Effects Of Captopril On Fibrosis In Lung Tissue Of Neonatal Rats With CLD Induced By Hyperoxia

IntroductionChronic lung diseae (CLD) of prematurity is a major long - term pulmonary consequence of preterm birth, in which pulmonary immaturity, oxygen tox-iciy, baro/volutrauma as a consequence of mechanical ventilation and/or infections are involved. Advances in neonatal medicine, particularly the use of ante-antal 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 in our country year by year, it was reported that the incidence of CLD has reached to 30% - 40% abroad. Among which 10% - 15% will die from right heart failure or severe respiratory syncytial virus broncholitis within one year, suvivors still depend on oxygen in long term or on mechanical ventilation repeatedly, which reduce the life quality significantly. Its pathogenic mechanisms are not fully understood, so there are not effective control methods or preventive measures.So far the mechanism research on CLD has been one of the hot topics all a-round the world, the accurate mechanism hasnt been identified although a great deal of clinical researches and animal experiments have been done on oxidative strees, 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 demonatrated to cause lung fibrosis in a-dults. Past researches demonstrated that the content of collagen in CLD BALF increased, and its level was related to future expection to a large degree. The results of body inspection of CLD showed that the collagen deposition was in positive linear correlation to the degree of fibrosis.Recently the role of fibroblast in lung fibrosis has been emphasized. The fi-broblast can synthesize extracellular matrix and cytokines, priming Inflammatory reaction, adjust the function of ype II epithelial cells, promote lung fibrosis. For the past few years, along with the lucubrating of the Renin - Angiotesin System (RAS) , more and more scholars in the world noticed that Angiotensin II which is catalyzed by Angiotensin - Converting enzyme ( ACE) can promote cellular proliferation and secretion of extracellular matrix. Angiotensin II can stimulate lung fibroblast to proliferate, synthesizeing more DNA, and secreting more collagen, thereby to resulting in lung fibrosis. Scholars also noticed that Captopril can eliminate kinds of free radical, decrease vascular permeability, relieve inflammatory exudation. So Captopril has antiinflammatory and antifibrosis function.Based on the characters that the sequnce and the relative timing of alveo-larization in rat resembles human lung development, in this study we prepared an animal modle of CLD with newborn rats induced by continously inhaling high concentration of oxygen (hyperoxia). Using immunoassay, immunohistochemis-try and reverse transcription polymerase chain reaction ( RT - PCR) , the dynamic changes of lung development and fibrosis were observed;the ACE, Ang II and collagen, ICAM - 1 ,TGF - Piprotien level and mRNA expression in the lung tissue were investigated;we try to provide more information of phathophysi-ological mechanism of CLD and try to use Captopril to protect neonatal rats from lung fibrosis induced by hyperoxia.Materials and Methods1. Animal modleWithin 12 hours of birth, pups were randomly redistributed to the newly delivered mothers. 360 term neonatal Wistar rats were randomly assigned into Aircontrol, Model, normal saline control and Captopril - treated groups ( n = 60each). The Air control group was exposed to air ( FiO2 =0. 21 ). The rest three grops were continuously exposed to hyperoxia. Hyperoxia exposures were done in plexiglas chamber into which oxygen was continously delivered to a-chieve a constant level of 90% -95% , oxygen monitored daily with an oxygen monitor ( OM -25 ME, US A). Oxygen and room air were filtered through sodal-ime to keep CO2 levels below 0. 5% ( Dapex Gas Monitor, USA). Temperature and humidity were maintailned at 25X1 -27^ and 70% -80% respectively. Chamber was opened for lh daily to switch nursing mother and change water, add food, clean dirty cages, record survival and body weight daily. Nursing mothers were rotated between oxygen exposed and room air litters every 24h to a-void oxygen toxicity in the mothers and to eliminate maternal effects between groups. . During exposure the Captopril treated group received Captopril ( 20 -40mg/kg/d ) by intragastric administration. The Normal saline control group was administrated with normal saline instead. The Model group did no any treatment.2. Tissue preparationEeach group of hyperoxia as well as air room 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 Ependorf tubes, and then put in 80^1 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 wax within 24 hours.3. Experiment Methods3. 1 The appearance, survial rate and weight were monitored everyday in hyperoxia groups, normal saline control, Captopril - treated groups and air controls , life table for survival analysis, point survival and survival curve were compared.3. 2 The changes of the lung pathologyMicroscope exam of section stained with HE : lung histological study.3. 3 The measurement of ACE, Ang H , collagen I , collagen M , ICAM -1, TGF - (^and theirs mRNA expression in the lung tissue3. 3. 1 Use automatic biochemistry analyzer(7170,Japen) to detect ACE activity (activity unit)3.3.2 Radio - immunity technique;the detection at concentration of Angn.3. 3. 3 Enzyme linked immunosorbent assay (ELISA);the detection at the protien levels of collagen I x collagen IK XICAM - 1 and TGF - Pjin the lung tissue3. 3. 