Nitric oxide synthase gene expression in hyperoxia-induced lung injury in premature rat

Objective Preterm infants are prone to develop oxygen-induced lung injury. However, little information was available on the potential effect of nitric oxide synthase(NOS)in mediating hyperoxic damage within premature infants lung. This study aimed at using a hyperoxia-induced lung injury preterm rat model, to investigate the effect of hyperoxia on NOS gene expression. Methods Preterm rats were delivered by hysterotomy at 21 days gestation and randomly divided into hyperoxia group and room-air control and, continuous exposed to oxygen (oxygen 0.95) and room air for 7 days, respectively. The animals were sacrificed on day 7 of life with an overdose of intraperitoneal pentobarbital. Their lungs were examined for immunohistochemical staining of endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS). The mRNA levels of eNOS and iNOS were measured by reverse-transcription polymerase chain reaction (RT-PCR). eNOS and iNOS protein expressions were measured by Western blotting. In addition, the lung wet/dry weight ratio, the detection of protein and cellular content of bronchoalveolar lavage fluid (BALF), histological examination of oxygen induced lung injury were performed. Results Compared to the room-air control, the hyperoxia group developed severe lung damage. The histological changes consisting of edema, hemorrhage and inflammatory were found. There were significant increases in lung wet/dry weight ratio (5.57±0.29 vs 5.29±0.25, t=2.29, P0.05) and the protein content (1.49 g/L vs 0.32 g/L, t=28, P0.05) and the cell count (139.70×10 7/L vs 16.30×10 7/L, t=21, P0.05) in BALF. The eNOS mRNA expression in the hyperoxia group was significantly elevated (1.02±0.06 vs 0.70±0.12, t=6.36, P0.01). The mean iNOS mRNA expression also showed an increase in the hyperoxia group, although the difference was not statistically significant. There was a significantly higher pulmonary eNOS and iNOS protein expressions in the hyperoxia group than the control group (8.77±0.75 vs 4.52±1.02, t=8.14, P0.01 and 4.61±0.65 vs 3.24±0.55, t=3.21, P0.05). Immunohistochemical staining for eNOS and iNOS was strongly positive in the hyperoxia group. Conclusion Hyperoxic exposure of preterm rat pups caused up-regulation of NOS gene expression, which persisted even in established oxygen-induced lung damage. The endogenous NOS may play an important role in the pathophysiology of the pulmonary oxygen injury in the premature animal. Whether this up-regulation is beneficial or harmful to the animal in the pathogenesis of oxygen-induced lung injury requires further studies.