The protective effects of bone morphogenetic protein-7 against epithelial injury and matrix metalloproteases upregulation induced by silica in vitro
The protective effects of bone morphogenetic protein-7 against epithelial injury and matrix metalloproteases upregulation induced by silica in vitro
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DOI：
10.1177/0960327116674527
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发表时间：
2017-09-01
影响因子：
2.8
通讯作者：
Tian, L.
中科院分区：
文献类型：
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作者：
Liang, D.;An, G.;Tian, L.
目的：我们在体外研究骨形态发生蛋白-7（BMP-7）对二氧化硅诱导和巨噬细胞介导的纤维化模型的影响及其可能机制。方法：将大鼠肺泡II上皮（RLE-6TN）细胞与小鼠巨噬细胞样细胞（RAW264.7）上清液一起孵育，并用0、25、50和100 g/mL二氧化硅处理。使用蛋白质印迹法检测上皮标记物（表面活性蛋白-C 和 E-钙粘蛋白）和间充质标记物（纤连蛋白（FN）和波形蛋白（Vim））。中和BMP-7后，通过羟脯氨酸（Hyp）含量和胶原蛋白I、III蛋白水平以及Smad信号通路蛋白，包括磷酸化Smad1/5（P-Smad1/5）和磷酸化Smad2/3（P-Smad2/3）来评估纤维化的进展。 I 型胶原蛋白也通过免疫荧光进行鉴定，并用 SB-431542、LDN-193189 或抗 BMP-7 中和抗体进行预处理。另外，采用Western blotting检测基质金属蛋白酶2（MMP-2）和MMP-9的水平。结果：RLE-6TN细胞模型建立成功，Vim、FN、MMP-2和MMP-9表达上调，同时二氧化硅浓度增加。中和 BMP-7 可刺激 P-Smad1/5 的减少和 P-Smad2/3 的增加，以及通过 Smad 信号通路刺激 I 型胶原、III 型胶原、FN 和 Hyp 的增加。此外，用LDN-193189或抗BMP-7中和抗体预处理，I型胶原蛋白的表达增加，但在SB-431542干预下降低。结论：激活的BMP/Smad和抑制的转化生长因子/Smad途径可以通过MMP依赖性机制抑制二氧化硅诱导的纤维化。 BMP-7有望成为延迟间质变化的优化策略。
Objective: We investigate the effects of bone morphogenetic protein-7 (BMP-7) on models with silica-induced and macrophage-mediated fibrosis and its possible mechanisms in vitro.Methods: Rat alveolar II epithelial (RLE-6TN) cells were incubated with the supernatant of mouse macrophage-like cells (RAW264.7) and treated with 0, 25, 50, and 100 g/mL silica. Using Western blotting, the epithelial markers (surfactant proteins-C and E-cadherin) and the mesenchymal markers (fibronectin (FN) and viminten (Vim)) were detected. After neutralizing the BMP-7, the progress of fibrosis was assessed by the content of hydroxyproline (Hyp) and collagen I, III protein levels as well as the Smad signaling pathway proteins, including phosphorylated Smad1/5(P-Smad1/5) and phosphorylated Smad2/3(P-Smad2/3). Collagen I was also identified by immunofluorescence and pretreated with SB-431542, LDN-193189, or anti-BMP-7-neutralizing antibody. In addition, the levels of matrix metalloproteinase-2 (MMP-2) and MMP-9 were detected using Western blotting.Results: The model of RLE-6TN cells was established successfully, the expressions of Vim, FN, MMP-2, and MMP-9 were upregulated, while the concentration of silica is increased. Neutralizing BMP-7 stimulated the decrease of P-Smad1/5 and the increase of P-Smad2/3, as well as the collagen I, collagen III, FN, and Hyp via Smad signaling pathway. Furthermore, pretreated with LDN-193189 or anti-BMP-7-neutralizing antibody, the expression of collagen I was increased, yet it was decreased with SB-431542 intervention.Conclusion: The activated BMP/Smad and suppressed transforming growth factor-/Smad pathways could suppress silica-induced fibrosis via a MMP-dependent mechanism. BMP-7 is expected to be the optimized strategy of delaying the interstitial changes.