丹参红花提取物促进心脏成纤维细胞自噬改善心肌梗死后心肌纤维化的机制研究

Myocardial fibrosis, the dominant abnormality of adverse ventricular remodelling post myocardial infarction (MI) pathologically, remains treated insufficiently. In traditional medicine, promoting blood circulation for removing blood stasis is considered as a promising therapy of myocardial infarction. Salvia miltiorrhiza and carthamus tinctorius extract (SCE), as the representative medication guided by the principle forementioned, was demonstrated to have beneficial property of anti-fibrosis post MI, which was associated with enhancing autophagy activation and inhibiting collagen synthesis of cardiac fibroblasts (CFs) in our previous pilot study. Activation of Wnt/β-catenin pathway plays a pivotal role of myocardial fibrosis. Dishevelled, the key regulate target of β-catenin pathway, can be degraded in some cancer cell lines, when autophagy is activated. In summary, we assume that SCE is able to inhibit myocardial fibrosis post MI via activating autophagy, subsequently downregulating Dishevelled of CFs. To validate the hypothesis, homozygous knock out (Atg7+/-) mice and GFP-LC3 transgenic mice are used to induce MI by permanent ligation of left anterior descending artery. In vivo, after administration of SCE, echocardiography and hemodynamic analysis will be performed to investigate the impaction on cardiac remodeling under knock down of autophagy related gene. Fibrosis and autophagy of CFs will also be detected by histological and immunological techniques. Mechanism of SCE inhibiting fibrosis via autophagy activation will be further investigated in vitro. The results will help to reveal novel mechanism of SCE alleviates fibrosis post MI and provide theoretical evidence for anti-fibrotic drugs involving new target furthermore.

心肌纤维化是心肌梗死（MI）后心室重构的主要病理改变，其目前仍缺乏有效的防治措施。中医药中的活血化瘀疗法对此具有良好的应用前景。我们前期研究发现丹参红花提取物（SCE）能改善MI后的心功能并减轻纤维化，该作用与促进心脏成纤维细胞（CFs）的自噬并抑制胶原合成相关。预实验还提示SCE能促使CFs中散乱蛋白（Dvl）减少。Dvl是促进心肌纤维化的重要通路Wnt/β-catenin途径中关键的调控分子，而促进自噬可抑制Dvl及该通路的活化。因此，我们提出以下假设“SCE通过促进CFs自噬并抑制Dvl的机制减轻MI后心肌纤维化”。我们将通过GFP-LC3转基因小鼠和自噬相关基因缺陷小鼠的MI模型明确SCE减轻MI后纤维化的效应以及自噬在其中发挥的作用；并进一步对SCE通过自噬介导抑制Dvl的机制进行探讨。本项目将有助于揭示SCE治疗MI后纤维化的新机制，还可为开发针对新靶点的药物提供实验依据。

背景：心肌梗死后心肌纤维化导致心室重构、损伤心功能。丹参红花提取物（SCE）在临床上主要用于心绞痛、心肌梗死等缺血性心血管疾病，但在心肌梗死后心肌纤维化中的作用及机制尚未明确。.主要研究内容：结扎小鼠左前降支近段建立心肌梗死模型，观察SCE对生存率、左心功能及心肌纤维化的影响。QPCR和WB检测心肌炎症和TGF-β通路下游基因的表达变化。新生大鼠原代心肌成纤维细胞分离培养后采用TGF-β刺激，观察SCE对心脏成纤维细胞胶原生成、基质金属蛋白酶表达、表型转化及TGF-β通路下游基因的表达变化。ChIP检测SCE干预后心肌成纤维细胞中Smad3启动子区组蛋白甲基化水平。.结果：SCE能显著改善梗死模型小鼠生存率、左心室功能以及减轻心肌纤维化，减少心梗边缘区心肌col1a1、col3a1、acta2、mmp2、mmp9、α-sma、collagen Ⅰ、collagen Ⅲ的表达，抑制急性期的炎症因子IL-1β、TNF-α和IL-6的表达。SCE可使Smad3的表达降低，而其他TGF-β相关基因smad2、smad4、smad7、msg1、strap、smurf1、smurf2等的表达无变化。SCE显著降低TGF-β刺激后大鼠心脏成纤维细胞col1a1、col3a1、acta2生成及Smad3基因和蛋白表达水平。SCE显著下调原代心脏成纤维细胞Smad3启动子区H3K4me3和H3K36me3水平。.科学意义：本研究的结果提示延长使用SCE能显著抑制心肌梗死后心脏重构，改善心功能，为临床使用SCE治疗缺血性心衰提供动物实验依据。本研究首次揭示了SCE抑制心肌纤维化的表观遗传学新机制，也首次提出Smad3启动子区组蛋白甲基化是心肌纤维化的重要调控靶点，为将来进一步深入研究表观遗传学在心脏重构中的调节作用以及开发调控Smad3组蛋白甲基化的药物来阻止心肌纤维化，甚至治疗慢性心力衰竭奠定基础。