P38 siRNA reduced lung ischemia-reperfusion injury of pulmonary microvascular endothelium after lung transplantation through NF-κB pathway inhibition

Abstract

Objective Using small interfering RNA (siRNA) against p38 and simulated lung transplantation model, we discussed the effect of p38 siRNA on hypoxia/reoxygenation injury of pulmonary microvascular endothelial cells (PMVECs) after lung transplantation. Methods We transfected the PMVECs with p38 siRNA or non-targeting (NT) siRNA. After 48 h, these cells were exposed to simulated ischemia-reperfusion. At 2 h and 4 h of reperfusion, we detected lactate dehydrofenase (LDH) leakage rate, malondialdehyde (MDA) levels, superoxide dismutase (SOD) activity, cell apoptosis, and the serum levels of interleukin (IL)-1, IL-6 and tumor necrosis factor (TNF)-α. Protein levels of p38, NF-κB and AP-1 were detected. Untreated PMVECs served as the negative control. Results As compared with NT siRNA, p38 siRNA reduced LDH leakage rate (22.3±5.7 vs. 45.1±6.2 and 46.3±7.3 vs. 75.6±12.4), decreased MDA levels (4.1±2.2 vs. 7.1±2.1 and 3.9±0.5 vs. 6.1±1.2), increased SOD activity (12.8±3.2 vs. 9.4±1.1 and 10.8±1.2 vs. 7.0±1.1), and inhibited apoptosis (2.8±0.6 vs. 4.1±1.4 and 3.1±1.1 vs. 5.8±1.3). p38 siRNA reduced the levels of IL-1 (288±89 vs. 592±95 and 380±94 vs. 775±175) and IL-6 (38±5 vs. 70±12 and 80±20 vs. 118±17), however, had no influence on TNF-α level. Silencing p38 gene decreased phosphorylation of p65 and inhibitor of nuclear factor kappa-B kinase β, and increased inhibitor of nuclear factor kappa-B expression. However, p38 siRNA had no effect on the phosphorylation of c-Jun and c-Fos. Conclusion Through inhibiting the NF-κB classic activation pathway, p38 siRNA reduced oxidative stress, inflammation and apoptosis of rat PMVECs, protected membrane integrity, and reduced hypoxia/reoxygenation injury. Key words: Pulmonary microvascular endothelial cells; Ischemia-reperfusion; p38; Nuclear factor kappa B; Activator protein-1