| IntroductionChronic lung disease ( CLD ) of prematurity is a major long - term pulmonary consequence of preterm birth, in which pulmonary immaturity, oxygen toxicity, baro/volutrauma as a consequence of mechanical ventilation and/or infections are involved. Advances in neonatal medicine, particularly the use of antenatal corticosteroids, mechanical ventilation and surfactant replacement therapy, have resulted in increasingly numbers of very preterm and very - low - birth weight survivors who are at risk of developing CLD, the incidence of CLD of prematurity continous to be a rising tendency, reaching 30% ~40% abroad, among which 10% ~ 15% will die from right heart failure or severe respiratory syncytial virus bronchiolitis within one year, survivors still depend on oxygen in long term or on mechanical ventilation repeatedly, which reduces the life quality significantly. There is no ideal prevention and perfect therapeutic strategies due to unclear mechanism.So far the mechanism research on CLD has been one of the hot topics all a-round the world, the accurate mechanism hasn't been identified although a great deal of clinical researches and animal experiments have been done on oxidative stress, inflammation, apoptosis and arrested lung development induced by the lackness of some growth factors to clarify the incidence and the development of CLD. However no matter what the mechanism is, arrested lung development and lung fibrosis are characterized pathological changes in CLD, and abnormal collagen deposition in interstitium has been demonstrtated to cause lung fibrosis in a-dults. Past researches demonsrated that the content of collagen in CLD BALF increased , and its level was related to future expection to a larger degree. The results of body inspection of CLD showed that the collagen deposition was in positive linear correlation to the degree of fibrosis. Due to the clinical limits, both the dynamic changes of pathological descriptions, and the comparision of the collagen deposition and lung morphology have not been researched deeply, and the expression of collagen gene is still argued. Past researches about collagen deposition mainly focused on the synethesis, but not on the degradation insight of CLD.Recently the role of ECM remodeling in lung fibrosis has been emphasized. The reduction of ECM degradation caused by the MMPs/TTMPs maybe the important factors to lung fibrosis. In lung fibrosis rats induced by blemycin, MMPs/TIMPs are implicated; some researches showed that the increase of MMP - 8 and MMP - 9 in BALF at the early stage of CLD was probably related to the alvelor injury and the remodeling at the early stage, but the studies are limited to the early stage of CLD, and some results are even conflicted, the interaction has not been clarified among the dynamic changes of MMPs/TIMPs, the pathological changes, and the collagen changes during ECM remodeling in CLD of prematurity.Plasmin system is also an important factor to modulate ECM degradation and MMP system, in which urokinase plasminogen activator ( uPA) and the plasminogen activator inhibator - 1 ( PAI - 1) convert plasminogen to plasmin so that ECM degradation is affected directly and MMPs are activated. Some experiments discovered that the lung fibrosis of mice with PAI -1 gene defect was less sereve; uPA gene treatment reduced the collegen in lung materially, so uPA/ PAI -1 is thought to be relevant with lung fibrosis closely. It is reported abroad that RDS of prematurity is related to lackness of plasmin, but there is few on the dynamic change and mechanism of plasmin in CLD of prematurity and animal models.Based on the characters that the sequence and the relative timing of alveo-larization in rat resembles human lung development, in this study we prepared an animal model of CLD with newborn rats induced by continuously inhaling highconcentration of oxygen (hyperoxia). Using special staining technic, immunoassay , immunohistochemistry and reverse transcription polymerase chain reaction (RT -PCR) , the dynamic changes of lung development and fibrosis were observed; the collagen protein level and mRNA expression in the lung tissue were investigated; MMP - 8, MMP - 9, TIMP - 1, TIMP - 2, u - PA and PAI - 1 protein and mRNA expression were detected in rat lung tissue to discuss MMPs and plasmin system in lung remodeling in CLD from degradation. We try to provide more information of pathophysiological mechanism of CLD.Materials and Methods1. Animal modelWithin 12 hours of birth, pups were randomly redistributed to the newly delivered mothers. One -half of the newborn pups were placed in hyperoxia, and one - half remained in room air. Hyperoxia exposures were done in Plexiglas chamber into which oxygen was continuously delivered to achieve a constant level of 90 ~95% oxygen monitored daily with an oxygen monitor ( OM -25 ME, USA). Oxygen and room air were filtered through Sodalime to keep CO2 levels below 0.5% (Dapex Gas Monitor, USA). Temperature and humidity were maintained at 25 ℃ ~27℃ and 70% ~80% respectively. Chamber was opened for 0. 