Male Sprague-Dawley rats were purchased from Sasco Company (Sasco Korea, pathogen free, 250~300 g). Goat anti-human cPLA₂ polyclonal antibody and anti- COX-II antibody were purchased from Santa Cruz Biochem (Santa Cruz, CA, USA). Anti-TNF-α-polyclonal antibody was obtained from Cell Signaling (Danvers, MA, USA). L-α-dipalmitoyl-[2-palmitoyl-9,10-³H(N)-phosphatidylcholine was obtained from Dupont NEN Research Products (Boston, MA, USA). Moxifloxacin hydrochloride was graciously provided by Bayer (Bayer Leverkusen, Germany). Not otherwise mentioned, all other reagents were purchased from Sigma Aldrich Company (St. Louise, MO, USA).

Animals were fed ad libitum standard chow and water till 24 hours before experiments. ALI was induced in rats by being given oleic acid intravenously (30µL of oleic acid with 270µL of 10% bovine serum albumin) as previously described11,14.

Rats were divided into three treatment groups

1) Sham group: rats were given 300µL of 10% bovine serum albumin intravenously via femoral vein; 2) Oleic acid control group (OA group): rats were given 30µL of oleic acid with 270µL of 10% bovine serum albumin intravenously; 3) Moxifloxacin group (MO group): rats were given moxifloxacin hydrochloride (100 mg/kg) with 30µL of oleic acid in 270µL of 10% bovine serum albumin. Moxifloxacin hydrochloride was given intraperitoneally just after the injection of oleic acid.

BAL was performed 5 hours after oleic acid injection. The rats were anesthetized with enflurane and tracheotomized and cannulated. BAL was performed with 8.0 mL of PBS for each rat and approximately 6.0 mL of BAL was recovered. BAL fluid was collected into 10 mL sterile tubes, centrifuged to collect cell free supernatant and stored at -20℃ for protein assay. The sediments were resuspended with PBS for counting of leucocytes.

Determination of BAL protein was performed as previously described by Brown et al.15 with modification. Stored BAL supernatant was used for protein assay.

TenµL of resuspended sediment solution was applied on hemocytometer and leukocytes were counted. Around 100µL of BAL suspension was cytospinned and slides were prepared before Wright staining. Percentage of neutrophils was calculated by differential count and total numbers of neutrophils were calculated.

The MPO activity (U/g of wet lung) was determined to estimate the accumulation of neutrophils in the lung. The assay was performed as described by Goldblum et al.16 with modification. Lungs were weighed and homogenized with Polytron homogenizer (Polytron, Switzerland) in 4.0 mL of 20 mM PBS at pH 7.4. An aliquot was mixed with an equal volume of 1% hexadecytrimethyl ammonium bromide in PBS and rehomogenized for 90 s in ice cold water. Then it was centrifuged 15,000 g for 5 minutes at 4℃. The supernatant (0.1 mL) was added to 2.9 mL of PBS (50 mM potassium phosphate, pH 6.0) containing 0.0005% H₂O₂ and 0.168 g/L of o-dianisidine. The samples were analyzed for change in absorbance on spectrophtometer at 460 nm over 3 minutes. MPO activity was expressed as U/g of wet lung by multiplying 13.5 to the changes of absorbance/min/g of wet weight tissue.

TBARS assay was carried out as described previously17 to measure lipid peroxidation as a marker of ALI. The same supernatant used for MPO assay was used. Aliquots of supernatant were mixed with equal volume of 3.5% sodium dodecyl sulfate (SDS) and 0.13 mL of 100% acetic acid in 15 mL-glass centrifuge tubes. The pH was adjusted to 3.5 with 1 N NaOH. Five mL of 0.6% thiobarbituric acid was added to each tube and they were heated in boiling water bath at 95℃ for 30 minutes. After cooling, 1.5 mL of n-butanol/pyrimidine (15:1) mixture was added to each tube and vortexed vigrously for 30 s followed by centrifugation at 4,000 rpm for 10 minutes. The absorbance of top layer of each sample was measured by spectrophotometer at 525 nm.

