Ansgenic mice that were not treated with doxycycline and wild-type mice (Fig. 1B). Furthermore, there was no difference in ApoA1 expression (-)-Calyculin A site between the transgenic mice not treated with doxycycline and wild-type mice (Fig. 1B). hApoA1 was detected in the BAL fluid of the doxycycline-treated transgenic mice, but not inApoA1 Attenuated Silica Induced Lung FibrosisApoA1 Attenuated Silica Induced Lung FibrosisFigure 4. Quantification of the lung collagen and TGF levels in the ApoA1 transgenic mice. (A) Masson’s trichrome staining of lung sections. Scale bar = 20 mm. (B) Soluble lung collagen was measured using a Sircol assay. **p,0.01 compared with the Silica group 12926553 (D15 and D30). (C) The level of the active TGF-b1 in the lung was measured by ELISA. **p,0.01 compared with the Silica group (D15 and D30). doi:10.1371/journal.pone.0055827.gEffect of ApoA1 Overexpression on Collagen and TGF-b1 in the LungMasson’s trichrome staining revealed a decrease in collagen deposition in the lungs of the ApoA1_D7 and D15 mice compared with that in the Silica group (Fig. 4A). Lung-soluble collagen was also significantly reduced in the lungs of both groups compared with the Silica group (Fig. 4B). The level of the active form of TGF-b1 in the lung was significantly increased following treatment with silica, but was significantly decreased in the ApoA1_D7 and D15 groups (Fig. 4C).form of caspase-3 protein in the ApoA1_D7 and D15 groups were significantly decreased compared with that in the Silica group (Fig. 6A). Similarly, a TUNEL assay revealed approximately 75 fewer apoptotic cells in the lungs of the ApoA1-overexpressing mice compared with the lungs of the Silica group mice (Fig. 6B). Double-labeled immunofluorescence demonstrated that the majority of apoptotic cells were alveolar epithelial cells and macrophages (Figure S2).Effect of ApoA1 on Silica-induced Increased Proinflammatory Mediators in Mouse LungTo determine whether ApoA1 inhibits the increase 
of the proinflammatory cytokines and chemokines that accumulate from macrophages and neutrophils stimulated by silica, mRNA levels of interleukin-1?(IL-1?, tumor necrosis factor-a (TNF-a), monocyte chemotactic protein-1 (MCP-1), macrophage inflammatory protein-2 (MIP-2) and chemokine (C-X-C motif) ligand 1 (CXCL1, synonym KC) in the lungs were measured. All of the measured levels of proinflammatory 1418741-86-2 Mediator mRNAs in the ApoA1_D7 and D15 group were significantly decreased compared with those 1516647 in the Silica group (Fig. 7).Effect of ApoA1 Overexpression on Lipid Mediator of InflammationGiven the important role of lipid mediators in the initiation, maintenance, and resolution of inflammation, the levels of antiinflammatory LXA4 were measured in the lungs of silica-treated ApoA1_D7 and D15 mice. ApoA1 overexpression was associated with increased levels of LXA4 in the lung parenchyma and BAL fluid (Fig. 5).Effect of ApoA1 on Silica-induced Apoptosis in Mouse LungTo determine whether ApoA1 inhibited silica-induced apoptosis, we measured the levels of caspase-3 expression in the lungs. Levels of caspase-3 protein were increased in the Silica group compared with the sham-exposed controls. The levels of activeEffect of Doxycycline on Silica-induced Lung Inflammation and Fibrosis in Mouse LungDoxycycline reportedly has an anti-fibrotic effect on bleomycininduced experimental fibrosis [21]. To determine whetherFigure 5. Quantification of LXA4 levels in the lung parenchyma(A) and BAL fluid(B) of the ApoA1 transgenic mi.Ansgenic mice that were not treated with doxycycline and wild-type mice (Fig. 1B). Furthermore, there was no difference in ApoA1 expression between the transgenic mice 
not treated with doxycycline and wild-type mice (Fig. 1B). hApoA1 was detected in the BAL fluid of the doxycycline-treated transgenic mice, but not inApoA1 Attenuated Silica Induced Lung FibrosisApoA1 Attenuated Silica Induced Lung FibrosisFigure 4. Quantification of the lung collagen and TGF levels in the ApoA1 transgenic mice. (A) Masson’s trichrome staining of lung sections. Scale bar = 20 mm. (B) Soluble lung collagen was measured using a Sircol assay. **p,0.01 compared with the Silica group 12926553 (D15 and D30). (C) The level of the active TGF-b1 in the lung was measured by ELISA. **p,0.01 compared with the Silica group (D15 and D30). doi:10.1371/journal.pone.0055827.gEffect of ApoA1 Overexpression on Collagen and TGF-b1 in the LungMasson’s trichrome staining revealed a decrease in collagen deposition in the lungs of the ApoA1_D7 and D15 mice compared with that in the Silica group (Fig. 4A). Lung-soluble collagen was also significantly reduced in the lungs of both groups compared with the Silica group (Fig. 4B). The level of the active form of TGF-b1 in the lung was significantly increased following treatment with silica, but was significantly decreased in the ApoA1_D7 and D15 groups (Fig. 4C).form of caspase-3 protein in the ApoA1_D7 and D15 groups were significantly decreased compared with that in the Silica group (Fig. 6A). Similarly, a TUNEL assay revealed approximately 75 fewer apoptotic cells in the lungs of the ApoA1-overexpressing mice compared with the lungs of the Silica group mice (Fig. 6B). Double-labeled immunofluorescence demonstrated that the majority of apoptotic cells were alveolar epithelial cells and macrophages (Figure S2).Effect of ApoA1 on Silica-induced Increased Proinflammatory Mediators in Mouse LungTo determine whether ApoA1 inhibits the increase of the proinflammatory cytokines and chemokines that accumulate from macrophages and neutrophils stimulated by silica, mRNA levels of interleukin-1?(IL-1?, tumor necrosis factor-a (TNF-a), monocyte chemotactic protein-1 (MCP-1), macrophage inflammatory protein-2 (MIP-2) and chemokine (C-X-C motif) ligand 1 (CXCL1, synonym KC) in the lungs were measured. All of the measured levels of proinflammatory mediator mRNAs in the ApoA1_D7 and D15 group were significantly decreased compared with those 1516647 in the Silica group (Fig. 7).Effect of ApoA1 Overexpression on Lipid Mediator of InflammationGiven the important role of lipid mediators in the initiation, maintenance, and resolution of inflammation, the levels of antiinflammatory LXA4 were measured in the lungs of silica-treated ApoA1_D7 and D15 mice. ApoA1 overexpression was associated with increased levels of LXA4 in the lung parenchyma and BAL fluid (Fig. 5).Effect of ApoA1 on Silica-induced Apoptosis in Mouse LungTo determine whether ApoA1 inhibited silica-induced apoptosis, we measured the levels of caspase-3 expression in the lungs. Levels of caspase-3 protein were increased in the Silica group compared with the sham-exposed controls. The levels of activeEffect of Doxycycline on Silica-induced Lung Inflammation and Fibrosis in Mouse LungDoxycycline reportedly has an anti-fibrotic effect on bleomycininduced experimental fibrosis [21]. To determine whetherFigure 5. Quantification of LXA4 levels in the lung parenchyma(A) and BAL fluid(B) of the ApoA1 transgenic mi.