5h daily to switch nursing mother and chang water, add food, clean dirty cages, record survival and body weight daily. Nursing mothers were rotated between oxygen exposed and room air litters every 24 h to avoid oxygen toxicity in the mothers and to eliminate maternal effects between groups.2. Tissue preparationEach group of hyperoxia as well as air control rats was anesthetized at each time point. The thorax was opened and lung tissue collected and stored respectively according to the following methods:2.1 The samples of lobes of the right lung are put in the Rnase -free Ep-pendorf tubes, and then put in 80℃ freezer.2. 2 The samples of the left lung are fixed in 4% formaldehydum polymeri-satum which contains 0. 1% DEPC, then were dehydrated, embedded in waxwithin 24 hours.3. Experiment Methods3. 1 The appearance, survival rate and weight were monitered everyday in hyperoxia groups and air controls, life table for survival analysis, point survival and survival curve were compared.3. 2 The changes of the lung pathology3. 2.1 Microscope exam of section stained with HE; lung histological stud-y.3. 2.2 Masson staining: deposition of collagen fibres in the lung. 3.2. 3 Gomori staining: deposition of reticular fibres in the lung. 3. 2. 4 Radical alveolar counts ( RAC) : value on the development of the lung.3.3 The measurement of collagen expression in the lung tissue3.3.1 Enzyme linked immunoadsorbent ( ELISA) : the detection at the protein level of ColⅢ　and ColⅣ.3.3.2 In situ hybridization; the detection at mRNA level of ColⅣ.3. 4 The measurement of MMP - 8 , MMP - 9 , TIMP - 1 and TIMP - 2 expression in the lung tissue3.4. 1 Immonohistochemistry: the detection at the protein level of MMP -8,MMP -9,TIMP -1 and TIMP -2.3.4. 2 RT - PCR: the detection at mRNA level of MMP - 8,MMP - 9, TIMP-1 and TIMP-2.3. 5 The measurement of u - PA and PAI - 1 expression in the lung tissue3.5.1 Western Blotting: the detection at the protein level of u - PA and PAI-1.3. 5. 2 RT - PCR: the detection at mRNA level of u - PA and PAI-1.4. Statistical analysisSPSS version 11.0 was used to perform statistical analysis, with all data expressed as ( x ± s ). Statistically significant differences in the mean values were analyzed using the Dunnett - Test for inter - group comparison. life table was used to value survival rate, u - Test for time comparison, log - rank test for curve comparison.Results1. Survival rate and general status of rats in two groups1.1 The death of hyperoxia rats increased with the time of oxygen exposure, and survival rate decreased; while there is no death after 3d in air group. From 3d, survival rate of hyperoxia decreased ( P < 0. 05 ) , and the survival curves were different in two groups from 3d (P<0.01),the hyperoxia is lower.1.2 The hyperoxia rats almost began to present dyspnea, pale, cyanosis of different degrees after 5 days of oxygen exposure, some even depended high oxygen after 7 to 10 days. The air group didn' t have the appearance above.1. 3 From 3 days of oxygen exposure, the weights began to decrease(P <0. 05 ) , and the difference lasted to 21 d and more evident compared to air controls (P<0.001).2. The pathological findings of the lung tissue following hyperoxia2.1 On day 1 of the experiment, it was observed in both room air and oxygen exposed group that the alveolar structure was irregular, terminal air space size was rather small, and the alveolar septum was thick. On day 3 , the alveolar structure of the two groups was more regular, the size of alveolus was equal, and alveolar septum was thinner, but in oxygen - exposed group, there was a few inflammatory cells exuded out, blooding, interstitial cells increased. On day 7, in room air rats, the alveolar size was equal, while the terminal air space size of the oxygen - exposed rat became large, there was inflammatory response and more interstitial cells. On day 14d and 21 d, the terminal air space size of oxygen - exposed rat grew significantly large, the quantity of alveolar reduced, with alveolus fusion, and interstitial cells increased, interstitium was thicker.2. 2 There was no difference between two groups for blue fibres, which was mainly deposited in bronchil and vascular wall, little in lung septum. On 7d, a little increased in lung interstitium, but the diffenrence was not significant. In hyperoxia group, there were more collagen fibres along alveolar, and lung collagen quantified by the area of blue increased over controls on 14d and 21 d (P < 0. 01) , which were distributed mainly in lung interstitium, paralleling morphog-ical results.2. 3 There was no difference between two groups for reticular fibres, which was distributed continously in bronchil and vascular wall and discontinously in septum. On 7d, a little increase in lung interstitium, but the diffenrence was not significant. A progressive increase of black reticular fibres was present as grid for hyperoxia rats on 14 and 21 d, distributed in lung interstitium, the area of black increased over controls ( P <0.01).2.4 RAC increased with days of rats in air controls, while they began to decrease from 7d for hyperoxia rats compared to air control ( P < 0. 05 ) , which was more evident on 14d and 21 d ( P < 0.01) .3. The dynamic changes of collagen expression in lung tissue3. 