Effect Of Rh-CCSP On Hyperoxia-induced Lung Injury And Its Mechanism

IntroductionThe development of neonatology has been increasingly effective in reducing the mortality of preterm infants at the expense of an increasing number of survivors with bronchopulmonary dysplasia (BPD) caused by lung immaturity. BPD is a common syndrome in newborns, especially in preterm infants, when treated with hyperoxia and mechanical ventilation. Unfortunately, there have been no effective medicines applied in clinic for the prevention and treatment of BPD because of its unclear pathogenesis.At present, new-type of BPD with the restricted lung development is considered most important in neonatal intensive care. BPD has been suggested to influence lung maturation by causing progressive and sustained lung inflammation, which would result in the depression of the development of alveoli, small respiratory tubes and small blood vessels. Apoptosis, together with the characteristic death of endothelial and alveolar epithelial cells are reported to participate in the pathogenic process of lung injury induced by high concentration of oxygen. When lung injury occurs, the denaturalization and destruction of typeⅡalveolar epithelium will lead to the decreased secretion of pulmonary surfactant protein (SP), disfunction of pulmonary surfactants and finally to inhibit the maturation of pulmonary alveoli. Therefore, the lung injury occurred during the formation stage of respiratory system may result in retardation of lung development.Clara cell secretory protein (CCSP), the major protein secreted by remote bronchiole Clara cells, is an inhibitor of secretary phospholipase A2, Which takes part in the pathophysiological processes such as inflammation and tissue reconstruction. CCSP decreases the degradation of pulmonary surfactant materials, the weakening of the pulmonary elastic recoil, and increases residual volume of lung by inhibition of phospholipase A2. Ramsay had reported that CCSP levels in bronchoalveolar lavage fluids were lower on days of life 1, 3, and 6 in infants who developed BPD than in those who did not develop BPD. Decreased CCSP expression in infants who subsequently developed BPD suggests that CCSP expression may be highly correlated with the development of BPD. However, many other studies have reported that exogenetic CCSP may reduce pulmonary injury by the inflammatory mechanism. So far, no studies on the relationship of apoptosis and BPD have been reported.In order to elucidate the relationship of CCSP and alveolar epithelial cell apoptosis, we investigated the pulmonary histomorphology, expression of CCSP, SP-A, TNF-αand cell apoptosis at different time points of BPD, so as to elucidate the influence and mechanism of hyperoxia on lung development and injury. In addition, rh-CCSP was administered to the model to compare lung development, SP-A expression and cell apoptosis to elucidate protective effect of rh-CCSP on hyperoxia-induced lung injury. The study is of theoretical significance to respiratory pathophysiology of newborns and provides theoretical and experimental evidence of rh-CCSP for prevention and treatment of BPD.ResultsMechanism of hyperoxia lung injury1. Mortality of rats exposed to hyperoxiaWith prolonged. hyperoxia exposure, the mortalities of both groups were increased. Compared with air group, the mortality of hyperoxia group increased more sharply after 7 days of exposure, and there was significant difference between them (P＜0.01).2. Pneumonedema after hyperoxia exposureThe wet to dry weight ratio of both groups had no difference on day 3 and day 14 of exposure (P＞0.05); but it showed a significant increase on day 7 and day 10 compared to the air control group (P＜0.05). The contents of MDA of BALF were also significantly increased on day 3 and day 7 compared to the air control group (P＜0.01).3. Lung morphology with hyperoxiaFrom birth until day 3, the terminal bronchiole of lung tissue in both groups branched into smooth-walled channel that ended in saccule, whose wall contained a lot of interstitial cells. The septa of the newborn lungs appeared and became much thicker. There was very little number of alveoli in this lung development stage. On day 7 and day 10, the number of alveoli was increased in the air group compared to the hyperoxia group. By day 14, alveoli number of air group continued to multiply resulting in a homogenous alveolar, and its wall was thick with many interstitial cells. But for the rats of hyperoxia group, saccule continued to dominate, the number of small diameter alveoli decreased sharply and distal air space enlarged obviously.4. Expression of CCSP, SP-A and TNF-αof lung tissues with hyperoxia exposureWith immunohistochemistry, it was found a focus of positive staining in Clara cells of bronchiole epithelium. The percentage of Clara cells in distal and respiratory