Attenuation of Lipopolysaccharide-Induced Acute Lung Injury by Cyclosporine-A via Suppression of Mitochondrial DNA

Abstract

BACKGROUND Lipopolysaccharide (LPS) is generally associated with sepsis, which causes multiple system injuries and systemic inflammatory response. Mitochondrial DNA (mtDNA) is of great importance in mediation of inflammation. The aim of this study was to investigate the protective profiles of Cyclosporine-A (CsA) in LPS-induced acute lung injury (ALI) and systemic inflammation by the inhibition of mtDNA and Toll-like receptor. MATERIAL AND METHODS Twenty-four C57BL/6 mice were randomly assigned to 4 groups: a sham group (n=6); an experiment group (ALI induced through intraperitoneal injection of 10 mg/ml LPS, n=6); a low-CsA group (injection of 2.5 mg/kg of CsA 15 min after injection of LPS, n=6); and a high-CsA group (injection of 25 mg/kg of CsA 15 min after injection of LPS, n=6). Lung tissue, bronchoalveolar lavage fluid (BALF), and blood samples were collected at 6 h for further analyses. RESULTS CsA treatment significantly attenuated LPS-induced lung histopathological changes (P<.05), myeloperoxidase (MPO) activity (P<.05) and lung wet-to-dry weight ratio (P<.05). In addition, injection of CsA decreased total cells (P<.05), neutrophils (P<.05), and total protein (P<.05) in BALF and inflammatory mediators, including tumor necrosis factor-a (TNF-a, P<.05) and interleukin-6 (IL-6, P<.05) in a dose-dependent manner. A significant decrease in mtDNA was observed in the CsA group when compared with controls (P<.05). Furthermore, we demonstrated that there was a significant difference between the high-CsA group and low-CsA group in lung injury score (P<.05), mtDNA (P<.05), and MPO (P<.05). CONCLUSIONS The evidence from this study suggests that CsA attenuated lung inflammation after LPS injection, and the protective mechanism may at least in part involve decreasing the release of inflammatory cytokines and mtDNA.

Figure 1

CsA Attenuates the Lung Wet-to-dry (W/D) Ratio. The lung wet-to-dry (W/D) ratio increase reflects pulmonary edema. CsA Significantly Attenuates the Lung Wet-to-dry Ratio. * P<0.05 vs. Control group, ^# P<0.05 vs. Experiment group. N=6.

Figure 2

CsA Attenuates the LPS-Induced Myeloperoxidase. MPO activity was evaluated to indicate polymorphonuclear leukocyte accumulation in the lung. CsA significantly decreased the LPS-induced myeloperoxidase and there was a significant difference between the high- and low-CsA groups. * P<0.05 vs. Control group, ^# P<0.05 vs Experiment group, ^@ P<0.05 vs. Low-CsA group. N=6.

Figure 3

CsA Attenuates the LPS-Induced Lung Injury. (A–D) Hematoxylin and eosin-stained images of lung tissues; (E) The lung injury score system was used to evaluate the severity of the lung injury. CsA significantly attenuated the LPS-induced lung injury score. * P<0.05 vs. Control group, ^# P<0.05 vs. Experiment group, ^@ P<0.05 vs. Low-CsA group. N=6.

Figure 4

CsA Decreased the LPS-Induced Induction of Inflammatory Cells and Total Proteins. (A–C) The total numbers of cell/neutrophils and the total protein were all significantly higher in the experiment group than in the control group. CsA significantly decreased the total number of cell/neutrophils in a dose-dependent manner. * P<0.05 vs. Control group, ^# P<0.05 vs. Experiment group, ^@ P<0.05 vs. Low-CsA group. N=6.

Figure 5

CsA Attenuates the LPS-Induced Mitochondrial DNA and Inflammatory Cytokines. (A–C) Plasma interleukin-6, tumor necrosis factor-α, and mitochondrial DNA levels were significant higher in the experiment group than in the control group. CsA significantly attenuated the LPS-induced TNF-α, IL-6, and mitochondrial DNA in a dose-dependent manner. * P<0.05 vs. Control group, ^# P<0.05 vs. Experiment group, ^@ P<0.05 vs. Low-CsA group. N=6.