1 The dynamic change at the protein level of ColⅢ and ColⅣThere was no chang of the contents compared to the air group from 1d to 7dof the experiment, however which was increased on 14d and 21 d for both typesof collagen, P <0.001 except for ColⅣ , P <0.05 on 14d.3. 2 The dynamic changes at the mRNA level of Col Ⅳ expressionIt was shown that the the positive expression of Col Ⅳ mRNA was mainly in the endothelial cells and the alveolar epithelial cells, whose mRNA expression showed no evident change between two groups on each time.4. The dynamic changes of MMPs and TIMPs expression in the lung tissue following hyperoxia in neonatal rats4. 1 The dynamic changes at the protein level of MMP -8 and MMP -9 expressionImmunohistochemical staining showed, in air lung tissues, MMP - 8 was expressed in epithelial cells and microvascular endothelial cells. In oxygen group, there was no chang of the expression intensity compared to the air group on day 1 and day 3 of the experiment, however which was decreased after 7d(P <0.05) ; It was shown that the positive expression of MMP -9 was mainly in the epithelial cells, which gradually increased in air control group. After 02 exposure, it showed a significant difference on 3d(P<0.05), but which was not obviously different after 7d between two groups( P >0.05).4. 2 The dynamic changes at the protein level of TIMP - 1 and TIMP - 2 ex-pressionTIMP -1 was expressed in epithelial cells, interstitial cells and pulmonary macrophages, the expression intensity of TIMP - 1 protein from pups exposed to O2 was stronger than that of air pups on 3d(P<0.05), this increase was maintained throughout the experimental period; While the expression of TIMP - 2 showed no significant difference between two groups at each time, which expressed mainly in vascular endothelial cells, basement membrane and epithelial cells(P>0.05).4. 3 The dynamic changes at the mRNA level of MMP - 8, MMP -9, TIMP- 1 and TIMP - 2 expressionExposure to O2 did cause change in MMP -8 mRNA level from 7d(P <0. 05) to 14 and 21 d (P <0. 01) , when level were decreased; MMP -9 mRNA increased only on 3d ( P < 0. 001); A significant increase in TIMP - 1 mRNA levels for the oxygen group on 3d ( P < 0.05 ) , 7d( P < 0.05 ) and 14d (P <0. 01), but there was no difference between two groups on 21d; While the expression of TIMP - 2 mRNA showed no significant difference between two groups at each time.5. The dynamic changes of u - PA and PAI - 1 expression in the lung tissue following hyperoxia in neonatal rats5. 1 The dynamic changes at protein level of u - PA and PAI - 1 expression A gradual increase of u - PA protein expression can be seen in air controls, while after O2 exposure, which increased on 3d(P<0.01), and gradually decreased after 7d; PAI - 1 protein expression showed a gradual decrease in air group, and there was an increased expression from 7d to 21 d in hyperoxia compared to controls (P < 0. 01).5. 2 The dynamic changes at mRNA level of u - PA and PAI - 1 expression The u - PA and PAI - 1 mRNA expression has a tendency of increase or decrease in controls with age. In hyperoxia u - PA increased on 3d(P<0.01), and gradually decreased after 7d(P<0.05); There was also an increased PAI- 1 expression from 7d to 21 d in hyperoxia compared to controls ( P<0.05).Conclusions1. Pathological findings charactered by the arresed alveolar development and deposited fibres in newborn rats after prolonged hyoeroxia are similar to those of CLD of prematurity.2. The protein of ColⅢ and Col Ⅳ increased at later stage, parallelling to the deposited fibres, improving the collagen accumulation at last in CLD induced by hyperoxia; While ColⅣ mRNA expression showed no increase, suggesting that posttranscriptional regulation ( especially degradation) by hyperoxia makes ColⅣ protein increase.3. At the late stage, the expressions of TIMP - 1 mRNA and protein are not the same, implicating posttranscriptional regulation by hyperoxia makes TIMP -1 protein increase.4. During the middle and late stage, the mRNA and protein expression of MMP-9 showed no change, while TIMP - 1 mRNA (except 21 d) and protein expression increased, when the ColIV accumulation happened, suggesting the imbalance of MMP - 9/TIMP - 1 may play an important role during the remodeling of CLD induced by hyperoxia.5. During the middle and late stage, the expressions of MMP - 8 mRNA decreased, which led to its protein decrease; TIMP - 1 mRNA (except 21 d) and protein increased; TIMP - 2 mRNA and protein showed no change after hyperoxia , which made the imbalance of MMP - 8/TIMPs in protein and mRNA levels, and may decrease the collagen degradation, in parallel to Col M accumulation in lung institium, suggesting ECM remodeling by MMP - 8/TIMPs parcipate in the development of CLD induced by hyperoxia.6. During the middle and late stage, the expression of uPA mRNA decreased and PAI -1 increased, so the protein expression of uPA decreased and PAI - 1 increased, too. And the imbalance of u - PA/PAI -1 was in parallel to collagen accumulation, suggesting u - PA/PAI - 1 may also play a role in lung remodeling of CLD induced by hyperoxia.7. The dynamic expression of u - PA/PAI - 1 was in parallel to MMPs/... |
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