益气通络颗粒及主要单体通过调节cAMP/PKA/Complex I通路治疗气虚血瘀证脑梗死的机制研究

Tonifying Qi and Promoting Blood prescription is main and effective medicine for Qi deficiency and blood stasis (QDBS) syndrome clinically. However, there is still deficient in systematic and in-depth study on the mechanism of these medicines. On the basis of previous genome research for QDBS animals’ model and in vitro study results that YQTL and theirs main active components regulated the level of ROS and ATP by acting on mitochondrial electron transport chain. Since ROS and ATP Play crucial roles in cells and are product of METC, we put forward the hypothesis that YQTL may improve activity of mitochondrial electron transport chain to regulating mitochondrial ROS and ATP level therefore protects brain cells from death in QDBS infarction rats. .In this study, we would apply Multiple Cerebral Infarction rats model as well as oxygen and sugar deprivation cells model to investigate the effect of YQTL and theirs main active components on apoptosis of nervous cells，the activity of mitochondrial electron transport chain and its products such as ROS and ATP. In the aspect of mechanism research, we would research the regulation role of cAMP/PKA/Complex signal pathway following YQTL and theirs main active components treatment in vivo and vitro. These studies will provide potential molecular mechanism on YQTL tonifying Qi and promoting blood, and it will bring theoretical evidence and support for the clinical application of YQTL, as well as some new viewpoints and means for study of QDBS syndrome and such medicines development.

益气活血方是临床上治疗气虚血瘀脑梗死的主要方剂，但关于其发挥药效的作用机制尚缺乏系统深入的研究。本课题针对气虚血瘀脑梗死这一临床多发疾病，在前期气虚血瘀基因组学的研究基础上，以气虚血瘀的作用分子线粒体电子传递链为切入点，结合前期体外实验结果“益气通络颗粒及其主要入脑单体成分能够调节神经细胞内电子传递链产物ROS及ATP水平”，提出假说：益气活血方益气通络颗粒通过作用于线粒体电子传递链，改善气虚血瘀证脑梗死大鼠线粒体ROS及ATP的产生，保护脑细胞免于死亡。.本课题借助气虚血瘀脑梗死大鼠模型以及神经细胞氧糖剥夺模型，从整体及细胞水平上观察益气通络颗粒及主要入脑单体成分对细胞凋亡、线粒体电子传递链活性及产物ROS、ATP的影响，评价益气通络颗粒益气活血的功效；并进一步探讨益气通络颗粒对线粒体复合体cAMP/PKA/Complex I 信号通路的调控，以期阐明益气通络颗粒益气活血的可能机制。

脑梗是最常见的致残因素之一，气虚血瘀证是脑梗最主要的证型。益气活血方是临床上治疗气虚血瘀证脑梗死的主要方剂，但关于其发挥药效的作用机制尚缺乏系统深入的研究。益气通络颗粒（YQTL）是经典名方补阳还五汤的改良方，本项目的主要研究内容是探讨YQTL治疗脑梗后认知障碍中线粒体形态、功能、ATP和ROS产生以及线粒体内cAMP/PKA/Complex I通路等方面的变化，以阐明缺氧情况下YQTL改善海马损伤致认知障碍的分子调节机制。我们通过使用水迷宫，蛋白组学，免疫组化，western blotting以及其它的分子生物学技术，得出两方面的重要结果：（1）发现YQTL降低了由气虚血瘀脑梗带来的认知障碍，而在这个过程中，YQTL显著影响了海马组织线粒体的结构、复合体活性以及ATP和ROS的产生，降低了海马神经元的凋亡；探讨其可能的机制是YQTL通过作用于线粒体内cAMP/PKA/Complex I通路保护缺氧海马组织的线粒体功能，调节ROS和ATP的生成，降低凋亡，因此，cAMP/PKA/Complex I通路可能是YQTL治疗缺血后认知障碍的潜在机制；（2）在此基础上，我们研究了YQTL的活性单体成分，结果显示黄芪甲苷（AST-IV）对氧糖剥夺（OGD）原代神经元具有较好的抗缺氧活性。通过使用PKA激酶抑制剂H-89，我们发现，AST-IV可能是通过直接作用于PKA而保护缺氧神经元的线粒体，调节ROS和ATP的产生，最终抑制了神经元的凋亡。总之，YQTL作为一个治疗脑梗的药物，其明显地改善了大鼠气虚血瘀脑梗后认知功能下降； YQTL及其活性成分，AST-IV，均通过激活原代神经元cAMP/PKA/Complex I通路，增强线粒体复合体活性，提高神经元的活力。本项目的科学意义在于揭示了YQTL如何调控线粒体的形态、功能、以及ROS和ATP的产生，降低凋亡，进而改善脑缺血后的认知障碍。