| Summary: | 博士 === 國立陽明大學 === 生理學研究所 === 94 === ABSTRACT
Although a link between toxic smoke and oxidant lung vascular injury has been indicated, the cellular mechanisms of smoke-induced injury to lung endothelial cells are completely unknown and the optimal therapy to treat smoke-induced lung injury is still absent.
In the first study series, we investigated oxidative stress and apoptosis induced by wood smoke extract (SE) in human pulmonary artery endothelial cells (HPAECs) and delineated their relationship. We found that SE increased intracellular reactive oxygen species (ROS), depleted intracellular glutathione (GSH), and up-regulated Cu/Zn superoxide dismutase (Cu/Zn SOD) and heme oxygenase-1 (HO-1) (two antioxidant-relative enzymes), but it failed to alter the expression of catalase and GSH peroxidase. Additionally, SE promoted apoptosis as indicated by the external exposure of membrane phosphatidylserine, the loss of mitochondrial membrane potential, an increase in the level of Bax (a pro-apoptotic protein), and enhanced DNA fragmentation. This apoptosis was associated with mitochondrial-to-nuclear translocation of apoptosis-inducing factor (AIF) and endonuclease G (EndoG) (two apoptogenic proteins), but was independent of caspase cascade activation. While N-acetyl-L-cysteine (an antioxidant and GSH precursor) effectively reversed the SE-induced increase in ROS and depletion of GSH, it also suppressed SE-induced nuclear translocation of either AIF or EndoG, and prevented the enhanced DNA fragmentation.
In the second study series, we investigated the anti-apoptotic function of HO-1 on SE-induced HPAEC death. We found that small interfering RNA knocked-down of HO-1 or inhibition of HO-1 activity by protoporphyrin-IX significantly deteriorated SE-induced cell apoptosis. Conversely, overexpression of HO-1 by adenoviral vector largely prevented the SE-induced nuclear translocation of AIF and significantly attenuated SE-induced cell apoptosis. While overexpression of HO-1 did not affect the SE-induced loss of mitochondrial membrane potential, it increased the interaction of HO-1 with AIF presumably at the cytosolic location. We conclude that 1) SE causes intracellular oxidative stress elevation and GSH depletion resulting HPAEC apoptosis, 2) SE induces caspase-independent HPAEC apoptosis that involves mitochondrial-to- nuclear translocation of AIF and 3) induction of HO-1 prevents nuclear translocation of AIF and alleviates SE-induced HPAEC apoptosis partly via its interaction with AIF. Thus, modulation of oxidative stress, the HO-1 expression and the caspase-independent apoptotic pathway are possible target choices for potential therapeutic regimes to treat smoke-induced lung injury.
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