bronchioles epithelium were decreased with hyperoxia compared to that in air group (P＜0.01). And statistically significant relationships were found in positive intensity of Clara cells between hyperoxia and air group (P＜0.01). With prolonged hyperoxia, the positive ratio of Clara cells and the expression of CCSP were reduced, and difference was considered statistically significant (P＜0.05).The expression of SP-A in two groups has no difference on day 7 (P＞0.05), but SP-A level became lower in hyperoxia group compared to air group on day 14, and difference was statistically significant (P＜0.01).The semiquantitative result by western blot assay indicated that expression of SP-A became higher on day 3 in hyperoxia group compared to air group, and difference was statistically significant (P＜0.05); but the expression of SP-A had no difference between two groups on day 5 and day 7 (P＞0.05).The expression of TNF-αwas higher in hyperoxia group compared with air group. TNF-αexpression reached a peak on day 3 after hyperoxia, and decreased by day 7, but still was higher in hyperoxia group as compared to air control group, and difference was statistically significant (P＜0.01).5. Cell apoptosis of lung tissues under hyperoxiaWith the assay of TUNEL, it showed that most apoptotic cells were alveoli and bronchi epithelial cells. The apoptotic indexes were significantly higher than those in air group on day 7 and day 14 after hyperoxia exposure (P＜0.01). Although cell apoptosis aggregated with the prolonged hyperoxia exposure, there was no statistically differences between the two groups at other different time points (P＞0.05).Effect of rh-CCSP on apoptosis of A549 cellsApoptotic indexes of A549 were significantly higher at 72h after hyperoxia exposure than those in air group (P＜0.01), but were significantly lower than those in hyperoxia group with rh-CCSP administration (P＜0.01). Apoptotic indexes increased with the prolonged hyperoxia exposure.Effect of rh-CCSP on lung injury induced by hyperoxia exposure1. RAC and morphological quantitative analysisCompared to hyperoxia group, RAC of hyperoxia+rh-CCSP group was reduced and Alveolar areal ratio was increased, and the difference was considered statistically significant (P＜0.01).2. Effect of rh-CCSP on the expression of SP-A after hyperoxia exposureOn day 7 of hyperoxia, there were no difference in the expression of SP-A among the four groups (P＞0.05); but on day 14, the SP-A expression level was significantly increased in hyperoxia+CCSP group than hyperoxia group (P＜0.05).3. Effect of rh-CCSP on pneumonocyte apoptosis after hyperoxia exposureWith the assay of TUNEL, it showed that most apoptotic cells were alveolar and bronchi epithelial cells. The apoptotic index was increased to a significantly greater extent for rh-CCSP group compared to hyperoxia group (P＜0.01).Conclusions1. The prolonged exposure under 85% hyperoxia can lead to lung injury and lung developmental arrest of neonatal models, which would result in the increase of wet to dry weight ratio, decreased number of alveoli, decrease of RAC, changes of lung morphology and increase of mortality.2. Hyperoxia exposure can promote the pneumonocyte apoptosis by increasing the expression of TNF-α. Maybe it is one of the critical mechanisms of BPD.3. Clara cells and typeⅡalveolar epithelium are the main target cells of oxidative damage. Hyperoxia injures Clara cells and inhibits the secretion of CCSP and finally aggravates the lung injury; the prolonged hyperoxia exposure leads to denaturalization and destruction of typeⅡalveolar epithelium, which would result in decreased secretion of SP-A, disfunction of PS and finally to inhibit maturation of pulmonary alveoli.4. Apoptosis of A549 significantly increases with the prolonged hyperoxia exposure, while rh-CCSP administration can effectively inhibit the apoptosis of alveolar epithelial cells after hyperoxia exposure.5. The hyperoxia exposure decreases expression of CCSP and inhibits the development of lung alveoli, while the administration of rh-CCSP increases the formation of lung alveoli and the secretion of SP-A, inhibits the apoptosis of alveolar epithelium after hyperoxia exposure, and finally alleviates lung injury.Creative pointsSo far, no studies on the relationship of CCSP, hyperoxia and BPD, as well as the influence of rh-CCSP on lung injury induced by hyperoxia have been reported domestically. In this study, we investigated the influence and mechanism of rh-CCSP on hyperoxia-induced lung injury, which could be medicated with promoting pneumonocyte proliferation and differentiation and inhibiting apoptosis process.Creative points①We performed the first study on the relationship ofCCSP, BPD and hyperoxia Induced lung injury in our country;②We studied the influence and mechanism of rh-CCSP administrated with nebulisation delivery for the first time in the world.