量在染毒后16h、24h较空白对照组显著降低。血浆AngⅡ含量与肺组织中AngⅡ变化趋势基本一致，但各时间点均无统计学差异；采用紫外线吸收法检测肺组织ACE酶活性随时间的变化：PFIB染毒后肺组织ACE活性在一定范围内波动，但各时间点无统计学差异。分析结果，肺组织AngⅡ含量和ACE酶活性的变化与肺损伤程度无明显的时间关联性，提示AngⅡ在大鼠PFIB急性吸入性肺损伤的发生、发展中可能不具有重要的病理学意义。
2、针对RAS发挥病理生理作用的最关键成分AngⅡ，选择AngⅡ的AngⅡ1型受体（angiotensionⅡ type1receptor，AT1-R）阻断剂氯沙坦，从时间-效应和剂量-效应两个方面考察AngⅡ在大鼠PFIB急性肺损伤中的作用：①时间-效应关系，PFIB染毒对照组大鼠的各项肺系数和BALF总蛋白含量较溶剂对照组显著升高，说明染毒模型建立成功。但不同时间给予氯沙坦（分别在PFIB染毒前1h、染毒前0.5h、染毒后0.5h、染毒后1h、染毒后2h、染毒后4h和染毒后8h腹腔注射氯沙坦10mg/kg）阻断AngⅡ受体进行预防或治疗，各组大鼠肺系数和BALF总蛋白含量与染毒对照组比较没有显著性差异。②剂量-效应关系，PFIB染毒对照组大鼠的各项肺系数和BALF总蛋白含量显著高于溶剂对照组，说明染毒模型建立成功。但给予不同剂量氯沙坦（分别在PFIB染毒前1h腹腔注射氯沙坦1.25、2.5、5、10和15mg/kg）阻断AngⅡ受体进行预防，各组大鼠肺系数和BALF总蛋白含量与染毒对照组比较没有显著性差异。以上结果表明，不同给药时间和不同给药剂量给予氯沙坦阻断AT1-R对PFIB急性肺损伤的影响微弱，进一步提示AngⅡ在PFIB急性吸入性肺损伤中可能不具有重要意义。
3、比较两种来源不同的AngⅡ受体阻断剂对PFIB急性吸入性肺损伤的防治效果：采用大样本量的小鼠为实验对象，观察国产和进口两种不同来源的氯沙坦对小鼠全身暴露吸入PFIB染毒后24h肺系数和72h死亡率的影响，实验结果表明染毒对照组和各个时间给药组（染毒前0.5h、染毒后0.5h和染毒后1h给药40mg/kg）比较均无显著性差异。这说明国产和进口来源的氯沙坦对小鼠PFIB急性吸入性肺损伤均无明显的防治作用。结果提示，AngⅡ在PFIB急性吸入性肺损伤中病理学意义有限。
综合分析，大鼠PFIB急性吸入性肺损伤过程中肺组织AngⅡ及其调节酶ACE与肺损伤程度无明显的时间相关性。时间-效应和剂量-效应关系研究揭示AT1-R阻断剂氯沙坦对大鼠PFIB急性肺损伤无明显影响。选择多个时间点腹腔注射两种不同来源的氯沙坦对小鼠PFIB急性吸入性肺损伤均无明显的改善作用。结果表明，AngⅡ在PFIB急性吸入性肺损伤中的病理作用微弱，RAS在PFIB急性肺损伤的发生、发展过程中可能不发挥主要作用，PFIB通过AngⅡ激活NF-κB启动炎症级联的通路可能并不存在。以上结果可能与不同ALI模型对RAS的影响不同有关，也可能与RAS复杂的生物学作用有关，其具体机理有待进一步研究。
二、PFIB对PMVEC功能的影响
肺气血屏障（blood-gas barrier）的结构和功能受损在各种原因所致ALI中居中心地位。其中，肺毛细血管内皮细胞（pulmonary microvascular endothelialcell，PMVEC）作为各种致病因素作用的靶细胞首先受到攻击，也是PMN扣押和游出血管时首先接触的细胞，所以PMVEC在ALI时的结构和功能变化受到越来越多的学者关注。
