Prevention of lung ischemia–reperfusion injury by short hairpin RNA–mediated caspase-3 gene silencing

Abstract

Lung ischemia-reperfusion injury remains a significant problem after lung transplantation. Caspase-mediated apoptotic pathways play an important role in lung ischemia-reperfusion injury, and caspase-3 is presumed to be the "effector" protease in the apoptotic cascade. Silencing gene expression of caspase-3 by short hairpin RNA (shRNA) can downregulate the caspase cascade. Therefore, we evaluated the therapeutic efficacy of caspase-3 shRNA in a rat model of lung ischemia-reperfusion injury. Lung ischemia-reperfusion injury was induced in rats by clamping the hilum of the left lung for 1 hour. In vivo delivery of caspase-3 shRNA was performed by intratracheal administration 48 hours before ischemia. As controls, animals received either scrambled shRNA or RNase-free 5% dextrose in water solution. Real-time polymerase chain reaction, Western blotting, and immunohistochemistry were used to assess the gene silencing efficacy. The therapeutic effects of shRNA were evaluated by lung function analysis and the ratio of wet/dry weight. In this study, we have shown that ischemia-reperfusion injury is associated with an increased level of lung caspase-3 messenger RNA. Animals treated with caspase-3 shRNA showed a significant downregulation in lung expression of caspase-3 at transcripts and protein levels. Lung function was protected by caspase-3 shRNA therapy, inasmuch as levels of partial pressure of oxygen and carbon dioxide were significantly increased and reduced, respectively. In summary, we have demonstrated the therapeutic potential of shRNA to knock down the expression of caspase-3 and prevent lung apoptotic injury. Our findings may have some potential therapeutic relevance for treating lung ischemia-reperfusion injury after transplantation.