Ce. *p,0.05 compared with the Silica group (D30). doi:10.1371/journal.pone.0055827.gApoA1 Attenuated Silica Induced Lung FibrosisFigure 6. Quantification of silica-induced apoptosis in the lungs of the ApoA1 transgenic mice. (A) Immunoblot analysis showed that active form of caspase-3 protein expression was significantly decreased in the ApoA1_D7 and D15 groups compared with the Silica group. **p,0.01 compared with the Silica group (D30). *p,0.05 compared with the Silica group (D30). (B) Tissues stained by the TUNEL method were observed at 6100 magnification, and the number of TUNEL-positive cells in a minimum of 20 fields per lung was counted (n = 8 mice/group). **p,0.01 compared with the Silica group (D15 and D30). doi:10.1371/journal.pone.0055827.gtreatment with doxycycline contributed to the anti-Tubastatin-A price fibrotic effect on ApoA1 transgenic mice, silica was administered intratracheally to the UBC-GFP transgenic mice with or without doxycyclinecontaining water. The histologic findings showed severe peribronchial nodules, GNF-7 site inflammatory cell accumulation, and interstitial edema in both the silica-exposed doxycycline mice and those fed drinking water that did not contain doxycycline (Figure S3A). The total numbers of inflammatory cells, macrophages, neutrophils, and lymphocytes in the BAL fluid were not different between the two groups (Figure S3B). Silica induced increased amounts of lung-soluble collagen, and the granuloma fractions were not decreased by doxycycline treatment (Figure S3C, D).DiscussionThe results of the present study show that the overexpression of hApoA1 in a murine silicosis model, which histologically mimics human silicosis, reduced the area occupied by silicotic nodules, the number of inflammatory cells, and the presence of collagen. The beneficial effect of ApoA1 overexpression was associated with decreases in silica-induced active form of TGF-b1 synthesis and apoptotic activity and an increase in endogenous LXA4 synthesis.The silica treatment increased the number of neutrophils and macrophages in the BAL fluid, which may be the source of the TGF-b1 and other inflammatory mediators. Silica has been shown to induce apoptosis in alveolar macrophages, and this may play an important role in the pathogenesis of silicosis [22,23]. Our findings showing a decrease in caspase-3 protein expression and the number of TUNEL-positive cells suggest that ApoA1 inhibited silica-induced apoptosis in the lung. In a previous study, we reported that local treatment with ApoA1 was effective against the development of bleomycin-induced lung 1516647 injury/fibrosis [5]. However, the ability of intratracheal bleomycin to induce experimental lung fibrosis has been reported to be self-limiting after 28 days [6,8]. Moreover, at 14 days after intratracheal bleomycin administration, when the mice were studied, the induced model might have more closely resembled lung injury than fibrosis. In contrast, because silica is not readily cleared from the lung, the pro-fibrotic stimulus is more persistent and fibrosis is easily identified as fibrotic nodules in areas of silica deposition. Studies using mouse models have shown that the initial inflammatory reaction occurs within the first seven days after crystalline silica delivery via the intratracheal route [24,25]. SomeApoA1 Attenuated Silica Induced 
Lung FibrosisFigure 7. Levels of proinflammatory mediator mRNAs in the lungs of ApoA1 transgenic mice. IL-1b (A), TNF-a (B), KC (C), MCP-1 (D) and MIP-2 (E) mRNA expres.Ce. *p,0.05 compared with the Silica group (D30). doi:10.1371/journal.pone.0055827.gApoA1 Attenuated Silica Induced Lung FibrosisFigure 6. Quantification of silica-induced apoptosis in the lungs of the ApoA1 transgenic mice. (A) Immunoblot analysis showed that active form of caspase-3 protein expression was significantly decreased in the ApoA1_D7 and D15 groups compared with the Silica group. **p,0.01 compared with the Silica group (D30). *p,0.05 compared with the Silica group (D30). (B) Tissues stained by the TUNEL method were observed at 6100 magnification, and the number of TUNEL-positive cells in a minimum of 20 fields per lung was counted (n = 8 mice/group). **p,0.01 compared with the Silica group (D15 and D30). doi:10.1371/journal.pone.0055827.gtreatment with doxycycline contributed to the anti-fibrotic effect on ApoA1 transgenic mice, silica was administered intratracheally to the UBC-GFP transgenic mice with or without doxycyclinecontaining water. The histologic findings showed severe peribronchial nodules, inflammatory cell accumulation, and interstitial edema in both the silica-exposed doxycycline mice and those fed drinking water that did not contain doxycycline (Figure S3A). The total numbers of inflammatory cells, macrophages, neutrophils, and lymphocytes in the BAL fluid were not different between the two groups (Figure S3B). Silica induced increased amounts of lung-soluble collagen, and the granuloma fractions were not decreased by doxycycline treatment (Figure S3C, D).DiscussionThe results of the present study show that the overexpression of hApoA1 in a murine silicosis model, which histologically mimics human silicosis, reduced the area occupied by silicotic nodules, the number of inflammatory cells, and the presence of collagen. The beneficial effect of ApoA1 overexpression was associated with decreases in silica-induced active form of TGF-b1 synthesis and apoptotic activity and an increase in endogenous LXA4 synthesis.The silica treatment increased the number of neutrophils and macrophages in the BAL fluid, which may be the source of the TGF-b1 and other inflammatory mediators. Silica has been shown to induce apoptosis in alveolar macrophages, and this may play an important role in the pathogenesis of silicosis [22,23]. Our findings showing a decrease in caspase-3 protein expression and the number of TUNEL-positive cells suggest that ApoA1 inhibited silica-induced apoptosis in the lung. In a previous study, we reported that local treatment with ApoA1 was effective against the development of bleomycin-induced 
lung 1516647 injury/fibrosis [5]. However, the ability of intratracheal bleomycin to induce experimental lung fibrosis has been reported to be self-limiting after 28 days [6,8]. Moreover, at 14 days after intratracheal bleomycin administration, when the mice were studied, the induced model might have more closely resembled lung injury than fibrosis. In contrast, because silica is not readily cleared from the lung, the pro-fibrotic stimulus is more persistent and fibrosis is easily identified as fibrotic nodules in areas of silica deposition. Studies using mouse models have shown that the initial inflammatory reaction occurs within the first seven days after crystalline silica delivery via the intratracheal route [24,25]. SomeApoA1 Attenuated Silica Induced Lung FibrosisFigure 7. Levels of proinflammatory mediator mRNAs in the lungs of ApoA1 transgenic mice. IL-1b (A), TNF-a (B), KC (C), MCP-1 (D) and MIP-2 (E) mRNA expres.