研究表明，肺损伤时气血屏障的细胞连接破坏、细胞骨架重排是炎性细胞渗出的关键环节，结果导致液体、蛋白、炎性介质等进入肺泡，这些构成了ALI的主要病理基础。PFIB染毒除了引起肺气血屏障主要细胞的结构发生病理改变，还影响细胞的分泌功能。一般情况下，血管内皮细胞受到炎症刺激时其胞内转录因子NF-κB被激活，促使多种炎性介质mRNA表达，进而诱发合成并释放肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α），同时诱导细胞间粘附分子-1（intercellular adhension molecular-1，ICAM-1）、血管细胞粘附分子-1（vascular celladhesion molecule-1，VCAM-1）、E-选择素等粘附分子的表达，促进PMN趋化和聚集，分泌基质金属蛋白酶（matrix metalloproteinases，MMPs）如基质金属蛋白酶-2（matrix metalloproteinase-2，MMP-2）、基质金属蛋白酶-9（matrixmetalloproteinase-9，MMP-9）等降解基底膜、破坏正常肺泡结构，并引发更多的炎性细胞聚集和活化。
本课题的研究目的是，拟在前期PFIB染毒对肺气血屏障形态学研究的基础上，观察体外培养的PMVEC经PFIB染毒后分泌功能的变化特点，重点观察细胞因子（TNF-α、IL-1β）、粘附分子（ICAM-1）和基质金属蛋白酶（MMP-2、MMP-9）的表达情况，探讨PMVEC在PFIB急性吸入性肺损伤过程中形态结构改变与功能变化的关联性，以丰富对PFIB染毒后气血屏障结构和功能的变化规律及PFIB急性吸入性肺损伤发病机制的认识，为更有效和针对性地防治PFIB急性吸入性肺损伤提供重要依据。
实验结果如下：①PFIB刺激PMVEC染毒后0.5h即迅速合成大量TNF-α，至2h达峰值。PMVEC合成大量TNF-α的同时缓慢释放到细胞外，至染毒后4h培养上清液中TNF-α含量才达峰值。②PFIB染毒后2h内刺激PMVEC合成大量的IL-1β，至2h达峰值，2h后伴随着PMVEC细胞凋亡数的增多合成的IL-1β也慢慢减少，但合成的IL-1β始终未释放到细胞外。③PFIB染毒后迅速促进PMVEC大量合成ICAM-1，但始终未有ICAM-1大量释放到细胞外。④染毒后PMVEC的培养上清液中MMP-2含量渐升高，至染毒后2h显著性增高，然后渐降低。PFIB染毒刺激PMVEC培养上清液中MMP-2迅速表达生物学活性。⑤PFIB染毒并未刺激PMVEC生成和释放大量MMP-9，提示PFIB急性吸入性肺损伤时PMVEC可能不是MMP-9的主要来源。
综合前期研究结果分析，一方面PFIB染毒促进PMVEC大量凋亡，使肺气血屏障的完整性遭到破坏；另一方面刺激存活的PMVEC合成并释放大量TNF-α，表达ICAM-1。在上述因子与其它因素的综合作用下，PMN激活并粘附于PMVEC，释放活性氧自由基（reactive oxygen species，ROS）与蛋白酶类，对PMVEC及其基底膜造成进一步损伤。此外，PFIB染毒刺激PMVEC迅速释放大量MMP-2，后者可加速肺泡微血管内皮细胞连接的破坏、细胞骨架的重排，并破坏细胞外基质。以上因素综合作用的结果是肺气血屏障完整性受到破坏，失去对大分子的屏障作用，血浆蛋白漏出形成渗透梯度，血浆水分也随之进入血管外造成水肿，炎症介质等进入肺泡内，这些也是ALI主要的病理基础。
As an important chemical raw materials or toxic byproducts in fluoroplastic industry,perfluoroisobutylene (PFIB) is a colorless and odorless fluoric gas at roomtemperature with low molecular weight. It can penetrate ordinary gas masks and notherapeutic measures are available up to now. It is one of the common toxic chemicalswhich can induce acute lung injury/acute respiratory distress syndrome(ALI/ARDS).
