九龙藤总黄酮调控PI3K/Akt信号通路抗心肌缺血再灌注损伤的作用及机制研究

Reducing MIRI is urgent problem in prevention and treatment of coronary heart disease. Research shows that PI3K/Akt pathway regulates apoptosis, affects metabolism, adjusts autophagy, which plays an important role in MIRI. Previous studies have demonstrated that total flavonoids of Bauhinia championii scavenged free radicals, improved myocardial energy metabolism by increasing ATPase and reducing LDH, reduced inflammation and myocardial injury by inhibiting TNF-α, CK-MB, and iNOS, as well as increasing eNOS, inhibited apoptosis by up-regulating Bcl-2, downregulating Bax and NF-kappa beta protein expression, and enhanced cardiac function by increasing HR, ± dp/dtmax, and decreasing LVEDP. These roles involve multiple PI3K/Akt pathway targets, such as mPTP, ATPase, TNF-α, eNOS, Bcl-2 family, NF-κB, etc. Therefore, we speculate that total flavonoids of Bauhinia championii produce anti MIRI effects through the intervention of PI3K/Akt pathway. This project will study the effects of total flavonoids of Bauhinia championii on PI3K/Akt pathway and its specific targets by adopting multiple technologies, such as cell biology, animal disease models, molecular biology, immunology, and so on. These studyings will be conducted in in vitro and in vivo, including signaling pathway, cell, gene and protein levels. According to this project, we hope to provide new ideas on prevention and treatment of coronary heart disease.

减轻MIRI是冠心病防治中亟待解决的问题。研究表明，PI3K/Akt通路调控凋亡、影响代谢、调节自噬，在MIRI中发挥重要作用。课题组前期研究发现，九龙藤总黄酮清除自由基；增加ATPase，减少LDH，改善心肌能量代谢；抑制TNF-α、CK-MB、iNOS，提高eNOS，减少炎症反应和心肌损伤；上调Bcl-2、下调Bax、NF-κβ蛋白表达，抑制凋亡；增加HR、±dp/dtmax，降低LVEDP，改善心功能。上述作用涉及多个PI3K/Akt通路靶点如mPTP、ATPase、TNF-α、eNOS、Bcl-2家族、NF-κβ等。据此，我们推测九龙藤总黄酮通过干预PI3K/Akt通路发挥抗MIRI作用。本项目拟通过细胞生物学、动物疾病模型、分子生物学、免疫学等技术，从体内、外层次，信号通路、细胞、基因和蛋白水平研究九龙藤总黄酮对PI3K/Akt通路的作用和具体效应靶点，为冠心病的防治提供新思路。

探讨天然药物活性成分对MIRI的作用是冠心病防治研究的热点，本项目采用先进的植化技术从豆科羊蹄甲属植物龙须藤中提取、分离、纯化九龙藤总黄酮（Bauhinia championii flavones，BCF）；建立心肌细胞H/R模型，以流式细胞术测定细胞凋亡率和坏死率，免疫荧光法观察自噬小体变化，FQ-PCR、WB检测PI3K/Akt通路靶点、坏死关键调控蛋白、凋亡、自噬相关基因、蛋白的表达以及自噬通量变化，验证BCF对H/R的干预作用；在此基础上，通过使用抑制剂或转染的方式，探讨PI3K-Beclin1 -mTOR对H/R受损心肌细胞的影响及BCF的作用。建立大鼠MIRI模型，研究BCF对MIRI不同时段自噬的影响；以伊文思蓝和TTC双染法测定心肌梗死面积，MS4000系统观测心功能，ELISA测定血清中心肌损伤标志物、抗氧化物、炎性因子的活性，TUNEL检测心肌凋亡，透射电镜观察心肌自噬小体，FQ-PCR、WB检测PI3K/Akt通路靶点以及凋亡、自噬相关基因、蛋白表达与磷酸化水平。.研究结果表明：BCF预处理降低CK-MB水平、减轻∆Ψm和 MPTP开放、改善心功能、减少心肌梗死面积；降低iNOS、LDH、ROS、MDA、增加T-AOC、ATP、SOD、GSH-Px活性；减少IL-6和TNF-α水平、下调 TLR4、NF-κB和p-NF-κB 蛋白表达；上调Cyt-c、Bcl-2、下调cleaved caspase-3、Bax 基因、蛋白表达，抑制凋亡；下调RIPK3蛋白表达，减轻坏死；上调PI3K和Akt 基因、蛋白表达与磷酸化水平，激活PI3K/Akt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，PI3K/Akt通路抑制剂—LY294002或Wortmannin可减弱BCF对损伤心肌细胞的保护作用，而BCF亦可部分逆转Rapamycin对自噬的正性调控作用；沉默或过表达Beclin 1基因均减弱BCF抑制自噬的作用。.通过研究，阐明九龙藤总黄酮通过激活PI3K/Akt通路、抗氧化、抗炎、抑制凋亡及再灌注期自噬过度表达而减轻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。