cPLA₂ activity was determined by the hydrolysis of radioactively labelled phospholipid (³H-dipalmitoylphosphatidylcholine, ³H-DPPC) as previously described by Katsumata et al.18 with modification. Lung tissues were homogenized in a polytron in ice-cold buffer (1:5, wt/vol) containing Tris-HCl (pH 7.6), EDTA (1 mM), EGTA (1 mM) and DTT (5 mM) to abolish sPLA₂ activity. The phospholipid substrate solution (pH 9.0) was composed of ³H-DPPC, lecithin (0.1 mM), glycine (100 mM), deoxycholate (2.5 mM), BSA (10 g/L) and 1.75 mM ethanol. The reaction was initiated by the addition of 100µL of lung homogenate to the phospholipid substrate solution and incubated at 37℃ in a shaking water bath for 60 minutes. Crotalus adamanteus PLA₂ was used as a positive control. The reaction was stopped by adding 200µL of 5% Triton X-100 containing 200 mM EDTA and 5 g of anhydrous Na₂SO₄. For the extraction of hydrolysis product, 5.0 mL of 0.1% glacial acetic acid-hexane was added to each sample. The extract was scanned with β-scintillation counter. 1 unit of PLA₂ activity was defined as 1µM production of fatty acid per minute.

Lungs were fixed by inflation with paraformaldehyde (pH 7.4, 4%) at pressure of 20 cm H₂O and sliced before putting into paraformaldehyde, then degassing and fixed. Tissue slices were stained with hematoxylin-eosin for examination of lung histology.

Microtomer sections were subjected to staining for fluorescence and diaminobenzidine (DAB) for cPLA₂ in rat lung tissues. Goat anti-human-cPLA₂ polyclonal was used as first antibody. The antibody complex was visualized by propidium iodide (fluorescense) and DAB staining.

Proteins of TNFα and COX-2 were identified in homogenized lung tissue (100µg of protein) using standard western blotting. Specific polyclonal antibodies, anti-TNF and anti-COX-II were diluted 1:1,000 (Tris buffer, Saline Tween 20 [TBST] buffer with 0.1% BSA).

Data were expressed as mean±standard error (SE). Data were analyzed using a one-way analysis of variance with Tukey-Kramer multiple comparisons test. A p-value of <0.05 was considered statistically significant.

The assay of BAL protein revealed the protective effect of moxifloxacin on lung leak in rats. As shown in Table 1, OA had increased the protein content (mg/two lungs) in BAL of rats given OA, as compared with that of sham-treated rats (p<0.001). Moxifloxacin had decreased the protein content in BAL of rats given OA (p<0.01).

As noted in Table 2, moxifloxacin reduced the accumulation of neutrophils in the interstitium of the lung and suppressed the migration of neutrophils into the alveoli of the OA group (p<0.001). As expected, the OA caused dramatic mobilization of neutrophils manifested by the drastic increase of lung MPO (p<0.001). The accumulation of neutrophils in the lung interstitium caused the migration of neutrophils into the alveoli as a process of alveolar flooding. Moxifloxacin was effective to decrease the effects of OA on the mobilization of neutrophils (p<0.001).

As an index of lung injury, the TBARS in the lung was measured. The MDA content (nmol/g of lung) was higher in the lungs of OA given rats compared with that of sham treated rats (p<0.001). The MDA content in the lungs of moxifloxacin given to rats was lower than that of the rats given OA reflecting the protective effect of moxifloxacin (p<0.001) (Table 3).

As shown in Table 4, the cPLA₂ activity was higher in the lungs of OA-treated rats compared with that of sham-treated rats (p<0.001). The cPLA₂ activity was lowered in the lung tissue by moxifloxacin in OA-treated rats (p<0.05).

While examining the histopathology of the lung of rats given OA, many phagocytes, mainly neutrophils, were observed. Perivascular cuffing and alveolar flooding were also observed, as these were the results of the OA injection (Figure 1A and B). Moxifloxacin ameliorated these inflammatory reactions dramatically, even if the remnants of migrated phagocytes, mainly alveolar monocyte/macrophages remained (Figure 1C). Along with these histopathological changes, the immunohistochemistry revealed the inhibitory effects of moxifloxacin on cPLA₂ in the lung. The DAB and fluorescent staining of cPLA₂ in the lung demonstrated the diminished expression of cPLA₂ by moxifloxacin (Figures 2, 3). Note the deep staining of cPLA₂ in the intra-alveolar neutrophils with DAB in the OA-treated rats. Interestingly, this phenomenon disappeared by moxifloxacin almost completely. In the lung tissue, the fluorescent staining of the alveolar septa was shown in OA-treated rats, and again, moxifloxacin suppressed this phenomenon (Figure 3).

The immunoprecipitation of TNFα and COX-2 was carried out to estimate the anti-inflammatory effects of moxifloxacin associated with the pro-inflammatory cytokine and rate-limiting enzyme of inflammation. The OA increased the expression of TNFa and COX-2, which was normalized by moxifloxacin (Figure 4).