| Perfluoroisobutylene (PFIB), as a main by-product during the manufacturing and the processing of the floropolymer industry, has around tens of thousands tons output every year, and along with the extensive usage of polytetrafluoroethylene (PTEE) products, PFIB can also be produced in poisonous smoke of a fire disaster.PFIB is ten times more toxic than phosgene and no masks can effectively protect people from it. Inhalation a little amount of PFIB can immediately result in acute pulmonary edema, and even death.The pathologic physiology of PFIB-inhalation induced by acute lung injury (ALI) and pulmonary edma is characterized by the low lung conformance, the enhancement of the lung split stream and the ratio of the ventilation/blood imbalance. The clinical symptoms of which are the refractory hypoxemia, the change of the breathing frequency, and the distress of the respiratory system. A chest X-ray examination can usually display diffusive infiltration shadow in both lungs, which might progress into multi-organ failure in the advanced stage, and finally cause death in the most severe cases.Up to the present, the mechanism of ALI induced by the kind of toxic gases is still incompletely clear, and the specific medicine and therapeutic measure are also totally unavailable yet. The research results from laboratories of both domestic and international, including our laboratory have shown that aggregation, sequestration of polymorphonuclear leukocyte (PMN) and excessive inflammatory reaction induced by those PMNs in the lung is a decisive factor in early responses to PFIB inhalation, and other factors (such as pulmonary macrophage) are probably more important in advanced stage. There is, currently, no specific therapeutic medicine or preventive and therapeutic measure on the clinical treatment against PFIB intoxication. High-dose hormone stosstherapy, symptomatic treatment and supportive treatment are usually applied. But the therapeutic efficacy is no good. Mortality is still high.The purpose of this study is to try, as systematically and thoroughly as possible, to go in depth in the mechanisms of PFIB intoxication, the induction of ALI and pulmonary edema, the initiation and deterioting processes, and many other aspects of which, so that it can possibly shed light on the clinical prevention and treatment and the development of related medicine.First of all, the patterns of the pathological changes were observed at different time points after PFIB inhalation, which were done from the conventional pathological section, the ultramicrostructure of the main cells of the air-blood barrier, and the cell conjunction and the cytoskeletal protein levels were also observed by the indirect immunohistofluorescence staining method.To fully imitate the typical clinical courses of the PFIB inhalation intoxication, Wistar rat (180-220 g) was exposed to a sublethal concentration of PFIB (140 mg/m3) for 5 min. Four lung coefficients (Lung wet-to-dry ratio, wet lung-to-body ratio, dry lung-to-body ratio, and water content in the lung) and albumin protein and total protein in BALF didn't change within the first 4 h after the exposure of PFIB (the earlier injury period), remarkably increased at the time point of the 8 h, reached peak at 24 h (pulmonary edema period), and recovered at the time point of the 72 h (convalescent period).On the conventional HE staining of the lung slices, the normal texture was characterized by the alveoli constitution integrity, no oedema, no bleeding, no protein leaking out, and no inflammatory cell infiltration. There was no remarkably change within the first 4 h after the PFIB exposure, but the destruction of the alveoli constitution integrity began to be observed, oedema emerged as indexed by the red blood cells, the protein leaking out, and the inflammatory cell infiltration since the time point of 8 h, peak at 24 h, and gradually recovered to normal condition.The ultrastructural pathological changes of alveolar typeâ… epithelial cell (AT-â… ), alveolar typeâ…¡epithelial cell (AT-â…¡), pulmonary microvascular endothelial cell (PMVEC), pulmonary macrophage (PM) were observed by transmission electronic microscope. The results were as follows.The normal AT-â… is kind of squamous flat cell, the cell nucleus of which is small and compact, cytoplasm thin, organelle rare, mitochondrion and rough endoplasmic reticulum accidentally seen. A few thick short microvilli are seen at dissociative side, and connected with AT-â…¡by the tight junction. Thirty minutes after the PFIB inhalation, the AT-â… cell nucleus began to be wrinkled, chromatin compacted, mitochondrion swollen, and cell matrix shallow. With the time going on the cell nucleus was wrinkled more seriously, the chromatin divided into some small pieces gradually and marginalized, the nucleolus split gradually, the double membranes of mitochondrion destroied gradually, the cristae swollen so seriously that it was even broken, a great deal of vacuolus appeared in the cell matrix. All the above-mentioned pathological changes were the most seriously at the time point of 24 h after PFIB exposure. Most cells break up, double membrane of mitochondrion was destroied seriously, and cristae even disappeared. After 24 h, all symptoms were ameliorated.The normal AT-â…¡is cube-like, contains abundant organelles including lamellar body, which can be dyed black by osmic acid. The pathological changes of cell morphology and mitochondrion after PFIB inhalation were similar to that of AT-â… . The microvillus began to be fallen off at the time point of 30 min after PFIB inhalation, the most serious changes were observed at the time of 24 h, followed a gradual amelioration.The normal PMVEC is flat-like, contains various organelles. The pathological changes of cell morphology and mitochondrion after PFIB inhalation were similar to that of AT-â… . The basal lamina began to be swollen at