The Research Of Mitochondrial Apoptosis Pathway In Neonatal Rats Of Hyperoxia-induced Lung Injury

The pathogenesis of hyperoxia-induced lung injury has not been fully clarified, oxidative stress and reactive oxygen species (reactive oxygen specie, ROS) generation play an important role in the development of hyperoxia-induced lung injury. Mitochondria is the major cellular sites for energy, which is the center of metabolism and energy conversion, and tricarboxylic acid cycle, transport of respiratory electron chain, oxidative phosphorylation are carried out in the init. It's the main organelles that generate intracellular reactive oxygen species and the target organelles of oxidative damage. In addition to generate ATP for cells needed, mitochondrial play an important role in the regulation of redox state of cell and osmotic pressure, calcium homeostasis, maintain of PH and transduction of apoptosis signal. With the increasing study of hyperoxia-induced lung injury mechanism, people has paied much more attention to the role of apoptosis in lung injury. Investigate pathogenesis of mitochondrial apoptosis pathway in hyperoxia-induced lung injury and prevent apoptosis provide a new strategy and direction in prevention reducing and cure of neonatal lung injury.ObjectivesTo observe dynamic expression and association between Cytc and caspase-3 in the mitochondrial apoptosis pathway of neonatal rats'model of hyperoxia-induced lung injury. To explore possible mechanism and significance of mitochondrial apoptosis pathway in hyperoxia-induced lung injury, provide experimental and theoretical foundation for the reducing and cure of hyperoxia-induced lung injury in neonatal.Methods1. The neonatal Sprague-Dawley rats were divided randomly into the air group and hyperoxic group immediately after birth, 24 in each group. The hyperoxic group were exposured to hyperoxia (≥95%) to make of hyperoxia-induced lung injury model, and the air group were placed in the same room air. Take samples of two groups when rats exposed to at least 1th,2th,3th,4th,6 in each Subgroups.2. To observe each group rats'lung tissue morphological changes with microscopy and ultrastructure changes of alveolar typeⅡepithelial cell with transmission electron microscopy; lung cell apoptosis were observed by terminal deoxynucleotid transferase-mediated X-dUTP nick end labeling(TUNEL) and calculated the apoptosis index The expressions of cytoplasm and mitochondria cytochrome C were observed using western blot techniques, and expression of caspase-3 were detected by immunohistochemistry.Results1.Observation of lung histological changes of rats in each group with microscope:the lung tissue in noraml rats showed clear and alveolar structure, uniform size, no fluid exudation. Compared with air groups, 1th and 2th of hyperoxic group showed no significant changes, 3th,4th of hyperoxic group displayed thinner walls of alveoli,uneven alveolar size and alveolar inflammatory changes, such as dead cells and inflammatory cell in space of alveoli, interstitial pulmonary edema, small vessel expansion, congestion and so on.2. Observation of ultrastructure changes of alveolar typeⅡepithelial cell with transmission electron microscopy: Compared with the air group, AECⅡshowed varying degrees of plate layer bodies vacuolization, mitochondrial swelling, cristae damage, matrix density changes, even changes of nuclear chromatin in hyperoxic groups,2th, 3 th, 4 th of hyperoxic group showed obvious.3. TUNEL detect apoptosis: A few apoptotic cells were observed in the air group. Compared with the air group, apoptosis index(%) of hyperoxic group at 1th, 2th (5.22±0.33,8.01±0.86) had no significant changed(P> 0.05). Apoptosis index of hyperoxic group at 3th, 4th (15.17±2.29, 23.48±4.07) were higher than air group (8.25±1.57, 10.91±1.74)(P <0.01). Positive staining was mainly scaterred in alveolar epithelial cells, capillary endothelial cells and bronchial epithelial cells.4. Cytc protein level expression in lung cytoplasm and mitochondrial:Cytc protein in lung cell cytoplasm of hyperoxic group at 2th ,3 th and 4th(0.82±0.14, 1.37±0.11,1.20±0.11)were higher than air group(0.23±0.04, 0.24±0.03, 0.24±0.02)(P<0.01 ); Cytc protein in mitochondrial of hyperoxic group at 2th ,3 th and 4th (0.72±0.04, 0.41±0.06, 0.23±0.05) were lower than air group(1.31±0.13,1.34±0.06,1.29±0.05)(P<0.01).5.caspase-3 protein level expression: positive immune stainning of caspase-3 maily scaterred in the cytoplasm of alveolar epithelial cells, endothelial cells, bronchial epithelial cells and the interstitial cell of lung. The caspase-3 gray value of hyperoxic group at 3th ,4 th(94.96±8.95, 91.27±10.64)were lower than air group(102.65±8.44,102.2±7.29)(P<0.05). Note: the lower the gray value, the stronger the expression.6. Correlation between apoptosis index and Cytc protein,caspase-3 expression: significantly positive correlations were found between apoptosis index and Cytc protein in lung cell cytoplasm(r=0.58,P<0.01), and significantly negative correlations were found between apoptosis index and Cytc protein in mitochondria(r=-0.921,P<0.01), and significantly negative correlations also found between apoptosis index and Caspase-3 gray value(r=-0.573,P<0.01). Conclusions1. Neonatal rats exposure to hyperoxia in short-term can be induced acute lung injury which is characterized by inflammatory changes. With prolonged exposure to hyperoxia condition, ultrastructural of AECⅡwere changed, and lung cell apoptosis index increasing.2. Hyperoxia exposure can cause Cytc releases from mitochondria to cytoplasm. Cytc possibly results activation of caspase-3 leading to mitochondrial pathway-mediated apoptosis, at last hyperoxia-induced lung injury.3. Cytc release, caspase-3 expression were related to apoptosis index. and dynamic changes of mitochondrial morphology.