Measurement of anti-oxidative effects of LVFX using EPR spectroscopy.

<p>The scavenging activities of LVFX against ROS derived from the UV/H₂O₂ system or neutrophils stimulated with 100 ng/ml PMA were determined using ESR spin trapping with DMPO. (A) The reaction mixtures, which contained 500 μM H₂O₂, 100 μM DTPA and 4.5 mM DMPO, were incubated with each FQ and were immediately transferred to an ESR flat cell and irradiated at 254 nm for 30 s. After UV-irradiation, the ESR flat cells immediately placed in a JES-TE 200 ESR spectrometer. ESR spectrum of DMPO spin adducts of OH radicals and (B) the quantitation of the OH radicals concentration were shown. (C) The scavenging activity of 200 μM FQs; lomefloxacin (LFLX), levofloxacin (LVFX), Ciprofloxacin (CPFX), pazfloxacin (PZFX), and norfloxacin (NRFX) against OH radicals. (D) ESR spectrum of DMPO spin adducts ROS derived from neutrophils containing OH radicals, and (E) the quantitation of the ROS concentration were shown. Each bar represents the mean ± SD (n = 3). *p<0.05, **p<0.01 vs control.</p

The effect of LVFX on oxidative stress in influenza virus-infected mice.

<p>The effect of LVFX on the accumulation of oxidative stress in the blood or lungs were determined (A) by measuring hydroperoxide levels (dROMs test) or (B) immunostaining for 8-OHdG (upper panel) and NO₂-Tyr (lower panel) level at the day 7 after influenza virus infection. The fluorescence intensity of (C) 8-OHdG and (D) NO₂-Tyr were quantified by the imageJ software. Each bar represents the mean ± SD (n = 5–9). **p<0.01, ***p<0.001 vs control.</p

The effect of LVFX on lung damage and viral titer in influenza virus-infected mice.

<p>(A) Section of lung tissue were prepared at the day 7 after influenza virus infection, and subjected to histopathological examination with HE staining (upper). The alveolar space was observed in lungs from control mice, however the influenza virus infection caused a marked increase in the infiltration of inflammatory cells into the alveoli or peribronchial areas, and the alveolar space was completely filled with these cells. Treatment with LVFX at a dose of 100 mg/kg decreased the overall infiltration of inflammatory cells. The histopathological severity of lung section was determined and average score showed in lower of HE staining image. Histological score shown PBS (n = 4), 25 mg/kg LVFX (n = 5) and 100 mg/kg LVFX (n = 5). (B) The effect of LVFX on viral load at day 4 and 7 were determined using a plaque forming assay. Each bar represents the mean ± SD (n = 3–5). *p<0.05, **p<0.01 vs PBS.</p

The effect of LVFX on NO and IFN-γ production in BALF of influenza virus-infected mice.

<p>(A) The effect of LVFX on the accumulation of NO in BALF was determined by measuring NOx (NO₂^- and NO₃^-). (B) IFN-γ levels in the BALF were determined by ELISA on day 7 after influenza virus administration. Each bar represents the mean ± SD (n = 4–5). *p<0.05, **p<0.01 vs control.</p

Therapeutic effect of LVFX on influenza virus-infected mice.

<p>Influenza virus infected mice were produced by the intratracheal administration of influenza virus under anesthesia on day 0. After infection, the mice were treated with LVFX on day 2 after infection by means of an intraperitoneal injection of LVFX. (A) Survival curves for control (n = 3), 100 mg/kg LVFX (not infected, n = 3), PBS (n = 10), 25 mg/kg LVFX (n = 10) and 100 mg/kg LVFX (n = 10) treatment during an influenza virus infection. (B) Weight loss during an influenza virus infection. Each bar represents the mean ± SD. **p<0.01 vs vehicle. Experiment was repeated three times.</p