No effect on lung development in neonatal rats and the mechanism of inhalation

PART ONE THE EFFECTS OF INHALED NITRIC OXIDE ON LUNG DEVELOPMENT OF NEONATAL RATSObjective:The aim of the part was to investigate the effects of inhaled NO with high concentration and low concentration on the alveolarization and lung vascular development of neonatal rats, so as to explore the impacts of inhaled NO on the lung development.Methods:①Neonatal rats were placed in the chamber containing NO and<1ppm NO2from birth to postnatal day7with different concentration (5ppm,20ppm). Control animals were placed in a chamber at room air. Rat pups were sacrificed at postnatal day7and postnatal day14(This part of rats inhaled NO at the beginning of the first7days of birth. Inhaled room air during the subsequent7days).②30rats were divided randomly into6groups including4experimental groups and2control groups with5rats in each group. NO5-7group:inhaled NO5ppm7days and were sacrificed on postnatal day7; NO20-7group: inhaled NO20ppm7days and were sacrificed on postnatal day7; NO5-14group:inhaled NO5ppm7days and were sacrificed on postnatal day14; NO20-14group:inhaled NO20ppm7days and were sacrificed on postnatal day14; C7group:inhaled room air7days and were sacrificed on postnatal day7; C14group:inhaled room air14days and were sacrificed on postnatal day14.③Body weights and lung weights of rats were weighed at the end of experiment. Lung weights to body weights ratios were counted.④Lung tissues were stained with HE. Radial alveolar counts (RAC) and mean linear intercept (MLI) were used to detect the changes of lung morphology.⑤CD34was used as vascular endothelial marker. All the specimens’ sections were stained by immunohistochemical method. The vessels marked by CD34were counted and the vessel density was counted by the method of Kunig.⑥The data were statistically analyzed with SPSS software.Results①There were no significant differences between experimental groups and control groups on body weight, lung weight, and lung weight to body weight ratio.②ompared with C7group, The RAC of NO20-7group was significantly increased (P<0.05). The MLI of NO20-7group was significantly decreased (P<0.05). There were no statistical difference between others experimental groups and corresponding control groups on RAC and MLI.③The vessel density of NO5.7group was significantly increased as compared with that of C7group (13.2±5.4vs9.5±3.3, P<0.05).The vessel density of NO20-7group was significantly increased as compared with that of C7group (13.7±4.7vs9.5±3.3, P<0.05)Conclusions①The RAC of NO20-7group was significantly increased and the MLI of NO20-7group was significantly decreased as compared with that of C7group. The results indicated that inhaled NO with high concentration enhanced alveolarization of neonatal rats.②The vessel density of NO5.7group and NO20-7group were significantly increased. The results indicated that inhaled NO with both low concentration and high concentration improved,vascular development of neonatal rats.③There were no observed differences on RAC, MLI, and vessel density between NO5-14group, NO20-14group, and C14group. The rats had some changes at the7day on alveolarization and lung vascular development after7days inhaled NO. However, no changes were found at the14day on alveolarization and lung vascular development after7days inhaled NO. These results indicated that inhaled NO may accelerate the lung development of neonatal rats at the early stage of lung development. We speculated that alveolarization and lung vascular development of rat mainly concentrate in the first postnatal week. PART TWO THE EFFECTS OF INHALED NITRIC OXIDE ON VEGFAND FLK-1EXPRESSION IN NEONATAL RATS LUNGObjective:The aim of this part was to investigate the effects of inhaled NO on VEGF and FLK-1expression in neonatal rats lung, so as to explore the molecular mechanism of NO inhalation on lung development.Methods①Establishment of animal model and experimental animal grouping (See part one).②Immunohistochemical staining (IHC) was used to detect the VEGF and FLK-1expression on vascular endothelial and airway epithelial.③Western blotting was used to detect VEGF and FLK-1expression in lung tissue.④ELISA was used to detect the concentration of VEGF in lung tissue.⑤Computing-image analysis system was used to detect the changes of VEGF and FLK-1staining intensity in IHC and optical density value changes in western blotting.