枸杞中绿原酸对转化生长因子-β1诱导心肌成纤维细胞纤维化的影响

Influence of Chlorogenic Acid from Lycium barbarum on Cardiac Fibroblast Fibrosis Induced by Transforming Growth Factor-β1

目的:考察枸杞中分离得到的绿原酸对转化生长因子-β1(transforming growth factor-β1,TGF-β1)诱导心肌成纤维细胞(cardiac fibroblasts,CFs)纤维化的干预效果。方法:质量浓度10 ng/mL TGF-β1诱导CFs建立心肌纤维化细胞模型,用不同质量浓度绿原酸处理纤维化细胞,细胞毒性实验(cell counting kit-8,CCK-8)检测绿原酸对CFs增殖的影响;酶联免疫吸附试验(enzyme linked immunosorbent assay,ELISA)检测纤维化标志蛋白胶原蛋白(collagen,Col)I、Col Ⅲ和免疫荧光检测α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)的表达;实时荧光定量聚合酶链式反应(quantitative real-time polymerase chain reaction,qPCR)检测Twist1、Acta2、Snail2及纤维化蛋白S100A4等mRNA表达水平;Westernblot检测TGF-β1/Smads信号通路相关蛋白及其磷酸化表达水平。结果:CCK-8检测表明枸杞中分离得到的绿原酸能抑制细胞异常增殖;ELISA和免疫荧光检测表明,枸杞中分离得到的绿原酸能下调纤维化标志蛋白Col I、Col Ⅲ和α-SMA的表达;qPCR检测表明枸杞中分离得到的绿原酸能高度显著下调纤维化转录调控因子Acta2、Twist1、Snail1、Snail2、Smad4及纤维化蛋白S100A4的mRNA表达(P<0.001);Western blot检测表明枸杞中分离得到的绿原酸能高度显著下调TGF-β1/Smads信号通路相关蛋白的表达(P<0.001)。结论:在本研究质量浓度范围内,枸杞中分离得到的绿原酸抑制TGF-β1诱导的CFs纤维化过程,其机制可能与TGF-βl/Smads信号通路有关。

Objective: To investigate the protective effect of chlorogenic acid from Lycium barbarum on cardiac fibroblast(CF) fibrosis induced by transforming growth factor-β1(TGF-β1). Methods: A TGF-β1 concentration of 10 ng/mL was used to induce CF fibrosis. The CFs were further treated with different concentrations of chlorogenic acid, and their proliferation was detected by cell counting kit-8(CCK-8) assay. The expression of the fibrosis markers collagen(Col) I and Col Ⅲ was detected by enzyme linked immunosorbent assay(ELISA) and α-smooth muscle actin(α-SMA) by immunofluorescence.The mRNA expression levels of Acta2, Twist1, Snail1, Snail2, Smad4 and S100A4 were detected by quantitative real-time polymerase chain reaction(qPCR). Western blot analysis was used to evaluate the expression levels TGF-β1/Smads signaling pathway-related proteins and their phosphorylation levels. Results: Chlorogenic acid from Lycium barbarum could inhibit abnormal cell proliferation, and down-regulate the expression of Col I, Col Ⅲ and α-SMA, the mRNA expression of Acta2, Twist1, Snail1, Snail2, Smad4 and S100A4(P < 0.001) and the expression of the TGF-β1/Smads signaling pathwayrelated proteins(P < 0.001). Conclusion: Within the concentration range tested in this study, chlorogenic acid from Lycium barbarum inhibits the fibrosis process of CFs induced by TGF-β1, and the mechanism may be related to the TGF-β1/Smads signaling pathway.