Figure 6

Pulmonary inflammation triggered by necrotic cells and the release of mitochondria. (A, B) Primary lung cells were either treated with DOTAP liposomes (50 μg/ml), PEI (10 μg/ml) and Chitosan (50 μg/ml) for 2 h to induce necrosis or treated with freeze-thaw process. Mitochondria were extracted from lungs of C57BL/6 mice. Treated cells and extracted mitochondria were injected into C57BL/6 mice (cells: 10⁶, mitochondria: 200 μg/mouse) for 24 h of exposure. HE staining of representative mouse lung sections was performed and the numbers of esterase-positive neutrophils in ten HPFs were counted. Scale bar, 50 μm. (C) Cells stained with Mito-tracker Green (mitochondria; green) and Hoechst 33342 (nucleus; blue) were treated with cationic carriers for 30 min at the concentrations above and observed for leakage of mitochondria. Scale bar, 25 μm. (D) Mice were injected with DOTAP liposomes (25 mg/kg), PEI (5 mg/kg) or Chitosan (25 mg/kg) and the mtDNA in serum was determined after 4 h of exposure by qPCR. n = 3 per group. (E) Primary lung cells were treated with cationic carriers at the concentration above for 4 h and supernatant was collected and concentrated for mtDNA determination. Data are mean ± SEM; n = 3.^*P< 0.05,^**P< 0.01,^***P <0.001 compared with control group by Student's t-test.