芪苈强心胶囊抑制阿霉素诱导的心肌成纤维细胞活化的作用机制研究

Mechanism of Qili Cardiotonic Capsule Inhibiting Doxorubicin-InducedActivation of Myocardial Fibroblasts

目的:探讨芪苈强心胶囊对阿霉素诱导的心肌成纤维细胞(CFs)活化的影响及与PAI-1/TGFβ1/Smad3通路的关系。方法:将细胞分为对照组、阿霉素模型组、芪苈强心低剂量组及芪苈强心高剂量组,实时荧光定量聚合酶链反应(qPCR)和western blot法检测4组细胞中Ⅰ型胶原(Collagen Ⅰ)、Ⅲ型胶原(CollagenⅢ)、纤溶酶原激活物抑制剂-1(PAI-1)、转化生长因子β1(TGFβ1)及Sma和Mad相关蛋白3(Smad3)的mRNA和蛋白表达水平;同时,转染PAI-1过表达质粒(PAI-1 OE),分为阴性对照组、阿霉素组、阿霉素+芪苈强心及阿霉素+芪苈强心+PAI-1 OE组,qPCR和western blot法检测4组细胞中Collagen Ⅰ、CollagenⅢ、TGFβ1和p-Smad3含量。结果:阿霉素模型组的Collagen Ⅰ、CollagenⅢ、PAI-1、TGFβ1、p-Smad3蛋白相对表达量均明显升高,芪苈强心低、高剂量组均有下调,差异均有统计学意义(P<0.01)。在PAI-1过表达实验中,与阿霉素+芪苈强心组相比,阿霉素+芪苈强心+PAI-1 OE组的Collagen Ⅰ、CollagenⅢ、TGFβ1及p-Smad3蛋白含量明显上升,差异均有统计学意义(P<0.01),提示过表达PAI-1后可以显著影响芪苈强心胶囊对TGFβ1和p-Smad3的抑制作用。结论:芪苈强心胶囊通过抑制PAI-1/TGFβ1/Smad3通路进而抑制阿霉素诱导的CFs活化。

Objective:To investigate the effect of Qili cardiotonic capsule on doxorubicin-induced activation of cardiac fibroblasts(CFs)and its relationship with PAI-1/TGFβ1/Smad3 pathway.Methods:The cells were divided into control group,adriamycin model group,Qili cardiotonic low-dose group and Qili cardiotonic high-dose group.Real time fluorescence quantitative polymerase chain reaction(qPCR)and Western blot methods were used to detect type I collagen(collagen I),type III collagen(collagen III),plasminogen activator inhibitor-1(PAI-1),and transforming growth factorβ1(TGFβ1),as well as mRNA and protein expression levels of Sma and Mad related protein 3(Smad3)in the cells of the four groups.Meanwhile,PAI-1 overexpression plasmid(PAI-1 OE)was transfected and divided into 4 groups:negative control group,adriamycin group,adriamycin+Qili cardiotonic group and adriamycin+Qili cardiotonic+PAI-1 OE group.The contents of collagen I,collagenⅢ,TGFβ1 and p-Smad3 in cells of the four groups were detected by qPCR and western blot.Results:The relative protein expressions of collagen I,collagenⅢ,PAI-1,TGFβ1 and P-SMad3 in adriamycin model group were significantly increased,while those in Qili cardiotonic low and high dose groups were down-regulated,with statistical significance(P<0.01).In the PAI-1 overexpression experiment,compared with adriamycin+Qili cardiotonic group,the protein contents of collagen I,collagenⅢ,TGFβ1 and p-SMad3 in adriamycin+Qili cardiotonic+PAI-1 OE group were significantly increased,with statistical significance(P<0.01).These results suggested that overexpression of PAI-1 could significantly reverse the inhibitory effect of Qili cardiotonic capsule on TGFβ1 and p-Smad3.Conclusion:Qili cardiotonic capsule inhibits doxorubicin-induced CFs activation by inhibiting PAI-1/TGFβ1/Smad3 pathway.