多西环素对TGF-β1诱导肺成纤维细胞α-SMA、FN、Col Ⅰ表达的影响
doi: 10.16766/j.cnki.issn.1674-4152.002274
Effect of doxycycline on the expression of alpha-SMA, fibronectin and collagen I in MRC-5 cell line induced by TGF-beta1
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摘要:
目的 研究多西环素(Doxy)对转化生长因子-β1(TGF-β1)诱导人胚肺成纤维细胞(MRC-5)向肌成纤维细胞转化的影响，探索Doxy抗肺纤维化的作用。 方法 体外培养MRC-5，采用不同浓度的Doxy(0、2.5、5.0、10.0、20.0、40.0、80.0 μg/mL)干预，72 h后采用MTT法检测细胞活性以确定Doxy浓度并进行分组。将该实验分为对照组(MRC-5)、阳性对照组(TGF-β1 10 ng/mL)、Doxy 2.5 μg/mL组、Doxy 5.0 μg/mL组、Doxy 10.0 μg/mL组、Doxy 20.0 μg/mL组，干预72 h后采用蛋白免疫印迹法(Western blotting)、细胞免疫荧光检测α-平滑肌肌动蛋白(α-SMA)、纤维连接蛋白(FN)、Ⅰ型胶原(Col Ⅰ)蛋白的表达，采用实时荧光定量法(qRT-PCR)检测α-SMA、FN、Col Ⅰ mRNA相对量的表达。采用单因素方差分析进行统计分析。 结果 Doxy浓度为40.0 μg/mL及80.0 μg/mL时MRC-5细胞的活性开始出现下降，分别为0.93±0.03、0.83±0.08，且在80.0 μg/mL处理时MRC-5细胞的活性与对照组相比出现明显的抑制(P＜0.05)。qRT-PCR显示阳性对照组α-SMA、FN、Col Ⅰ mRNA的相对表达均明显高于对照组(1.62±0.04 vs. 0.94±0.05，4.33±0.28 vs. 0.98±0.06，2.25±0.97 vs. 1.04±0.04，均P＜0.05)，而Doxy 10.0 μg/mL组、Doxy 20.0 μg/mL组α-SMA、FN、Col Ⅰ mRNA的表达较阳性对照组均明显下降(0.70±0.15、0.41±0.11 vs. 1.62±0.04;1.19±0.85、0.79±0.06 vs. 4.33±0.28;0.94±0.12、0.52±0.07 vs. 2.25±0.97，均P＜0.05)。Western blotting及免疫荧光也发现了同样的变化趋势。 结论 Doxy可通过抑制TGF-β1诱导的肺成纤维细胞分化发挥抗纤维化作用。 -
关键词:
- 多西环素 /
- 转化生长因子-β1 /
- 肺间质纤维化
Abstract:Objective To explore the effect of doxycycline (Doxy) on Transforming growth factor -β1 (TGF-β1) induced conversion in lung fibroblast, and to provide a theoretical basis for the treatment of pulmonary fibrosis. Methods Human embryonic lung fibroblast (MRC-5) were cultured in vitro, treated with different concentrations of doxycycline (0, 2.5, 5.0, 10.0, 20.0, 40.0, 80.0 μg/mL) for 72 hours, 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay was used to detect the cell survival to determine the concentration of doxycycline. TGF-β1 was used to induce the conversion in MRC-5 cell, and treated with different concentrations of doxycycline (0, 2.5, 5.0, 10.0, 20.0 μg/mL) for 72 hours. Western blotting, RT-PCR, immunity fluorescence were used to detect the expression of α-smooth muscle actin (α-SMA), fibronectin (FN), collagen Ⅰ (Col Ⅰ). Results The MRC-5 cell viability decreased which the concentration of Doxy was 40.0 μg/mL and 80.0 μg/mL, relative to the control group were 0.93±0.03 and 0.83±0.08. qRT-PCR showed that the relative expressions of α-SMA, FN, and Col Ⅰ mRNA in the TGF-β1 10 ng/mL group were significantly higher than those in the control group (1.62±0.04 vs. 0.94±0.05, 4.33±0.28 vs. 0.98±0.06, 2.25±0.97 vs. 1.04±0.04, all P < 0.05), while the expressions of α-SMA, FN, and Col Ⅰ mRNA in the Doxy 10.0 μg/mL group and Doxy 20.0 μg/mL group were significantly lower than those in the TGF-β1 10 ng/mL group (0.70±0.15, 0.41±0.11 vs. 1.62±0.04; 1.19± 0.85, 0.79±0.06 vs. 4.33±0.28, 0.94±0.12, 0.52±0.07 vs. 2.25±0.97, all P < 0.05). Western blotting and immunofluorescence also found the same trend. Conclusion Doxy can exert anti-fibrosis effect by inhibiting the conversion of lung fibroblast induced by TGF-β1. -
Key words:
- Doxycycline /
- Transforming growth factor -β1 /
- Pulmonary fibrosis
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图 1 不同浓度的多西环素对MRC-5细胞活性的影响(n=6)
注：与对照组比较，aP＜0.05。
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图 2 多西环素对TGF-β1诱导的MRC-5细胞α-SMA、FN、Col Ⅰ mRNA的影响(n=6)
注：与对照组比较，aP＜0.05；与阳性对照组(TGF+Dox 0 μg/mL)比较，bP＜0.05。
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图 3 多西环素对TGF-β1诱导的MRC-5细胞α-SMA、FN、Col Ⅰ蛋白表达的影响(Western blotting结果，n=6)
注：与对照组比较，aP＜0.05；与阳性对照组(TGF+Dox 0 μg/mL)比较，bP＜0.05。
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图 4 多西环素对TGF-β1诱导的MRC-5细胞α-SMA、FN荧光表达的影响(免疫荧光结果)
注：目的蛋白为绿色，细胞核为蓝色。
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