Di Liang1, Yan Wang1, Zhonghui Zhu1, Gengxia Yang2, Guoliang An1, Xiaoli Li1, Piye Niu1, Li Chen1, Lin Tian3. 1. School of Public Health, Capital Medical University, Beijing 100069, China; Beijing Key Laboratory of Environmental Toxicology, Capital Medical University, Beijing 100069, China. 2. Oncology Minimally Invasive Interventional Center, Beijing Youan Hospital, Capital Medical University, Beijing 100069, China. 3. School of Public Health, Capital Medical University, Beijing 100069, China; Beijing Key Laboratory of Environmental Toxicology, Capital Medical University, Beijing 100069, China. Electronic address: tian_lin@163.com.
Abstract
OBJECTIVE: To investigate the anti-fibrotic effects and possible mechanisms of bone morphogenetic protein-7 (BMP-7) on silica induced fibrosis in RLE-6TN cells, and compare the preventive treatment of experimental silicosis with BMP-7 with therapeutic treatment of silicosis in vitro models. METHODS: RLE-6TN cells were incubated with the supernatant of RAW264.7, treated by 50 μg/mL silica in either presence or absence of BMP-7 in different phases. Morphological changes and the cellular wound-healing assays were used to evaluate the process of EMT. By using Western Blotting, the epithelial marker E-cadherin (E-cad), and the mesenchymal markers Vimentin (Vim), Snail, and fibronectin (FN) were detected as well as the Smad signaling pathway proteins, including phosphorylated Smad1/5(P-Smad1/5), phosphorylated Smad2/3(P-Smad2/3), and non-phosphorylated Smad1, Smad8, and Smad2. The progress of fibrosis was assessed by the content of hydroxyproline (Hyp) and collagen I and III protein levels. In addition, MTT assay was used to explore the toxic effects of silica as well as BMP-7. RESULTS: The EMT model of RLE-6TN cells was established successfully, the cells had a fibroblast-like morphology with increasing migration activity. The expressions of Vim, Snail, FN, collagen I and collagen III were up-regulated with the increase of silica concentration. BMP-7 could attenuate the decrease of P-Smad1/5 and the increase of P-Smad2/3, collagen I, collagen III, and FN via Smad signaling pathway. BMP-7 inhibited the mesenchymal-like responses in RLE-6TN cells, including cell migration, expression of fibrosis markers, and secretion of Hyp. Furthermore, the anti-fibrotic effects in the prevention group were more effective than treatment group. CONCLUSION: The restoration of BMP signaling with BMP-7 is associated with inhibiting silica-induced fibrosis through the mechanisms of activated BMP-7/Smad and suppressed TGF-β/Smad pathways. Preventive treatment of pulmonary fibrosis progression with BMP-7 may expect to be the optimized strategy than therapeutic therapy of fibrosis.
OBJECTIVE: To investigate the anti-fibrotic effects and possible mechanisms of bone morphogenetic protein-7 (BMP-7) on silica induced fibrosis in RLE-6TN cells, and compare the preventive treatment of experimental silicosis with BMP-7 with therapeutic treatment of silicosis in vitro models. METHODS: RLE-6TN cells were incubated with the supernatant of RAW264.7, treated by 50 μg/mL silica in either presence or absence of BMP-7 in different phases. Morphological changes and the cellular wound-healing assays were used to evaluate the process of EMT. By using Western Blotting, the epithelial marker E-cadherin (E-cad), and the mesenchymal markers Vimentin (Vim), Snail, and fibronectin (FN) were detected as well as the Smad signaling pathway proteins, including phosphorylated Smad1/5(P-Smad1/5), phosphorylated Smad2/3(P-Smad2/3), and non-phosphorylated Smad1, Smad8, and Smad2. The progress of fibrosis was assessed by the content of hydroxyproline (Hyp) and collagen I and III protein levels. In addition, MTT assay was used to explore the toxic effects of silica as well as BMP-7. RESULTS: The EMT model of RLE-6TN cells was established successfully, the cells had a fibroblast-like morphology with increasing migration activity. The expressions of Vim, Snail, FN, collagen I and collagen III were up-regulated with the increase of silica concentration. BMP-7 could attenuate the decrease of P-Smad1/5 and the increase of P-Smad2/3, collagen I, collagen III, and FN via Smad signaling pathway. BMP-7 inhibited the mesenchymal-like responses in RLE-6TN cells, including cell migration, expression of fibrosis markers, and secretion of Hyp. Furthermore, the anti-fibrotic effects in the prevention group were more effective than treatment group. CONCLUSION: The restoration of BMP signaling with BMP-7 is associated with inhibiting silica-induced fibrosis through the mechanisms of activated BMP-7/Smad and suppressed TGF-β/Smad pathways. Preventive treatment of pulmonary fibrosis progression with BMP-7 may expect to be the optimized strategy than therapeutic therapy of fibrosis.