益气活血中药优化梗死后纤维化心肌ECM重建策略的效应机制研究

Myocardial fibrosis is a key factor which affects the life quality and long-term prognosis of patients with myocardial infarction.How to prevent myocardial fibrosis after infarction is a tough but hot orientation in current medical research.This research here is to study the acting mechanisms of myocardial fibrosis after myocardial infarction in vivo and in vitro using herbs of benefiting Qi and activating blood circulation which have certified clinical effects. In vivo study, recruiting the model of rat myocardial infarction, a Label-free quantitative proteomic analysis of ExtraCellular Matrix(ECM) components will find novel fibrosis markers and figure out their organization network as the pathologic evolvement goes on. Also the perspective therapeutic effects of herbs benefiting Qi and activating blood circulation will be evaluated from histopathological, molecular pathology and expressions of ECM represented by CILP and SPARC.Then AMPK mediated Integrin β1 signaling pathways during repair stage in myocardial fibrosis after infarction would be elucidated. In vitro study, using cardiac fibroblasts from neonatal rats stimulated by pro-fibrogenic factors Ang II, the biological behaviour of fibroblasts including cell morphology, phenotype differentiation, cell proliferation, cell migration, and secretion of specific ECM proteins, together with AMPK inhibited Integrinβ1 signaling pathways will be elaborated on the molecular mechanism of aforementioned herbs about the regulation of ECM system reconstruction. There is great significance to seek effective therapeutic targets to regulate myocardial remodeling and improve the prognosis of patients with myocardial infarction.

心肌纤维化是影响心梗患者生活质量和预后的关键因素，如何有效防治梗塞后纤维化是当前世界医学研究的难点和热点。本课题以临床疗效确切的益气活血中药（人参、水蛭）为治疗手段，从整体、细胞、分子层面研究其干预梗死后纤维化主要病理产物-细胞外基质（ECM）的作用机制。在体研究通过建立大鼠AMI模型，采用Label-free定量蛋白质组学技术分析纤维化组织中ECM时间衍变规律及蛋白互作关系，从AMPK介导Integrinβ1相关信号通路评价益气活血中药调节CILP、SPARC等为代表的ECM表达，优化心梗纤维化修复中ECM重建策略的作用机制。体外研究运用AngII诱导乳鼠心肌成纤维细胞，观察益气活血中药组分对成纤维细胞形态以及表型转化、增殖、迁移、分泌等生物学行为的影响，阐释AMPK抑制Integrinβ1信号系统调控ECM网络重建的分子机制。为中医药防治梗死后心肌纤维化、改善心梗患者预后提供科学依据。

【项目背景】.心肌纤维化是影响心梗患者预后的关键因素，如何有效防治梗塞后纤维化是当今医学难题。本课题以益气活血中药（人参、水蛭）主要成分为治疗手段，从整体、细胞和分子层面研究其干预心梗后心肌纤维化的疗效机制。.【研究内容】.在体研究通过建立大鼠AMI模型，采用蛋白质组学技术分析纤维化组织中ECM时间衍变规律，从AMPK相关信号通路评价益气活血中药优化ECM重建策略的作用机制。体外研究运用AngII诱导乳鼠心肌成纤维细胞，观察益气活血中药组分对成纤维细胞转化、增殖、迁移和分泌等生物学行为的影响，阐释AMPK抑制Integrinβ1信号系统调控ECM网络重建的分子机制。.【重要结果】.①通过蛋白质组学技术发现，心梗后心肌纤维化发生的可能作用靶点为巢蛋白-1、光蛋白聚糖、骨甘氨酸等ECM差异蛋白。调控ECM差异蛋白表达的作用机制可能涉及FAK、PI3K/Akt、Integrin β1等信号通路。.②人参皂苷Re和水蛭素能够改善心梗后心功能，减轻心肌损伤，减少胶原沉积，延缓心肌纤维化进展；其机制与调节ECM差异蛋白代谢，调控心肌AMPKα-Integrinβ1信号通路有关。.③人参皂苷Re和水蛭素能够抑制Ang II诱导的心肌成纤维细胞增殖、分化、迁移、分泌；其机制与调节AMPK-Integrinβ1信号通路表达有关。.【关键数据】.①心肌梗死2周后心肌中骨甘氨酸显著上调，巢蛋白-1显著下调；4周后光蛋白聚糖显著上调，巢蛋白-1、胶原VI型α2链显著下调。KEGG分析显示，FAK、PI3K/Akt、Integrin β1在ECM受体通路显著富集。.②人参皂苷Re和水蛭素能够升高LVEF，降低CK-MB、cTnT、BNP、MDA的水平，促进p-AMPKα/AMPKα蛋白表达，下调Integrinβ1、p-FAK/FAK、TGFβ1的水平。.③人参皂苷Re和水蛭素能够增加G0G1期细胞百分比，降低S期细胞百分比；减少穿越Transwell小室的细胞数量，延长划痕宽度；降低α-SMA、HYP、Collagen I的合成；促进p-AMPK/AMPK的表达，抑制Integrin β1合成。.【科学意义】.益气活血中药可从整体、细胞、分子水平调节心梗后心肌纤维化的病理过程，优化纤维化ECM重建策略，对于改善心梗患者的预后具有重要意义。