At present, the study on the pathogenesis of PFIB-induce ALI suggests that directoxidative damage to the lung tissue may be one of the important mechanisms. Besides,polymorphonuclear leukocyte (PMN) also plays a pivotal role, in which alveolarmacrophage (AM) is involved as an initiator of PMN sequestration in the lung. PFIBcan indirectly activate nuclear factor-κB (NF-κB) in AM which induces theexpression of many kinds of proinflammatory cytokines and chemokines. Theseproinflammatory cytokines and chemokines have strong activation and chemotaxiseffect on PMN. But the mechanism by which PFIB activates NF-κB remains unclearand the pathogenesis of PFIB-induced ALI remains to be fully elucidated.
The role of angiotensin Ⅱin PFIB-induce ALI
The renin-angiotensin system (RAS) plays an important role in the onset anddevelopment of ALI/ARDS. As the main effector of RAS, Angiotensin II (Ang II) notonly regulates the blood circulation, but also acts as a proinflammatory factor whichcan initiate inflammatory response in the lung by activating NF–κB and thereforeup-regulating the expression and excretion of IL-8, IL-1and TNF-. Angiotensinconverting enzyme (ACE) and angiotensin converting enzyme2(ACE2) acts aspositive and negative regulatory enzyme, respectively, to influence the process of ALI.Reducing the production of Ang Ⅱ or blocking AngⅡ receptor can alleviate ALI. Upto now it has not been documented in the literature that whether Ang Ⅱ is involved inPFIB inhalation-induce ALI by activating NF–κB and initiating the inflammatorycascade. One of the purposes of the present study is to preliminarily investigate theabove questions.
1. Correlation between the time-course of lung injury induced by PFIB inhalation and the level of AngⅡ and its regulatory enzyme ACE in the lung tissue: as indicated bylung coefficients (including wet lung-to-body weight ratio, dry lung-to-body weightratio, water content in the lung, and lung wet-to-dry weight ratio), total proteincontent in bronchoalveolar lavage fluid (BALF) and lung histopathology examination,the severity of lung injury worsen gradually after PFIB exposure, which peaked at16h and relieved at24h post exposure, demonstrating that the modeling of PFIBinhalation-induced ALI was successful. The content of Ang Ⅱ in lung tissueincreased firstly and then decreased, with significant decrease at16h and24h postexposure compared to the control group. The time-course of AngⅡin Plasma afterPFIB exposure showed similar changing trend as that in lung tissue with nosignificant difference compared to the control group. The activity of ACE in the lungtissue fluctuated after PFIB exposure. These results revealed that there were noobvious time-course correlation between the content of AngⅡ and the activity ofACE in the lung tissue and the degree of lung injury, suggesting AngⅡ may not havesignificant pathological significance in PFIB inhalation-induced ALI.
2. The role of AngⅡ in PFIB inhalation-induced ALI was determined by usinglosartan, an antagonist of AngⅡ type1receptor (AT1R) as the tool drug. The lungcoefficients and total protein content in BALF in the rats exposed to PFIB weresignificantly higher than those in the control rats, indicating the model ofPFIB-induced ALI was successfully established. However, injection of the rats withdifferent doses (1.25,2.5,5,10and15mg/kg injected1h before PFIB inhalation) oflosartan at different time points (1h,0.5h before PFIB and1h,2h,4h,8h afterPFIB inhalation) did not change the lung coefficients and total protein content inBALF. These results indicated that antagonizing of AT1-R does not have significanteffect on PFIB-induced ALI. These results further suggest that AngⅡ does not playimportant role in PFIB-induced ALI.
3. We also determined the effect of two kinds of losartan from different sources(domestic and imported) on the prevention and treatment of PFIB-induced ALI. Ourresults showed that treatment of the rats with either type of losartan (0.5h before and0.5h or1h after PFIB inhalation at the dose of40mg/kg) did not have significanteffect on24h-post-inhalation lung coefficients and72h-post-inhalation mortality.These results indicated that domestic or imported losartan does not have significant preventive or therapeutic effect on PFIB-induced ALI and AngⅡ is not involved inthe pathogenesis of PFIB-induced ALI.
