Prevention Of Lung Ischemia-reperfusion Injury By Short Hairpin RNA-mediated Caspase-3 Gene Silencing

Partâ… Expression of caspase-3 in a rat model of lung ischemia-reperfusion injuryObjective:To evaluate the level of caspase-3 during reperfusion at different time point in a rat model of lung ischemia-reperfusion injury.Methods:Both real-time PCR and western blot were used to examine caspase-3 expression in lungs at different time of reperfusion. Blood samples were obtained from the left pulmonary vein to assess lung function. Blood gas values, especially partial pressure of oxygen and carbon dioxide (PaO2 and PaCO2) were measured. The ratio of wet/dry (W/D) was used to assess lung edema after ischemia-reperfusion injury. TUNEL methods were used to detect the apoptotic alveolar epithelial cells, especially typeâ…¡alveolar epithelial cell.Results:The level of caspase-3 in both mRNA and protein had started to increase at half an hour after reperfusion, and the peak of maximal caspase-3 activity occurred at 2 hrs after reperfusion. Two hours after reperfusion, the level of PaO2 was decreased mostly; otherwise the level of PaCO2 was increased respectively. W/D was also increased at the reperfusion time of 2 hrs. TUNEL staining indicated that animals subjected to reperfusion of 2 hrs showed more apoptotic cells than others.Conclusions:The level of caspase-3 expression was most intense in the time of 2 hrs after reperfusion, so was the apoptotic cells in lungs. Lung function was worse at the time of 2 hrs after reperfusion than others. Partâ…¡Designment of short hairpin RNA specific for silencing of caspase-3 and screening out effective interfering target sequenceObjective:To design oligonucleotide cDNA inserts encoding short hairpin RNA specific for caspase-3 and to screen out the effective interfering target sequence.Methods:Firstly, we had synthesized double-stranded DNA oligonucleotide containing several RNA interfering target sequences for caspase-3, then we had combined the dsDNA oligo and the specific plasmid together. After identification and ampliation, the effective shRNA specific for caspase-3 was distilled. NRK cells, using as the tool cells, was transfected by the caspase-3 shRNA using lipofectamine 2000. Then the level of caspase-3 expression was examined using western blot to screen out the effective interfering target sequence.Results:Four DNA plasmids, called SD-704,SD-705,SD-706 and SD-707 respectively, were compounded, which containing dsRNA oligonucleotide and the pGCsi plasmid. After examined by western blot and immunofluorescence, the effective caspase-3 shRNA was selected.Conclusions:The short hairpin RNA specific for caspase-3 was designed and screened out successfully, and it had been proved to silence the expression of caspase-3 in tool cells.Partâ…¢Transfection of caspase-3 shRNA into a rat model of lung ischemia-reperfusion injuryObjective:To evaluate the therapeutic efficacy of caspase-3 shRNA in a rat model of ischemia-reperfusion injury.Methods:Lung IRI was induced in rats by clamping the hilum of the left lung for 1hr. In vivo delivery of caspase-3 shRNA was performed by intratracheal administration 48 hrs before ischemia. As controls, animals received either scrambled shRNA or RNase-free D5W solution. Real-time polymerase chain reaction (PCR), western blot 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 (W/D).Results:We have shown that IRI is associated with increased level of lung caspase-3 mRNA. Animals treated with caspase-3 shRNA showed a significant down-regulation in lung expression of caspase-3 at transcripts and protein levels. Lung function was protected by caspase-3 shRNA therapy, as levels of partial pressure of oxygen and carbon dioxide (PaO2 and PaCO2) were significantly increased and reduced respectively.Conclusions:We have demonstrated the therapeutic potential of shRNA for knocking down the expression of caspase-3 and prevent lung apoptotic injury. Our findings may have some potential therapeutic relevance for treating lung IRI after transplantation.