⑥The data were statistically analyzed with SPSS software.Results①The effects of inhaled NO on VEGF expression in neonatal rats lung: Both control groups and experimental groups showed positive staining of VEGF on vascular endothelial and airway epithelial. However, the staining intensity of VEGF in NO20-7group was significantly increased as compared with that in C7group (P<0.05). The results of western blotting confirmed that the VEGF expression in NO20-7group was significantly increased. The results of ELISA showed that the concentration of VEGF in NO20-7group (144.00±6.44)pg/ml was significantly increased as compared with that of C7group (84.96±10.22)pg/ml (P<0.05). There were no statistical difference between others experimental groups and corresponding control groups on VEGF expression②The effects of inhaled NO on FLK-1expression in neonatal rats lung: Both control groups and experimental groups showed positive staining of FLK-1on vascular endothelial and airway epithelial. However, the staining intensity of FLK-1in NO5-7group and NO20-7group were significantly increased as compared with that in C7group (P<0.05). The results of western blotting confirmed that the FLK-1expression in NO5-7group and NO20-7group were significantly increased (P<0.05). There were no statistical difference between others experimental groups and corresponding control groups on FLK-1expression.Conclusions①Inhaled high concentration NO enhanced VEGF and FLK-1expression in neonatal rats lung. Combined with the morphology changes that we have investigated in part one, we can draw the conclusion that: inhaled NO may improve lung vascular development and alveolarizati on by enhancing the VGEF and FLK-1expression.②Inhaled low concentration NO enhanced FLK-1expression in neonatal rats lung. Combined with the morphology changes that we have investigated in part one, we can draw the conclusion that:inhaled NO may improve lung vascular development by enhancing the FLK-1expression.③Inhaled low concentration NO only can enhance FLK-1expression, but inhaled high concentration NO can enhance VEGF and FLK-1expression in neonatal rats lung. Inhaled low concentration NO only can improve lung vascular development, but inhaled high concentration NO can improve lung vascular and alveolus development. We can speculate that VEGF not only act on lung vascular endothelial to improve lung vascular development but also act on airway epithelial to improve alveolus development directly. The molecular mechanism is still unclear and needs further study.④There were no observed differences on VGEF and FLK-1expression among NO5-14group, NO20-14group, and C14group.This experiment studied the molecular mechanism about the improvement of lung growth after NO inhalation in neonatal rats. This it the first time to illustrate the molecular mechanism of the effect of inhaled NO on lung development. Inhaled NO improves lung development partly by enhancing VEGF and VEGFR-2protein expression. PART THREE THE MOLECULAR PATHWAY OF VEGF UPREGULATION BY EXOGENOUS NO IN NEONATAL RATS LUNGObjective:The aim of this part is to investigate the effects of inhaled NO on HBF-1α, HO-1, eNOS proteins expression in neonatal rat lung, so as to explore the molecular pathway of augment of VEGF synthesis by NO.Methods:①Establishment of animal model (See part one).②Experimental animal grouping:15rats were divided into3groups in this part with each5rats. C7group; NO5-7group; NO20-7group.③Immunohistochemical staining (IHC) was used to detect the eNOS expression on vascular endothelial and airway epithelial.④Western blotting was used to detect HIF-laand eNOS expression in lung tissue.⑤ELISA was used to detect the concentration of HO-1in lung tissue.⑥Computing-image analysis system was used to detect the changes of eNOS staining intensity in IHC and optical density value changes of HIF-lain western blotting. ⑦The data were statistically analyzed with SPSS softwareResults:The effects of inhaled NO on HIF-1αprotein expression:The results of western blotting indicated that there are no differences among NO20-7group, NO5.7group, and C7group on HIF-1αprotein expression.The effects of inhaled NO on HO-1protein expression:The results of ELISA indicated that the concentration of HO-1in lung tissue of NO20-7group was increased as compared with that in C7group (P<0.05). There are no differences between NO5-7group and C7group (P>0.05)The effects of inhaled NO on eNOS protein expression:Both control group and experimental groups showed positive staining of eNOS on vascular endothelial and airway epithelial. However, the staining intensity of eNOs in NO20.7group were significantly decreased as compared with that in C7group (P<0.05). The results of western blotting confirmed that the eNOS expression in NO20-7group were significantly decreased (P<0.05). There were no statistical difference between NO5.7group and C7group on eNOS expression.Conclusions:①The effect of inhaled NO with high concentration on upregulation of VEGF synthesis is mediated by HO-1.②The effect of inhaled NO with high concentration on upregulation of VEGF synthesis is not mediated by HIF-la. ③Inhaled NO with high concentration depresses the expression of eNOS. VEGF synthesis induced by exogenous NO is not through VEGF-NO signal pathway by enhancing the endogenous NO production.④The mechanism of eNOS expression depression by inhaling high concentration NO is not clear.⑤This is the first time to illustrate the molecular pathway of VEGF synthesis regulated by NO inhalation. The effect of inhaled NO on VEGF upregulation is mediated by HO-1.