Taking together, Ang Ⅱ and its regulatory enzyme ACE do not have significantcorrelation with the degree of PFIB-induced ALI. Time-and dose-dependencestudies showed that PFIB-induced ALI was not affected by AT1-R antagonist losartan.Intraperitoneal injection of two different kinds of losartan at multiple time points hadno significant improvement to ALI, indicating that Ang Ⅱ and RAS play a minorrole in the pathogenesis of PFIB-induced ALI. These results also suggest that Ang Ⅱ-activated NF-κB inflammatory cascade may not exist in PFIB-induced ALI. Theseresults may be related to different models of ALI and their effect on RAS. It may alsobe related to complex biological role of RAS. The role of RAS on ALI needs to befurther investigated.
The effect of PFIB on the function of PMVEC
Impairment of the structure and function of blood-gas barrier play a central role inALI caused by different factors. Pulmonary microvascular endothelial cell (PMVEC)is the first target attacked by the pathogenic factors. PMVEC is also the first cellcontacted by PMN released from vessel. Therefore, structural and functionalalteration of PMVEC in ALI has attracted more and more attentions. Studies haveshown that blood-gas barrier damage and cytoskeletal rearrangement are the key stepto the exudation of inflammatory cells, resulting in the entering of fluid, protein, andother inflammatory mediators into the alveoli. These constitute the main pathogenicmechanism of ALI. PFIB exposure not only causes main pathological changes ofblood-gas barrier, but also affects cell secretion function. Generally, when endothelialcells are stimulated with inflammatory factors, transcriptional factor NF-κB isactivated, leading to the expression of multiple inflammatory mediators includingTNF-α, ICAM-1, VCAM-1and E-selectin, which consequently promotes PMNchemotaxis and aggregation, secretion of matrix metalloproteinases (MMPs), e.g.,MMP-2and MMP-9. MMPs degrade the basement membrane and disrupt normalalveolar structure, resulting in more inflammatory cell recruitment and activation. Based on the morphology of blood-gas barrier of rat exposed to PFIB, the secondobjective of this study was to characterize the secretion function of in vitro culturedPMVEV exposed to PFIB with a focus on cytokines (TNF-α, IL-1β), adhesionmolecules (ICAM-1) and matrix metalloproteinase (MMP-2and MMP-9). Thecorrelation between the structural alteration and functional changes in PFIB-exposedPMVEC was explored in order to understand the mechanisms of structural alterationof blood-gas barrier and provide fundamental bases for the effective treatment ofPFIB inhalation-induced ALI.
Our results showed that TNF-α was rapidly expressed by PMVEC at0.5h post PFIBstimulation and the maximum value was achieved at2h post PFIB stimulation. Thenewly synthesized TNF-α was slowly released to outside of the cells. The maximumTNF-α in the supernatant was achieved at4h post stimulation. Within2h ofstimulation, PMVEC synthesizes large amount of IL-1β and peaks at2h.After2h ofstimulation, the synthesized IL-1β was decreased with the apoptosis of PMVEC.However, IL-1β was never released to the extracellular milieu. Large amount ofICAM-1was rapidly synthesized by PMVEC after PFIB stimulation, but was notreleased. After stimulation with PFIB, MMP-2in the supernatant of PMVEC culturewas gradually increased, peaked at2h and then decreased subsequently. Thebiological activity of MMP-2in the supernatant was also enhanced after PFIBstimulation. PFIB did not stimulate synthesis and secretion of MMP-9, indicating thatPMVEC is not the main source of MMP-9during PFIB inhalation-induced ALI.
Based on these results, we concluded that on one hand PFIB promotes apoptosis ofPMVEC and disrupts the blood-gas barrier. On the other hand, PFIB stimulates thesurviving PMVEC to synthesize large amount of TNF-α. Subsequently, PMN isactivated and adheres to PMVEC, leading to the release of ROS and proteases andfurther damages to the basement membrane. In addition, large amount of MMP-2secreted by PFIB-stimulated PMVEC can accelerate the destruction of alveolarcapillary endothelial cell connections, cytoskeleton rearrangement and destruction ofthe extracellular matrix. Consequently, the integrity of the blood-gas barrier isdamaged, leading to the loss of barrier function to the macromolecules, plasmaprotein leakage, osmotic gradient formation, plasma water entering to theextravascular milieu, formation of edema and entering of inflammatory mediators into the alveoli. These are also the main pathological basis of ALI.
引文
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