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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 observe the effect of BMP-7 on the secretion of extracellular matrix (ECM) of human renal tubular epithelial cells (HK-2) induced by transforming growth factor-β1 (TGF-β1) and the related signaling pathways, BMP-7 anti-fibrosis mechanism in-depth discussion. Methods: HK-2 cells were pretreated with different concentrations of BMP-7 and then stimulated with 5ng / ml TGF-β1. The expression of ECM was detected by real-time PCR and Western blotting. Western blot and EMSA were used to detect the correlation Signal pathway protein expression. Results: Real-time quantitative PCR and Western blotting showed that BMP-7 down-regulated the mRNA and protein expression of type I collagen, type III collagen and fibronectin in HK-2 cells induced by TGF-β1 in a concentration-dependent manner. TGF-β1 can promote HK-2 cells Smad2, Smad3 phosphorylation. However, Smad2 and Smad3 phosphorylation up-regulated by TGF-β1 was inhibited by BMP-7, the difference was statistically significant (P <0.05 and P <0.01). EMSA results show that BMP-7 can inhibit the TGF-β1 upregulated NF-κB activity. Conclusion: BMP-7 can inhibit the effect of TGF-β1 on ECM secretion induced by HK-2 by inhibiting the phosphorylation of Smad2 / 3 and inhibiting the DNA-binding activity of NF-κB.