芍药苷对异丙肾上腺素诱导心肌成纤维细胞增殖和胶原产生的影响及其机制研究

Effect and mechanism of paeoniflorin on cell proliferation and collagen production induced by isoproterenol in cardiac fibroblasts

目的观察芍药苷(PEF)对异丙肾上腺素(ISO)诱导心肌成纤维细胞增殖和胶原产生的影响,并探讨其机制。方法体外培养的心肌成纤维细胞,待细胞生长到融合状态时加入不同浓度的PEF(10或100μmol/L)预处理2 h,然后加入ISO(10-5mol/L)作用48 h。用四唑盐比色实验(MTT)检测细胞增殖活性;实时荧光定量PCR检测Ⅰ、Ⅲ型胶原和心肌营养素-1(CT-1)mRNA表达;酶联免疫吸附法(ELISA)检测上清液中Ⅰ、Ⅲ型胶原的含量;Western blot和DCFH-DA荧光法分别检测CT-1蛋白和细胞内活性氧(ROS)的水平。结果 ISO能明显诱导心肌成纤维细胞的增殖,并能显著增加Ⅰ、Ⅲ型胶原mRNA的表达和含量,同时CT-1的mRNA和蛋白表达以及细胞内ROS的水平也显著升高,而预先应用不同浓度的PEF处理后,上述效应明显减弱。结论 PEF能抑制ISO诱导的心肌成纤维细胞增殖和胶原产生,其机制可能与降低细胞内ROS的产生进而下调CT-1的表达有关。

【Objective】To observe the effect of paeoniflorin（PEF） on isoproterenol（ISO）-induced proliferation and collagen production in cardiac fibroblasts.【Methods】Cardiac fibroblasts were pretreated with various concentration of PEF（10 or 100 μmol/L） for 2 hours,followed by incubating with ISO（10-5 mol/L） for another 48 hours.Cell proliferation activity was detected by MTT;The mRNA expression of collagen type Ⅰ,type Ⅲ and cardiotrophin-1（CT-1） was determined by real-time quantitative reverse transcription-polymerase chain reaction（realtime RT-PCR）;The amount of collagen type Ⅰ and type Ⅲ in cell culture supernatants was measured by ELISA;The protein expression of CT-1 and levels of intracellular reactive oxygen species（ROS） were determined by western bolt and DCFH-DA fluorescent probe,respectively.【Results】ISO significantly induced cardiac fibroblasts proliferation,increased the mRNA expression and concentration of collagen type Ⅰ and type Ⅲ,concomitantly with enhanced CT-1 expression and intracellular ROS.However,these effects of ISO were markedly ameliorated by pretreatment with PEF.【Conclusion】PEF ameliorated ISO-induced cardiac fibroblasts proliferation and collagen production through down-regulating CT-1 expression by inhibiting intracellular ROS production.