Antioxidant activity of pomegranate juice reduces acute lung injury secondary to hyperoxia in an animal model.

BACKGROUND: Hyperoxia triggers the release of toxic reactive oxygen species (ROS). Pomegranate Juice (PJ) is a rich source of potent antioxidants. We assessed the effects of PJ supplementation on Acute Lung Injury (ALI) in adult rats exposed to hyperoxia for 5 days. METHODS: Adult rats were divided into four different groups: control, hyperoxia, hyperoxia + PJ and PJ. Animals were placed in chambers containing either room air or oxygen above 95% for a total of 5 days. Two different PJ concentrations were utilized and the control group received placebo water. Animals were euthanized and their lungs were excised. Assessment of lung injury was accomplished by: a) wet to dry ratio (W/D) method, b) measurement of albumin concentration in the bronchoalveolar lavage fluid (BALF), c) oxidative stress, d) histological evaluation of the lung e) apoptosis and f) transcriptional expression levels of the inflammatory mediators IL-1ß, IL-6 and TNF-alpha. RESULTS: An increase in the W/D and albumin leak was noted in Hyperoxia (p < 0.05). Those findings were attenuated by the higher dose of PJ supplementation. Hyperoxia increased ROS production. Again PJ significantly reduced oxidative stress. Lung sections showed significant reduction in inflammation, edema, and infiltrating neutrophils in Hyperoxia + 80 µmol/kg when compared with Hyperoxia. TUNEL demonstrated significant apoptosis in the Hyperoxia, which was diminished in the Hyperoxia + 80 µmol/kg. Furthermore, increase in IL-1ß and IL-6 was noted in Hyperoxia. Again, 80 µmol/kg of PJ significantly reduced the expression of inflammatory mediators. CONCLUSION: In this animal model, PJ supplementation attenuated ALI associated with hyperoxia.

Activated protein C attenuates acute lung injury and apoptosis in a hyperoxic animal model.

Evidence suggests that activated protein C (APC) attenuates acute lung injury (ALI) through antithrombotic and anti-inflammatory mechanisms. The aim of this study was to determine the effects of APC on ALI in adult rats exposed to hyperoxic environment. Rats were divided into control, hyperoxia, hyperoxia + APC, and APC. Hyperoxia and hyperoxia + APC were exposed to 1, 3, and 5 days of hyperoxia. Hyperoxia + APC and APC were injected with APC (5 mg/kg, i.p.) every 12 h. Control and hyperoxia received isotonic sodium chloride solution injection. Measurement of wet to dry ratio and albumin leak demonstrated significant improvement in hyperoxia + APC when compared with hyperoxia. Apoptosis, as measured by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay, was significantly reduced in hyperoxia + APC when compared with hyperoxia. Histological evaluation of lung sections showed significant reduction in inflammation, edema, and in the number of marginating neutrophils in hyperoxia + APC as compared with hyperoxia. Transcriptional expression of lung inflammatory mediators demonstrated a time-dependent surge in the levels TNF-alpha, IL-1beta, and IL-6 in response to hyperoxia that was attenuated with APC administration in the presence of hyperoxia. In this rat model, APC attenuates lung injury and the expression of inflammatory mediators in ALI secondary to hyperoxia.

Apoptosis and the activity of ceramide, Bax and Bcl-2 in the lungs of neonatal rats exposed to limited and prolonged hyperoxia.

BACKGROUND: The aim of the study is to examine the effect of limited and prolonged hyperoxia on neonatal rat lung. This is done by examining the morphologic changes of apoptosis, the expression of ceramide, an important mediator of apoptosis, the expression of inflammatory mediators represented by IL-1beta and the expression of 2 proto-oncogenes that appear to modulate apoptosis (Bax and Bcl-2). METHODS: Newborn rats were placed in chambers containing room air or oxygen above 90% for 7 days. The rats were sacrificed at 3, 7 or 14 days and their lungs removed. Sections were fixed, subjected to TUNEL, Hoechst, and E-Cadherin Staining. Sections were also incubated with anti-Bcl-2 and anti-Bax antisera. Bcl-2 and Bax were quantitated by immunohistochemistry. Lipids were extracted, and ceramide measured through a modified diacylglycerol kinase assay. RT-PCR was utilized to assess IL-1beta expression. RESULTS: TUNEL staining showed significant apoptosis in the hyperoxia-exposed lungs at 3 days only. Co-staining of the apoptotic cells with Hoechst, and E-Cadherin indicated that apoptotic cells were mainly epithelial cells. The expression of Bax and ceramide was significantly higher in the hyperoxia-exposed lungs at 3 and 14 days of age, but not at 7 days. Bcl-2 was significantly elevated in the hyperoxia-exposed lungs at 3 and 14 days. IL-1beta expression was significantly increased at 14 days. CONCLUSION: Exposure of neonatal rat lung to hyperoxia results in early apoptosis documented by TUNEL assay. The early rise in Bax and ceramide appears to overcome the anti-apoptotic activity of Bcl-2. Further exposure did not result in late apoptotic changes. This suggests that apoptotic response to hyperoxia is time sensitive. Prolonged hyperoxia results in acute lung injury and the shifting balance of ceramide, Bax and Bcl-2 may be related to the evolution of the inflammatory process.