Simvastatin protects against endotoxin induced acute lung injury and apoptosis via activation of survivin/NF-κB signaling pathway

[Speaker] Lana Nezic:1
[Co-author] Ranko Skrbic:1, Ljiljana Amidzic:2
1:Department of Pharmacology, Toxicology and Clinical Pharmacology, Faculty of Medicine University of Banja Luka, Bosnia and Herzegovina, 2:Department of Pathology, Clinical Center Banja Luka, Bosnia and Herzegovina

Background: It is considered that acute lung injury (ALI) is an uncontrolled inflammation, mainly attributed to the release of inflammatory mediators and extensive apoptosis.
Aim: As simvastatin exerts antiinflammatory properties by regulating cellular signaling pathways, we explored its effects on ALI development and apoptotosis of immune and epithelial cells in endotoxic shock.
Methods: Male Wistar rats were intraperitoneally injected with 0.25 of the medial lethal dose of endotoxin (lipopolysaccharide, LPS). Simvastatin was given orally (10, 20, 40 mg/kg, per group) as a five-day pre-treatment prior to a single dose of LPS, and 48 afterwards the animals were sacrificed. Lung tissue inflammatory damage was assessed by histopathological examination (haematoxylin-eosin staining), and expressed as a tissue damaged score (TDS). Apoptosis was analysed by TUNEL (DNA fragmentation assay) and expressed as an apoptotic index (AI), and immunohistochemically by detection of cleaved caspase-3, Bcl-XL, inhibitor of apoptosis, survivin as well as expression of nuclear factor (NF)- κB signalling pathway.
Results: TDSs showed that simvastatin ameliorated ALI induced by LPS in dose-dependent manner (simvastatin 20 mg/kg and 40 mg/kg vs LPS, 2.0±0.59 and 1.33±0.48 vs 3.35 ±0.42, p<0.01, respectively). LPS induced significant apoptosis (AI 43.8%±11.3), and increased expression of cleaved caspase-3 in epithelial and inflammatory cells (58.7% ±18.6). Consistently to attenuated lung inflammation, simvastatin dose dependently decreased AI (vs LPS, p <0.01, respectively), and cleaved caspase-3 expression of epithelial cells (vs LPS, p <0.05, respectively) without effects on its expression in macrophages. In parallel, LPS induced antiapoptotic Bcl-XL (34.5% ±10.1) and survivin (61.5%±19.4) in epithelium as well as in inflammatory cells and macrophages, indicating cell protective mechanism. Simvastatin 40 mg/kg induced the most intensive BclXL and survivin expression in epithelialium vs LPS (vs LPS, p <0.01), with modest effects on survivin in macrophages. Further analysis showed strong positive correlation of activated NF-κB (brown nuclear staining) and survivin expression in epithelial cells, but negatively correlated with cleaved caspase-3, indicating potential mechanism of simvastatin in cell survival.
Conclusion: Results showed that simvastatin attenuated inflammation induced ALI, preventing apoptosis and partly by activating epithelial cell survival via survivin/NF-κB signalling pathway.

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