基于TGFβ-Smad/TGFβ-TAK1通路的四妙勇安汤干预心肌纤维化的效应机制研究

Myocardial fibrosis is a common pathological feature of various heart diseases and is closely related to the prognosis of the disease. Classical TGFβ-Smad and the atypical TGFβ-TAK1-p38 pathway, the most important pro-fibrotic pathways, were considered as important drug targets and attracted more attentions. However, the potent inhibitors against single targets have obvious toxic and side effects. In consequence, there was no drug coming to clinical application yet. Simiao Yongan Decoction (SMYA), a formula with the effect of promoting blood circulation and detoxication, showed significant clinical curative effect, and delayed experimental myocardial fibrosis and improved cardiac function. Our preliminary data showed that these effects of SMYA were related to the regulation of TGFβ pathway. This project will focus on the regulatory mechanism of SMYA about TGFβ-fibrosis pathway. Cardiac fibrosis mice, which are induced by pressure overload, will be used to analyze the targets of SMYA; network- pharmacological methods, medicinal chemistry analysis and pharmacological verification of targets and effects will be applied together to find the target-component correlations. This project will be helpful to elucidate the integrative effect of SMYA on TGFβ-fibrosis pathways.

心肌纤维化是多种心脏病的共性病理特征，与疾病预后密切相关，促纤维化的经典TGFβ-Smad和非典型TGFβ-TAK1-p38通路是药物研发的热点。但针对单一靶点的强效抑制，毒副作用明显，尚无上市药物。四妙勇安汤(SMYA)具有活血解毒功效，临床疗效明确，可减缓实验性心肌纤维化、改善心功能，机制与调节TGFβ通路有关。本研究拟围绕TGFβ-纤维化通路，采用压力负荷型小鼠心肌纤维化模型分析SMYA的效应节点；采用网络药理学预测与药物化学分析、药效靶点验证相结合，建立“靶标-成分”关联；为阐明四妙勇安汤在TGFβ纤维化通路的整合效应机制奠定基础。

心肌纤维化是多种心脏病的共性病理特征，与疾病预后密切相关，促纤维化的经典TGFβ-Smad和非典型TGFβ-TAK1-p38通路是药物研发的热点。但针对单一靶点的强效抑制，毒副作用明显，尚无上市药物。四妙勇安汤(SMYA)具有活血解毒功效，临床疗效明确，可减缓实验性心肌纤维化、改善心功能，机制与调节TGFβ通路有关。本研究拟围绕TGFβ-纤维化通路探讨SMYA改善压力负荷型小鼠心肌纤维化的主要效应节点及其作用于TGFβ-纤维化通路的“靶点-成分”关联。结果显示SMYA可改善主动脉弓缩窄引起的小鼠心肌纤维化,提高左室射血分数，降低心脏指数；SMYA通过调节MMP-TIMPs平衡促进心肌胶原降解，且能显著下调模型小鼠心肌组织中胶原蛋白和α-SMA等纤维化相关基因的表达，能同时降低TGFβ1-Smad 和TGFβ1-TAK1-p38 通路上关键靶点蛋白的表达或磷酸化。进一步，将SMYA其入血成分中含量较高的绿原酸等11个化合物纳入效应成分的候选范围。建立了TGF-β诱导成纤维细胞模型，确定了SMYA候选效应成分干预心肌成纤维细胞的安全浓度范围。将不同浓度的绿原酸等11种候选化合物分别处理心肌成纤维细胞后发现4种候选化合物可显著降低Col3的蛋白表达，2种化合物可同时显著降低Col1和Col3的蛋白表达，且这两种可以同时降低I型和III型胶原的化合物分别来自四妙勇安汤中的金银花和玄参。因此，以这两个化合物的配伍效应为代表，初步探讨四妙勇安汤中的君臣配伍机制。此外，候选化合物可降低TGF-β-纤维化通路的关键分子, 在上述11个候选化合物中1个化合物可显著降低p-smad2的表达，3个化合物可显著降低p-smad3的表达，2个化合物可降低smad4的表达。通过本项目的研究，进一步证实了四妙勇安汤通过调节TGFβ-Smad/TGFβ-TAK1通路改善压力负荷型心衰小鼠心肌纤维化的效应，发现了11个可减少胶原纤维表达的候选活性成分，并阐释了部分成分在TGFβ-Smad/TGFβ-TAK1通路的效应节点，为四妙勇安干预心肌纤维化效应成分及机制的阐明提供了实验依据，为抗纤维化活性成分的筛选提供了候选化合物及化合物组合，有望为心肌纤维化的治疗提供新的候选药物。