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目的:观察柚皮素对支气管上皮细胞中Smad介导的转化生长因子β1(transforming growth factor β1,TGF-β1)通路的影响,并研究其可能的机制。方法:培养HBE细胞,分为对照组、TGF-β1组和柚皮素干预组。柚皮素干预组分别与25、50和100μmol/L的柚皮素共孵育2 h后,与TGF-β1组一起加入TGF-β1。2 h后用real-time PCR方法检测各组细胞纤溶酶原激活物抑制剂1(PAI-1)mRNA表达。另外在加入TGF-β1后30 min时,提取对照组、TGF-β1组和柚皮素高剂量组细胞总蛋白,用Western blot方法检测Smad2水平及磷酸化的Smad2水平,同时用免疫荧光法检测Smad2的核转位。结果:TGF-β1组的PAI-1mRNA水平较对照组明显增高,随着给予柚皮素剂量的增大,PAI-1 mRNA水平明显降低。TGF-β1组经TGF-β1刺激30 min后,磷酸化的Smad2明显增加,经柚皮素干预后则随着柚皮素剂量的增加,Smad2磷酸化明显受抑制。各组的总Smad2水平无明显变化。免疫荧光实验显示TGF-β1组的细胞核内Smad2明显增多,而柚皮素干预组(100μmol/L)的细胞核内Smad2含量明显减少。结论:柚皮素可能通过抑制Smad2磷酸化及减少核内Smad2含量来影响TGF-β1在支气管上皮细胞中的生物功能。
OBJECTIVE: To observe the effect of naringenin on Smad-mediated transforming growth factor β1 (TGF-β1) pathway in bronchial epithelial cells and to investigate its possible mechanism. Methods: HBE cells were cultured and divided into control group, TGF-β1 group and naringenin intervention group. Naringenin intervention groups were incubated with 25, 50 and 100μmol / L of naringenin for 2 h, then added with TGF-β1 group for TGF-β1 for 1.2 h and then detected by real-time PCR Enzyme Inhibitor 1 (PAI-1) mRNA expression. In addition, 30 min after the addition of TGF-β1, the total protein of high-dose group, TGF-β1 group and naringenin were extracted, Smad2 and phosphorylated Smad2 were detected by Western blot and immunofluorescence Smad2 nuclear translocation. Results: The PAI-1 mRNA level in TGF-β1 group was significantly higher than that in control group. PAI-1 mRNA level was significantly decreased with the increase of dosage of naringenin. TGF-β1 group stimulated by TGF-β1 for 30 min, phosphorylated Smad2 increased significantly after naringenin intervention with naringenin dose, Smad2 phosphorylation was significantly inhibited. The total Smad2 levels in each group had no significant change. The results of immunofluorescence showed that Smad2 in the nucleus of TGF-β1 group was significantly increased, whereas the Smad2 content in the nucleolus of the naringenin-treated group (100 μmol / L) was significantly decreased. Conclusion: Naringenin may affect the biological function of TGF-β1 in bronchial epithelial cells by inhibiting the phosphorylation of Smad2 and decreasing the content of Smad2 in the nucleus.