4 Immunohistochemistry: the detection at the protien expression levels of collagen I , collagen HI, ICAM -1 and TGF - Pjin the lung tissue3. 3. 5 RT-PCR;the detection at mRNA expression levels of ACE, Ang E , collagen I , collagen HI and TGF - (3jin the lung tissue4. Statistics analysis;SPSS version 13. 0 was used to perform statistics analysis, with all data expressed as (x s). Statistically significant differences in the mean values were analyzed using the Dunnet - Test for inter - group comparison. life table was used to value survival rate, u - Test for time comparison, log - rank for curve comparison.Results1. survival rate and general status of rats in four groups 1. 1 the death of hyperoxia goroup and normal saline control group rates increased with the time of oxygen exposure, and survival rate decreased;while there is no death after 3d in aic group. From 3d, survival rate of hyperoxia goroup and normal saline control group decreaed ( p < 0. 05), and the survival curves were different in four groups from 3d( p <0. 01) ,the hyperoxia goroup and normal saline control group is lower jhoweve the survival rate of Captopril -treated groups increased and the survival curves is higher than thoes of hyperoxia goroup and normal saline control group.1. 2 The hyperoxia goroup and normal saline control group rats almost be-gan to present dyspnea, pale, cyanosis of different degrees after 7-10 days of oxygen exposure, some even depended high oxygen after 14 -21 days. The air control group didnt have the appearance above, however the Captopril -treated group was better than hyperoxia goroup and normal saline control group withdraw from oxygen.1. 3 From 3 days of oxygen exposure, the weights began to decrease (p < 0. 05), and the differencelasted to 21 d and more evident compared to air controls (p <0. 01), the weights of Captopril - treated group are higher than hyperoxia goroup and normal saline control group.2. The pathological findings of the lung tissue following hyperoxia and Captopril administrationOn 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 four groups was more regular, the size of alveolus was equal, and alveolar septum was thinner, but in oxygen exposed two groups, there was a few inflammatory cells exuded out, blooding, interstitial cells increased. On day 7, in room air rats, the alvolar size was equal, while the terminal air space size of oxygen exposed rats becam large, there was inflammatory response and more interstitial cells. On day 14 and 21, the terminal air space size of oxygen exposed rats grew signiificantly large, the quantity of alveolar reduced, with alveolus fusion, and interstitial cells increased, interstitum was thicker. The pathological findings of the Captopril - treated group was better than thoes of oxygen exposed rats.3. The dynamic changes of ACE, AngH , collagen I , collagen Iff, ICAM -1, TGF - piand theirs mRNA expression in the lung tissueThere was no change of the contents compared to the four groups from 1 d to 7d of the experiment, however which were increased on 14d and 21 day for ACExAngH >ICAM - 1XTGF - Pjand both of collagen in the hyperoxia goroup and normal saline control group than the room air control group, the Captopril -treated group was decreased than the hyperoxia goroup and normal saline controlgroup (p<0. 05,orp<0. 01).4. The dynamic changes at the protien levels of collagen I , collagen M, ICAM - 1, TGF - Pj expression in the lung tissueImmunohistochemical staining showed : in air lung tissues, collagen I , collagen HI, ICAM - 1, TGF - (3jWas weakly expressed on trachea tube wall or vessel wall and connective tissue around them, the hyperoxia goroup and normal saline control group are the same as air control group on the days of 1 -7d, and the positive expressed cells increased on 14d (p <0. 05) , mainly expressed on interstitium and alveolar wall, the expression intensity is higher than room air control group, on day 21, the protien expression reached peak (p <0. 01), a great quantity interstitial cells expressed positive;The protien expressions of the Captopril - treated group were weaker than thoes of hyperoxia goroup and normal saline control group (p<0. 05, orp<0. 01), but higher than room air controls (p<0. 05).Conclusions1. Pathological findings charactered by the arresed alvolar development and deposited fibres in newborn rats after prolonged hyperoxia are similar to those of CLD of prematurity.2. RAS do exist in rat lung tissue, the protein level and mRNA expression of Ang II , collagen I , collagen HI and ACE activity in newborn rats lung tissue are significantly higher than room air control group after prolonged hyperoxial4d and 21 d, meanwhile the lung tissue present fibrosis. The consistency of chrono-logicaol order and fixing of protein and mRNA expression indicated that RAS participated the development of lung injury and lung fibrosis.3. The protein level and mRNA expression of ICAM - 1NTGF - p, in new-born rats lung tissue are significantly higher than room air control group after prolonged hyperoxia 14d and 21 d, meanwhile the lung tissue present fibrosis, it indicated that ICAM -1%TGF - pi participated the development of lung injury and lung fibrosis.4. After Captopril adminitration, the protein level and mRNA expression ofAng II , collagen I , collagen HI ,ICAM - 1, TGF - j^and ACE activity of Captopril - treated groups are significantly lower than thoes of hyperoxia goroup and normal saline control group, meanwhile the lung tissue present less fibrosis. It indicated that Captopril may have protective effects on lung injury and lung fibrosis induced by hyperoxia.