*3.5. 12-LO Is Also Necessary for the Production of G-CSF and Contributes to Neutrophilic Airway Inflammation*

Since 12-LO is an enzyme that produces 12(*S*)-HETE, which is a ligand for BLT2, we examined whether 12-LO activity was also necessary for the production of G-CSF. The levels of 12(*S*)-HETE in BALF were suppressed by the administration of the 12-LO inhibitor, baicalein (Figure 5A). Histopathological analysis showed that inflammation and mucus secretion in lung tissue were suppressed by baicalein treatment (Figure 5B,C). Under these experimental conditions, the protein level of 12-LO was also suppressed in lung tissue by baicalein (Figure 5D). Clearly, G-CSF, as well as MPO levels in BALF increased by LPS/OVA treatment, were markedly suppressed by baicalein administration (Figure 5E,F). The number of total cells and neutrophils in BALF was also significantly reduced by baicalein treatment (Figure 5G). IF staining also showed that the levels of G-CSF, MPO, and BLT2 in lung tissue were markedly decreased by baicalein treatment (Figure 5H,I). Taken together, these results showed that 12-LO was necessary for the production of G-CSF, thus contributing to neutrophilic airway inflammation.

**Figure 5.** 12-LO is also necessary for the production of G-CSF and contributes to neutrophilic airway inflammation. The 12-LO inhibitor baicalein (20 mg/kg) or DMSO was administered by i.p. injection 1 h before each challenge. The negative controls (NC) were untreated. (**A**) 12(*S*)-HETE levels in BALF were analyzed by ELISA. (**B**,**C**) H&E and PAS staining of mice lung tissues. Perivascular and peribronchial inflammation in addition to mucus secretion, was examined and scored (400×). Inflammation scores are shown as the mean ± SD (*n* = 5 per group). (**D**) Mouse lung tissue was homogenized, and the protein was isolated to assess the level of 12-LO by Western blotting. (**E**,**F**) G-CSF and MPO levels in BALF were analyzed by ELISA. (**G**) The number of immune cells in BALF was estimated using a CytoSpin and staining with Diff-Quik. The results are shown as the mean ± SD (*n* = 5 per group). (**H**,**I**) IF staining of mice lung tissue for BLT2 (green, Alexa Flour 488), G-CSF (red, Alexa Flour 647), and MPO (magenta, Alexa Flour 594). Nuclei were counterstained with DAPI

(blue; 400×). The IF images are representatives of three independent trials with similar results. All experiments were performed in triplicate. \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001, \*\*\*\* *p* < 0.0001 versus each control group.
