骨成形蛋白7拮抗转化生长因子β1致肾小管上皮细胞细胞外基质分泌的作用

Antagonistic effect of bone morphogenetic protein-7 on transforming growth factor-β1 induced extracellular matrix synthesis

目的:观察骨成形蛋白7(BMP-7)拮抗转化生长因子β1(TGF-β1)诱导人肾小管上皮细胞(HK-2)细胞外基质(ECM)分泌作用,并对相关信号通路进行研究,对BMP-7抗纤维化机制进行深入探讨。方法:将不同浓度BMP-7预处理HK-2细胞,随后予以5ng/mlTGF-β1刺激,利用荧光实时定量PCR和Western印迹检测ECM表达、利用Western印迹和凝胶迁移滞后实验(EMSA)检测相关信号通路蛋白表达。结果:荧光实时定量PCR和Western印迹结果表明,BMP-7可以浓度依赖方式下调TGF-β1刺激后HK-2细胞I型胶原,III型胶原,纤维连接蛋白在mRNA和蛋白水平表达。TGF-β1可促进HK-2细胞Smad2、Smad3磷酸化。但TGF-β1上调的Smad2、Smad3磷酸化可被BMP-7抑制,差异有统计学意义(P<0.05和P<0.01)。EMSA结果表明,BMP-7则可抑制TGF-β1上调的NF-κB活性。结论:BMP-7可通过抑制Smad2/3磷酸化、抑制NF-κB的DNA结合活性达到阻断TGF-β1诱导HK-2分泌ECM的效应。

Objective：To investigate the molecular details in antifibrotic effects of bone morphogenetic protein-7 （BMP-7） on TGF-β1 induced extra-cellular matrix （ECM） production. Methodology：In human proximal tubular cells （HK-2）, the expressions of fibronectin, collagen type Ⅲ and collagen type Ⅰ were measured by real-time PCR and western blot. The expressions of Smads were examined by Western blot and NK-B activity was detected by electrophoretic mobility shift assay （EMSA）. Results：BMP-7 could antagonized TGF-β1 induced collagen type Ⅰ, collagen type Ⅲ and fibronectin mRNA and protein expression in a dose-dependent manner. The application of BMP-7 in the presence of TGF-β1 resulted in reduced Smad 2/3 phosphorylation. BMP-7 could also significantly down-regulated TGF-β1 induced NF-κB activity. Conclusion：BMP-7 could counteract TGF-β1 induced fibrogenesis. The inhibitory effects of BMP-7 on TGF-β1 induced ECM production could be mediated by inhibiting Smad 2/3 phosphorylation and NF-κB activity.