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背景:转化生长因子β1通过Smads信号通路刺激心脏成纤维细胞增殖与分化是心肌纤维化最重要的发生机制之一。前期研究证实丹参酮ⅡA能有效抑制心肌纤维化,但是否通过阻断转化生长因子β1/Smads信号通路起作用尚不清楚。目的:观察丹参酮ⅡA对大鼠心脏成纤维细胞内转化生长因子β1信号转导的影响。方法:采用胰酶消化法和差速贴壁法获取新生SD大鼠心脏成纤维细胞,应用5μg/L转化生长因子β1刺激及不同浓度丹参酮ⅡA(10-6,10-5和10-4mol/L)。用反转录聚合酶链反应法和免疫蛋白印迹法分别检测转化生长因子β1刺激后6,12和24h纤维连接蛋白的表达,免疫蛋白印迹法检测转化生长因子β1刺激后15,30,60和120min的Smads蛋白表达。结果与结论:纤维连接蛋白mRNA和蛋白表达量在转化生长因子β1刺激6h后开始呈现上升趋势,至作用24h时分别增加1.3倍和1.8倍(P<0.01);磷酸化Smad2/3蛋白表达量在转化生长因子β1刺激15min后开始上升,1h达到高峰,2h后虽有所下降,但仍较刺激前增加3.9倍(P<0.01)。丹参酮ⅡA(10-5和10-4mol/L)预处理可下调纤维连接蛋白和磷酸化Smad2/3表达(P<0.05或P<0.01),而且效应呈剂量依赖性。由此可知,转化生长因子β1在一定范围内以时间依赖方式诱导纤维连接蛋白及其mRNA和磷酸化Smad2/3表达。丹参酮ⅡA抗心肌纤维化作用可能与其抑制转化生长因子β1诱导的Smad2/3磷酸化,阻断心脏成纤维细胞内转化生长因子β1/Smads信号通路有关。
BACKGROUND: Transforming growth factor β1 stimulates cardiac fibroblast proliferation and differentiation through Smads signaling pathway is one of the most important mechanisms of myocardial fibrosis. Previous studies confirmed that tanshinone Ⅱ A can effectively inhibit myocardial fibrosis, but whether through the block of transforming growth factor β1 / Smads signaling pathway is unclear. Objective: To observe the effect of tanshinone Ⅱ A on the signal transduction of transforming growth factor-β1 in rat cardiac fibroblasts. Methods: Newborn SD rat cardiac fibroblasts were obtained by trypsin digestion and differential adherent method. The cells were treated with 5μg / L transforming growth factor β1 and different concentrations of tanshinone Ⅱ A (10-6, 10-5 and 10-4 mol / L). The expression of fibronectin was detected by reverse transcription polymerase chain reaction (RT-PCR) and Western blotting respectively at 6, 12 and 24h after TGF-β1 stimulation. The expression of fibronectin was detected by Western blotting at 15, 30, 60 and 120min Smads protein expression. RESULTS AND CONCLUSION: The mRNA and protein expression of fibronectin began to increase after 6 h of TGF-β1 stimulation, and increased 1.3-fold and 1.8-fold (P <0.01) respectively after 24h of TGF-β1 stimulation. The phosphorylated Smad2 / 3 protein expression The expression of TGF - β1 increased 15 min after stimulation and peaked at 1 h. Although it decreased after 2 h, it was still 3.9 times higher than that before stimulation (P <0.01). Tanshinone IIA (10-5 and 10-4mol / L) pretreatment down-regulated the expression of fibronectin and phosphorylated Smad2 / 3 (P <0.05 or P <0.01), and the effect was dose-dependent. Thus, transforming growth factor β1 induced fibronectin and its mRNA and phosphorylated Smad2 / 3 expression in a time-dependent manner in a certain range. The anti-myocardial fibrosis effect of tanshinoneⅡA may be related to the inhibition of TGF-β1-induced phosphorylation of Smad2 / 3 and the block of transforming growth factor β1 / Smads signaling pathway in cardiac fibroblasts.