OBJECTIVE

Apoptosis has been shown to be involved in lung remodeling in both rat lung and human fetal lung explants. Mounting evidence suggests the pro- and anti-apoptotic members of the Bcl-2 family exert most of their function at the mitochondrial level and play pivotal roles in the process of apoptosis. Molecules such as Bax (death agonist) and Bcl-2 (death antagonist) act in competition, and their relative abundance and dimerization can determine cell death or cell survival. It is not known whether apoptosis is in part responsible for the impaired lung growth found in preterm developing lung exposed to intra-amniotic endotoxin priming plus hyperoxia, and imbalance of Bax/Bcl-2 contributes to it. The purpose of this study was to investigate the expression apoptosis index (AI), Bax and Bcl-2 in premature lungs of rats with intra-amniotic endotoxin priming and/or exposed to 60% O(2) and to elucidate the relationship between intrauterine inflammatory/chronic O(2) exposure and the pathogenesis of bronchopulmonary dysplasia (BPD).

METHODS

Timed pregnant Sprague Dawley (SD) rats were randomly divided into two groups: lipopolysaccharide (LPS) group and saline group. LPS or saline were intra-amniotically injected into the sacs on gestational age day 15 (70% of term). Six days after intra-amniotic injection, the preterm rats were delivered and randomized to put in 60% O(2) exposure or in room air. On days 1, 7 and 14 after birth, the lungs were removed and dissected from the main bronchi for analysis. Total RNA were extracted from the right frozen lung tissues. Lung AI was measured by terminal transferase nick end labeling (TUNEL) and Bax and Bcl-2 mRNA levels were measured by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR).

RESULTS

(1) To quantify the proliferation of preterm lungs after intra-amniotic endotoxin priming and/or exposed to 60% O(2), proliferating cell nuclear antigen (PCNA) in the four experimental groups were measured: PCNA increased along with the lung development from d1 to d14 in both control and endotoxin alone groups. However, in endotoxin alone group, PCNA was significantly lower than that of controls from d1 to d14. In hyperoxia alone group, PCNA begun to decreased on day 7, and became significantly lower than that of the control group on day 14 (P < 0.05). In endotoxin plus hyperoxia group, PCNA was significantly lower than that of controls on days 1 and 7. (2) To quantify the apoptosis of immature lungs after intra-amniotic endotoxin priming and/or exposed to 60% O(2), AI increased along with the lung development from d 1 to d 7 in both control group and endotoxin alone group. However, in endotoxin alone group, AI was significantly higher than that of controls from d1 to d7. In hyperoxia alone group, AI begun to increased on day 7, and became significantly lower than that of the control group on day 14 (P < 0.05). In endotoxin plus hyperoxia groups, AI was higher than controls on days 1 and 7, and became significantly higher than those of the other 3 groups on day 14. (3) RT-PCR showed that mRNA of Bax decreased while Bcl-2 mRNA increased from day 1 to day 14 in endotoxin alone group, and they were significantly higher than those of controls on day 1 (P < 0.05). Conversely, Bax mRNA increased while Bcl-2 mRNA decreased from day 1 to day 7 in hyperoxia treated groups, and was significantly different from that of the control group on days 7 and 14 in both hyperoxia treated groups.

CONCLUSIONS

These results suggest that intra-amniotic endotoxin presensitization and exposure to hyperoxia may inhibit proliferation of lung cells. Increased expression of Bax and Bcl-2 may be related to the evolution of lung injury and may contribute to the pathogenesis of BPD.