芦丁对TGF-β1诱导的心肌成纤维细胞纤维化的影响

Effect of rutin on TGF-β1 induced fibrosis of cardiac fibroblasts

关键词:  芦丁  心肌纤维化  心肌成纤维细胞  TGF-β1/Smad信号通路

Key words:  rutin  cardiac fibrosis  cardiac fibroblasts  TGF-β1/Smad signaling pathway

摘要:

[目的] 探究芦丁对转化生长因子-β1（TGF-β1）诱导的心肌成纤维细胞（CFs）纤维化及TGF-β1/Smad 信号通路的影响。[方法] 构建TGF-β1诱导的CFs细胞模型，设置组别为：对照组（完全培养基+细胞）、模型组（含10 ng/mL TGF-β1+细胞）、阳性对照组（10 ng/mL TGF-β1+10 μmol/L卡托普利和细胞）和给药组（10 ng/mL TGF-β1+200 μg/mL的芦丁和细胞）。采用蛋白免疫印迹法检测α-平滑肌肌动蛋白（α-SMA）、波形蛋白（Vim）、纤连蛋白（FN）、胶原蛋白Ⅰ（Collage Ⅰ）、胶原蛋白 Ⅲ（Collage Ⅲ）、TGF-β1/Smad通路相关蛋白的表达情况；采用实时荧光定量聚合酶链式反应（qPCR）法检测Acta 2、Vim、FN、Col Ⅰ 1a1、基质金属蛋白酶2（MMP-2）和基质金属蛋白酶9（MMP-9）的基因表达水平；采用免疫荧光技术检测关键蛋白α-SMA的表达及定位来探究芦丁对TGF-β1 诱导的CFs纤维化的影响。[结果] 用TGF-β1诱导CFs后，成功激活Vim、FN、Collage Ⅰ、Collage Ⅲ、TGF-β1/Smad信号通路相关蛋白的表达，200 μg/mL 芦丁干预后，抑制了Vim、FN、Collage Ⅰ、Collage Ⅲ、TGF-β1/Smad信号通路相关蛋白和mRNA的激活从而缓解了心肌纤维化的发生；免疫荧光实验结果显示：与对照组相比，TGF-β1模型组中α-SMA绿色荧光强度显著增强；200 μg/mL芦丁组与模型组相比，α-SMA绿色荧光强度明显减弱。[结论] 本研究结果表明200 μg/mL芦丁通过抑制TGF-β1 诱导的Vim、FN、Collage Ⅰ、Collage Ⅲ、α-SMA以及TGF-β1/Smad信号通路相关蛋白和mRNA的表达，从而缓解心肌纤维化。

Abstract:

[Objective] To explore the effect of rutin on transforming growth factor-β1（TGF-β1）-induced fibrosis of cardiac fibroblasts（CFs） and the TGF-β1/Smad signaling pathway. [Methods] The CFs cell model induced by TGF-β1 was constructed，and the groups were set as follows：control group（complete medium+cells），model group（containing 10 ng/mL TGF-β1+cells），positive control group（10 ng/mL TGF-β1+10 μmol/L captopril and cells） and administration group（10 ng/mL TGF-β1+200 μg/mL rutin and cells）. The expression of α-smooth muscle actin（α-SMA），Vimentin（Vim），Fibronectin（FN），Collagen I，Collagen Ⅲ and TGF-β1/Smad pathway-related proteins were detected by Western blot. The gene expression levels of Acta 2，Vim，FN，Col Ⅰ1a1，matrix metalloproteinase 2（MMP-2） and matrix metalloproteinase 9（MMP-9） were detected by quantitative real-time polymerase chain reaction（qPCR）. Immunofluorescence was used to detect the expression and localization of the key protein α-SMA to explore the effect of rutin on TGF-β1-induced CFs fibrosis. [Results] After CFs were induced by TGF-β1，the expression of Vim，FN，Collage I，Collage Ⅲ，TGF-β1/Smad signaling pathway-related proteins was successfully activated. After intervention with 200 μg/mL rutin，the activation of Vim，FN，CollageⅠ，Collage Ⅲ，TGF-β1/Smad signaling pathway-related proteins and mRNA was inhibited，thereby alleviating the occurrence of myocardial fibrosis. The results of immunofluorescence assay showed that compared with the control group，the green fluorescence intensity of α-SMA in the TGF-β1 model group was significantly enhanced. Compared with the model group，the green fluorescence intensity of α-SMA was significantly weakened in the 200 μg/mL rutin group. [Conclusion] The results of this study showed that 200 μg/mL rutin alleviated myocardial fibrosis by inhibiting the expression of Vim，FN，Collage I，Collage Ⅲ，α-SMA and TGF-β1/Smad signaling pathway-related proteins and mRNA induced by TGF-β1.