益母草碱调控miR-29的分子机制及其抗心肌纤维化的作用研究

Inhibition of myocardial fibrosis is vital to improve the prognosis of cardiovascular diseases and reduce the risk of death, however, there is a lack of effective treatment at present. Our research group discovered for the first time that leonurine, the active ingredient of medicines for blood-activating and stasis-dissolving, could significantly inhibit collagen deposition and myocardial fibrosis in mice suffered myocardial infarction and increase the survival rate; miR-29a/b/c levels elevated significantly in these effects; when miR-29a/b/c siRNA were transfected into cells in vitro respectively, the function of leonurine inhibiting related genes of collagen and myocardial fibrosis reduced or disappeared, indicating that miR-29 played a key role in the effect of leonurine inhibiting myocardial fibrosis. In view of this, we build the hypothesis that leonurine inhibites synthesis of collagen and suppresses myocardial fibrosis through regulating the expression of miR-29. In this study, we aim to establish animal and cell models of myocardial fibrosis, and study the molecular mechanism of leonurine regulating miR-29 from gene and protein levels, using gene chip, EMSA, DNA affinity chromatography, siRNA and other methods, combined with bioinformatics tools; and elucidate the role of the mechanism on inhibiting myocardial fibrosis, thus providing a theoretical basis for taking effective prevention strategy of myocardial fibrosis in clinical practice.

抑制心肌纤维化对改善心血管疾病预后和降低致死风险具有重要意义，但目前缺乏有效治疗手段。课题组首次发现活血化瘀类中药的活性成分益母草碱对心肌梗死小鼠具有显著抑制胶原沉积和抗心肌纤维化作用，并可提高生存率；该作用中miR-29a/b/c水平显著升高是主要变化；离体细胞分别转染miR-29a/b/c siRNA后，发现益母草碱抑制胶原和心肌纤维化相关基因作用减弱或消失，表明miR-29在益母草碱抗心肌纤维化效应中发挥关键作用。鉴于此，我们提出益母草碱通过调控miR-29的表达，进而抑制胶原合成，发挥抗心肌纤维化作用的科学假设。本项目拟建立心肌纤维化的动物和细胞模型，通过基因芯片、EMSA、DNA亲和层析、siRNA等方法，并结合生物信息学手段，从基因水平和蛋白水平系统研究益母草碱调控miR-29的分子机制；并阐明该机制在抗心肌纤维化中的地位和作用，从而为临床采取有效的防治策略提供理论依据。

心肌纤维化是多种心脏疾病终末期的病理表现之一，患者的病死率高，远期预后差，临床上尚缺乏有效治疗手段，因此抑制心肌纤维化对于延缓心脏疾病进展具有重要意义。本课题组前期研究发现益母草碱可以有效抑制小鼠心肌梗死后心肌纤维化，且可能与调控miR-29的表达有关，但益母草碱调控心肌纤维化的具体机制及其与miR-29之间的内在联系仍有待进一步研究。课题组根据申报书的研究计划已完成益母草碱调控miR-29的相关机制探索，在此基础上进行了适当拓展，更加全面地探讨了益母草碱调控心肌纤维化的分子机制。主要研究成果如下：①益母草碱可以抑制小鼠心肌梗死后心肌纤维化的进展；②益母草碱可以抑制血管紧张素II诱导的原代小鼠心肌成纤维细胞的分化、迁移和胶原合成；③益母草碱的抗心肌纤维化效应与miR-29a-3p/CollagenI/CollagenIII信号轴密切相关；④转录组基因测序进一步揭示益母草碱可以抑制小鼠心肌梗死后炎症相关基因的表达；⑤益母草碱可以抑制脂多糖诱导的急性心肌炎症。综上，本课题全面、深入地探索了益母草碱调控心肌纤维化的具体作用机制，即通过上调miR-29a-3p，进而抑制其下游多个促纤维化蛋白的表达，此外益母草碱的抗炎效应也发挥了重要作用。本研究的完成丰富了我们对益母草碱药理学效应的认识，也为心肌纤维化的治疗提供新思路和新方向。