枸杞多糖抑制TGF-β1诱导的心肌成纤维细胞活化作用机制

Inhibitory Mechanism of Lycium barbarum Polysaccharide against Cardiac Fibroblast Activation Induced by TGF-β1

目的:探究宁夏枸杞多糖对转化生长因子β1(transforming growth factor β1,TGF-β1)诱导的心脏纤维化细胞模型干预作用。方法:分离培养小鼠原代心肌成纤维细胞(cardiac fibroblasts,CFs),用质量浓度10 ng/mL TGF-β1作用于CFs,建立心脏纤维化细胞模型,采用宁夏枸杞多糖进行处理,观察细胞形态;免疫荧光检测α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)的表达水平;Western blot检测TGF-β1/Smads信号通路相关蛋白表达及其磷酸化水平;实时荧光定量聚合酶链式反应检测Snail、Twist和S100A4 mRNA的表达水平。结果:与对照组相比,模型组加入TGF-β1后,CFs细胞的形态由梭形变得狭长,细胞数目显著增多;α-SMA蛋白相对表达量升高;TGF-β1/Smads信号通路相关蛋白表达及其磷酸化水平升高;Snail、Twist和S100A4的mRNA表达水平高度显著升高(P<0.001);给予宁夏枸杞多糖处理,可高度显著抑制TGF-β1诱导的CFs增殖(P<0.001),减少CFs细胞中α-SMA及TGF-β1/Smads信号通路相关蛋白的表达,高度显著下调Snail、Twist和S100A4的mRNA表达(P<0.001)。结论:枸杞多糖对TGF-β1诱导的CFs纤维化具有改善作用,其作用机制与抑制TGF-β1/Smads信号通路有关。

Objective: To explore the ameliorative effect of Lycium barbarum polysaccharide(LBP) on TGF-β1-induced cardiac fibrosis in cardiac fibroblasts(CFs). Methods: Mouse primary CFs were cultured and treated with 10 ng/mL TGF-β1to induce fibrosis. After treatment with LBP, the morphology of cells was observed by microscopy. The expression of α-smooth muscle actin(α-SMA) was detected by immunofluorescence. Western blot was used to detect the expression and phosphorylation levels of proteins involved in the TGF-β1/Smads signaling pathway. The mRNA expression levels of Snail,Twist and S100A4 were detected by real-time quantitative polymerase chain reaction. Results: Compared with the control group(spindle-like in shape), the morphology of TGF-β1-induced cells was longer and narrower, and the number of cells increased significantly. The relative expression level of α-SMA, and the expression and phosphorylation levels of proteins involved in the TGF-β1/Smads signaling pathway increased, and the mRNA expression of Snail, Twist and S100A4 was significantly elevated(P < 0.01). After treatment with LBP, the proliferation of CFs induced by TGF-β1 was significantly inhibited(P < 0.001), the expression of α-SMA and proteins involved in the TGF-β1/Smads signaling pathway in CFs decreased, and the mRNA expression of Snail, Twist and S100A4 was down-regulated(P < 0.001). Conclusion: Lycium barbarum polysaccharide can ameliorate TGF-β1-induced cardiac fibrosis, in CFs and its mechanism may be related to the inhibition of the TGF-β1/Smads signaling pathway.