the time point of 30 min after PFIB inhalation. The most serious changes such as split, were observed at the time of 24 h, followed a gradual amelioration.The normal PM is relatively big, contains mitochondria, lysosome and cytorrhyctes in the cytoplasm. The increased grains in PM's cytoplasm were only observed at the time of 2 h after PFIB inhalation. The pathological changes of cell morphology and mitochondrion after PFIB inhalation were similar to that of AT-â… .The apoptosis of cell in rat lung texture was observed by hybridization in situ. The apoptosis cell increased at the time point of 30 min after PFIB inhalation. The maximum was achieved at the time point of 24 h, and they ameliorated gradually after 24 h.The change of zonula occludens-1 (ZO-1), one of tight junction proteins, reflects the injury of cell junction after PFIB inhalation. The pathological changes of ZO-1 were observed in the early stage of the air-blood barrier injury, whereas that of actin, one of cytoskeletal protein, in the midanaphase injury.As compared to the normal group, the positive fluorimetric stains of ZO-1 began to descend at the time point of 30 min after PFIB inhalation, the most serious at 24 h, then ascended gradually. The positive fluorimetric stains of actin were not remarkably changed within the first 4 h after PFIB inhalation, and began to descend remarkably at the time point of 8 h. The minimum was achieved at 24 h, then ascended gradually. The result of Western-blot was consistent with the observation above-mentioned.We wondered whether other factors participate to regulate in vivo the above pathological changes besides the PFIB direct hurts?The construction and function of the air-blood barrier become a focus in recent researches. The phosphorylation of myosin light chain (MLC) is the decisive step about the adjustment of lung microvascular endothelial barrier. The phosphorylated MLCs not only destroy cell junction, but also induce to reorganize the stress fiber of myosin, result in an increasing permeability of air-blood barrier. The phosphorylation of MLC has two main regulative paths. One is the MLCK path, the other is the ROCK path. The former promotes the phosphorylation of MLC, and the latter inhibits the activity of phosphatase of MLC. Because the former directly increases the phosphorylated MLC, it is studied more, and the position is more important.The positive fluorimetric stains of MLCK were increased gradually within the first 4 h after PFIB inhalation and descended remarkably at the time of 8 h. The minimum was achieved at the time of 24 h, then ascended gradually. These results maybe explain the changes of ZO-1 and actin after PFIB inhalation. If the MLCK depressant ML-7 (dissolve by 10% ethanol, first time 4.3 mg/kg i.p. soon after 1.4 mg/kg, i.p. b.i.d, consecutively administer 2 days) was given in advance, the pulmonary edema induced by PFIB can be repressed remarkably. The result of Western-blot was consistent with the above-mentioned.Because of the complex network in whole animal which is hard to clarify, PMVEC and AT-â…¡were separated and purified to observe their pathological changes after PFIB exposure.The survival rates of PMVEC and AT-â…¡were more than 95% within 72 h after PFIB (130 mg/m3, 5 min) exposure.The ultrastructural pathological changes of PMVEC were observed by transmission electron microscope. The cell morphology and the mitochondria began to be injured at the time of 1 h after PFIB exposure. The pathological changes were the most serious at 24 h. After 24 h, they were ameliorated gradually. The results were in concordance with that of animal experiment.The apoptosis of PMVEC and AT-â…¡were observed after PFIB exposure. The supernatant of AT-â…¡exposed by PFIB promoted normal PMVEC apoptosis and protected PMVEC from apoptosis which were examined with Annexin V-FITC apoptosis Detection Kit.The apoptosis of AT-â…¡and the PMVEC began to increase at the time of 1 h after PFIB exposure. The maximum was achieved at 24 h, and then ameliorated gradually.The normal AT-â…¡supernatant couldn't induce normal PMVEC apoptosis. But the AT-â…¡supernatant exposed by PFIB induced normal PMVEC apoptosis. The action of the AT-â…¡supernatant exposed by PFIB at the time of 24 h was the maximum.The PMVEC apoptosis was increased remarkably after PFIB exposure. The normal AT-â…¡supernatant and the AT-â…¡supernatant exposed by PFIB could prevent the PMVEC from apoptosis induced by PFIB. The protection of AT-â…¡supernatant at the time of 1 h after PFIB exposure was the best. They were attenuated gradually within 24 h, but the protection of AT-â…¡supernatant at the time of 24 h after PFIB exposure was still better than the normal AT-â…¡supernatant. The protection of AT-â…¡supernatant at the time of 48 h after PFIB exposure recovered gradually. The pulmonary surfactant (PS), one of special physiology anti-injury factors in lung, has important function on the defence and the protection of lung in recent researches. The PS mainly synthesizes in the endochylema of AT-â…¡, stores in the laminated body. The phospholipid is main composition of PS. The content of total phospholipid can indirectly reflect the change of PS.The content of total phospholipid was increased gradually within 4 h after PFIB exposure, and descended gradually after 4h. The minimum was at 24 h. It was ameliorated gradually after 24 h. The result can explain the protection of AT-â…¡supernatant to PMVEC.In general, the results described, completely and in detail, the changing patterns of the lung structure and the function of the air-blood barrier, along with its major cells of components after PFIB intoxication. The rule and action of myosin light chain kinase in ALI induced by PFIB was also clarified preliminarily, which can be of some help for the prevention and treatment of ALI induced by PFIB.
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