九龙藤总黄酮通过miR30a/Beclin1–MAPK调节自噬抗心肌缺血再灌注损伤的机制研究

Effects and mechanisms of traditional Chinese medicine with promoting blood circulation and removing blood stasis on myocardial ischemia reperfusion injury(MIRI)is a hotspot in current researchs. Reports showed that miR-30a and mitogen activated protein kinase (MAPK) played an important role in heart diseases. Previous studies have demonstrated that total flavonoids of Bauhinia championii inhibited apoptosis by up-regulating Bcl-2, down-regulating Bax, Cytc, CaSR, and Caspase-3, as well as reduced autophagy in reperfusion period by activing PI3K/Akt passway, raising the target of rapamycin, and down-regulating markers expression of autophagy, such as LC3-II and Beclin1. These roles involve the target gene of miR-30a , i.e. Beclin1, and multiple MAPK down-stream targets. Therefore, we speculate that total flavonoids of Bauhinia championii produce anti-MIRI effects through affecting miR-30a, interfering protein phosphorylations of MAPK pathway, and regulating autophagy. This project will observe the influence of total flavonoids of Bauhinia championii on signal cascades of miR-30a and MAPK in vitro and in vivo, while demonstrate its effects on MIRI via miR-30a/Beclin1-MAPK and regulating autophagy. Multiple technologies will be used, such as cell biology, molecular biology, gene knockout animal model technology, and so on. According to this project, we hope to provide ideas and references for research and development of new drugs on prevention and treatment of MIRI.

活血化瘀类中药对心肌缺血再灌注损伤（MIRI)的作用及机制是目前研究热点。报道表明miR-30a与丝裂原活化蛋白激酶（MAPK）在心脏病发挥重要作用。课题组研究发现，九龙藤总黄酮上调Bcl-2、下调Bax、细胞色素C、钙敏感受体、Caspase-3蛋白表达，抑制凋亡；通过激活PI3K/Akt通路，上调雷帕霉素靶蛋白、下调自噬标志物LC3-Ⅱ、Beclin1蛋白表达，抑制再灌注期自噬发生。上述作用涉及miR-30a的靶基因Beclin1与MAPK下游多个靶点。据此，我们推测九龙藤总黄酮通过作用miR-30a，影响MAPK通路蛋白磷酸化，调控自噬抗MIRI。本项目拟采用细胞生物学、分子生物学、基因敲除动物模型等技术，观察九龙藤总黄酮对miR-30a、MAPK体内外信号级联的影响，论证其通过miR-30a/Beclin1-MAPK调节自噬抗MIRI的作用，为防治MIRI新药的研发提供思路与参考。

探讨天然药物活性成分对MIRI的作用及机制是冠心病防治研究的热点。本项目通过体外建立心肌缺氧/复氧（H/R）模型，使用MAPK抑制剂为对照，以流式细胞术检测线粒体膜电位（∆Ψm），透射电镜检测自噬小体数量、免疫荧光法观察自噬小体变化，RT-qPCR、western blot 检测MAPK通路靶点及自噬相关基因、蛋白的表达以及自噬通量变化，观察九龙藤总黄酮（Bauhinia championii flavones，BCF）对MAPK通路信号分子及自噬的影响。在此基础上，通过转染相关质粒，探讨miR-30a/Beclin1对H/R受损心肌细胞自噬与MAPK通路的影响以及BCF的干预作用。体内实验建立大鼠心肌缺血再灌注损伤（MIRI）模型，以MAPK抑制剂为对照，以ELISA法测定血清中心肌损伤标志物酶活性，透射电镜观察心肌自噬小体，RT-qPCR、western blot 检测MAPK通路靶点以及自噬相关基因、蛋白表达与磷酸化水平；并利用“在体转染技术”沉默SD大鼠miR-30a基因、基因敲除技术敲除小鼠Beclin1基因，进一步验证miR-30a/Beclin1对MIRI诱导的自噬与MAPK通路的影响及BCF干预的作用。. 研究结果表明：BCF预处理降低∆Ψm与CK-MB水平，减轻MIRI；上调miR-30a基因；下调p38、ERK、JNK基因、蛋白表达与磷酸化水平，抑制MAPK通路；上调p62蛋白表达，下调ATG5、Beclin 1、LC3-II基因、蛋白表达，减少自噬小体数量，抑制自噬。我们发现：BCF与MAPK通路抑制剂均可减弱H/R受损心肌细胞自噬水平，减轻MIRI；此外，过表达miR-30a促进BCF的抑制自噬作用，而沉默miR-30a后，BCF的抑制作用自噬丧失。另一方面，沉默Beclin 1基因对BCF调控MAPK级联信号分子无影响，而过表达Beclin 1基因则逆转了BCF对MAPK级联信号分子的调控作用。综上所述，BCF抑制心肌再灌注自噬过度发生的机制可能与调控miR-30a/Beclin 1-MAPK信号轴有关。. 通过该项目研究，阐明九龙藤总黄酮通过miR-30a/Beclin1-MAPK信号级联，抑制过度自噬，减轻MIRI的作用及分子机制，为进一步开发防治冠心病新靶点药物提供试验依据。