Perfluorinated Isobutylene The Pathological Mechanism Of Acute Inhalation Lung Injury

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 ALIThe 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 PMVECImpairment 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.