Prevention of lung ischemia–reperfusion injury by short hairpin RNA–mediated caspase- 3 gene silencing Yong-Xing Zhang, MD, Hong Fan, PhD, Yu Shi, MD,

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Prevention of lung ischemia–reperfusion injury by short hairpin RNA–mediated caspase- 3 gene silencing Yong-Xing Zhang, MD, Hong Fan, PhD, Yu Shi, MD, Song-Tao Xu, PhD, Yun-Feng Yuan, MM, Ru-Heng Zheng, MD, Qun Wang, PhD The Journal of Thoracic and Cardiovascular Surgery Volume 139, Issue 3, Pages 758-764 (March 2010) DOI: 10.1016/j.jtcvs.2009.09.027 Copyright © 2010 The American Association for Thoracic Surgery Terms and Conditions

Figure 1 Western blotting and real-time polymerase chain reaction (PCR) analysis of caspase-3 expression. The efficacy of gene silencing was examined at the level of protein and mRNA, using Western blotting and real-time PCR. A, The protein expression of caspase-3 was reduced significantly in animals subjected to caspase-3 shRNA. The relative quantity of caspase-3 mRNA for animals treated with caspase-3 shRNA was significantly reduced, as shown in B (∗P = .008 shRNA vs scrambled, ∗P = .007 shRNA vs solution, P < .01). No significant difference between scrambled shRNA and RNase-free 5% dextrose in water solution groups was demonstrated. Data of the relative quantity of mRNA was expressed as means ± SEM, statistical significance as compared between caspase-3 shRNA and scrambled shRNA or RNase-free D5W solution. The Journal of Thoracic and Cardiovascular Surgery 2010 139, 758-764DOI: (10.1016/j.jtcvs.2009.09.027) Copyright © 2010 The American Association for Thoracic Surgery Terms and Conditions

Figure 2 Immunohistochemical analysis of caspase-3 protein expression. Immunohistochemistry was used to analyze the expression of caspase-3 at the level of protein. Positive staining of caspase-3 (brown deposit) was observed in the cytoplasm of the alveolar epithelial cells of IRI lungs, as shown in black arrows. A and B, Animals treated with RNase-free 5% dextrose in water (D5W) solution and scrambled shRNA, respectively. As shown, rats treated with caspase-3 shRNA (C) showed significant reduction of brown deposit in the cytoplasm. No significant differences between scrambled shRNA and RNase-free D5W solution groups were demonstrated. The Journal of Thoracic and Cardiovascular Surgery 2010 139, 758-764DOI: (10.1016/j.jtcvs.2009.09.027) Copyright © 2010 The American Association for Thoracic Surgery Terms and Conditions

Figure 3 Caspase-3 shRNA protects lung function from IRI. We measured levels of Pao2 and Paco2 and wet/dry weight (W/D) of the lung from the rats 2 hours after reperfusion. A, The level of Pao2 in animals treated with caspase-3 shRNA before blocking the hilum of the lung significantly prevented the decrease of Pao2 (∗P < .01). The level of Paco2 shown in B significantly increased in animals subjected to caspase-3 shRNA (∗P < .01). W/D also decreased for rats treated with caspase-3 shRNA (∗P < .01), which meant that the lung edema was alleviated after treatment with caspase-3 shRNA, as shown in C. The Journal of Thoracic and Cardiovascular Surgery 2010 139, 758-764DOI: (10.1016/j.jtcvs.2009.09.027) Copyright © 2010 The American Association for Thoracic Surgery Terms and Conditions

Figure 4 Electron microscopic observation of apoptotic expression. A and B, RNase-free 5% dextrose in water solution or scrambled shRNA treatment group. Animals were observed for the presence of swelling of mitochondrion, outer membrane breaks, and intracristal dilation, as shown by black arrows. A, In the caspase-3 shRNA treatment group, the expression of swelling of mitochondrion and intracristal dilation wasn't obvious. The Journal of Thoracic and Cardiovascular Surgery 2010 139, 758-764DOI: (10.1016/j.jtcvs.2009.09.027) Copyright © 2010 The American Association for Thoracic Surgery Terms and Conditions

Figure 5 Quantification of apoptotic cells during ischemia–reperfusion injury. TUNEL analysis was used to detect the apoptotic alveolar epithelial cells. TUNEL staining (brown deposit) indicated that animals treated with caspase-3 shRNA showed fewer apoptotic cells than those treated with scrambled shRNA or RNase-free 5% dextrose in water (D5W) solution (P < .05). The percentage of positive cells of caspase-3 shRNA (C) was 0.049 ± 0.006 (n = 6), inasmuch as 0.097 ± 0.010 (n = 6), and 0.098 ± 0.037 (n = 6) belonged to scrambled shRNA (B) and RNase-free D5W solution (A), respectively. The Journal of Thoracic and Cardiovascular Surgery 2010 139, 758-764DOI: (10.1016/j.jtcvs.2009.09.027) Copyright © 2010 The American Association for Thoracic Surgery Terms and Conditions