Background and objective: Hypoxia/reoxygenation (H/R) is a key factor in the pathogenesis of the most lung diseases where exсessive ROS production and prooxidant/antioxidant imbalance greatly contribute to disease progression. We have used severe hypoxia in sessions of repeated H/R of different duration as a model of lung pathologic states to investigate mitochondrial oxidative stress intensity, protein expression/activity of antioxidant enzymes manganese-superoxide dismutase (MnSOD), glutathione peroxidase (GPx), and antiapoptotic Bcl-2 as well as protein expression of their upstream regulators: p53 and nuclear factor- kappa B (NF-kB).
Methods: A total 86 rats were divided into five experimental groups and subjected to H/R [5 cycles of 10 min hypoxia (5.5 % O2 in N2) alternated with 10 min normoxia, daily]. Eight rats from each group were sacrificed on 1st -, 3rd - day, 1st and 2nd - week time points. Oxidative stress biomarkers (ROS formation, lipid peroxidation, H2O2 production, GSH/GSSG ratio, and mitochondrial aconitase activity as marker of compartment-specific superoxide anion production), indices of antioxidant status (MnSOD,GPx, glutathione –S-transpherase activities, and reduced glutathione level) were measured in lung mitochondria. Western blot was used to detect the protein levels of p53, Bcl-2, MnSOD, and GPx in mitochondria as well as the phosphorylated NF-kB p65 in the nucleus of lung cells. Expression of mRNA MnSOD was determined by real-time polymerase chain reaction.
Results: The short- (1-3 days) and long-term (1-2 wk) H/R differentially affects the oxidative stress level, p53 protein expression and its subcellular distribution as well as antioxidant capacity in lung mitochondria.The long-term H/R caused mitochondrial p53 protein translocation, a decrease in Bcl-2 protein content, and a significant increase in nuclear accumulation of the phosphorylated NF κB p65 protein. We observed an increase in GPx protein content/activity, in parallel with decrease in MnSOD protein level and activity. In the dynamics of MnSOD gene expression we found a phase time point dependence.
Conclusions: Long lasting H/R leads to mitochondrial prooxidant/antioxidant disbalance that resulted in redox alteration as consequence of oxidative stress propagation and apoptotic cascade activation. A close correlation between mitochondrial p53 Protein level and protein expression/activities of its targets MnSOD and GPx suggest participation of p53 in regulation of H/R-induced mitochondrial oxidative stress level.
Published on: May 19, 2017 Pages: 35-43