基于miRNA-214调控细胞自噬探讨活血涤痰法干预高血压心室重构的作用机制

Previous studies had showed that miRNA-214 had been involved in the development of hypertension. However, the mechanism of miRNA-214 involved in the ventricular remodeling of hypertension has not been elucidated. In this study, spontaneously hypertension ventricular remodeling model rats and AngⅡinduced cardiomyocytes were used as the research object. We will study the mechanism of miRNA-214 on reverse ventricular reconstruction through the activating blood and wiping out phlegm method prescription intervention. The expression of miRNA-214 targeting PTEN gene-mediated PI3K/AKT/mTOR signaling pathway will be studied by morphology immunohistochemistry/immunofluorescence staining, laser confocal microscopy and molecular biology techniques and through overexpression or deletion the miRNA-214 and PTEN expression and signal pathway specific blocking agent. Our studys clarify the signal transduction mechanism of miRNA-214 regulation cell autophagy, reveal the activating blood and wiping out phlegm method to intervene in hypertension ventricular remodeling through regulating the expression miRNA-214 targeting PTEN gene-mediated PI3K/AKT/mTOR signaling pathway. It can help to elucidate the ventricular remodeling mechanism of the activating blood and wiping out phlegm method in hypertension.

基于前期研究显示miRNA-214参与高血压的发生发展过程，但其参与高血压心室重构的机制尚未阐明，本项目选择自发性高血压心室重构模型大鼠和AngⅡ诱导心肌细胞为研究对象，通过动物与细胞相结合，以逆转心室重构为切入点，选择miRNA-214调控细胞自噬为主线，采用活血涤痰法方药进行干预；运用形态学、免疫组织/细胞荧光染色、激光共聚焦、分子生物学技术等方法研究高血压心室重构中miRNA-214靶向PTEN基因介导PI3K/AKT/mTOR信号通路的表达特点，引入miRNA-214、PTEN过表达和缺失表达技术及信号通路特异性阻断剂；阐明miRNA-214调控细胞自噬的信号转导机制，揭示活血涤痰法干预高血压心室重构是通过miRNA-214靶向PTEN基因介导PI3K/AKT/mTOR信号通路而实现的，有助于阐明活血涤痰法干预高血压心室重构的科学内涵。

基于前期研究显示miRNA-214参与高血压的发生发展过程，但其参与高血压心室重构的机制尚未阐明，本项目选择自发性高血压心室重构模型大鼠和AngⅡ诱导心肌细胞为研究对象，通过动物与细胞相结合，以逆转心室重构为切入点，选择miRNA-214调控细胞自噬为主线，采用活血涤痰法方药进行干预；运用形态学、免疫组织/细胞荧光染色、激光共聚焦、分子生物学技术等方法研究高血压心室重构中miRNA-214靶向PTEN基因介导PI3K/AKT/mTOR信号通路的表达特点；研究结果显示：（1）在自发性高血压模型大鼠中，活血涤痰法方药能够降低血压、改善心室重构，进一步研究显示能够降低心肌细胞凋亡指数；透射电子显微镜观察发现，活血涤痰法方药可抑制自噬流，自噬体明显增多，表现为细胞自噬水平升高，可能与抑制miRNA-214表达、导致靶基因PTEN表达增加相关。（2）动物实验证实活血涤痰法方药能够增强自噬相关基因及蛋白Atg7、Atg5、Beclin1、LC3-II表达及LC3-II/LC3-I表达水平、降低p62 表达，可能与增强PI3K、AKT及mTOR的磷酸化蛋白及基因表达，激活PI3K/AKT/mTOR信号通路，导致细胞自噬增加。（3）体外实验证实miRNA-214调控的靶基因为PTEN；AngⅡ诱导心肌细胞肥大模型显示，心肌细胞凋亡减弱，细胞增殖增强，自噬水平降低，可能与miRNA-214表达增强、导致靶基因PTEN表达下降、抑制PI3K/AKT/mTOR信号通路相关；（4）细胞学实验证实，活血涤痰法含药血清对AngII诱导的心肌细胞肥大模型的细胞凋亡具有促进作用、抑制细胞增殖，增强细胞自噬，可能与抑制miRNA-214表达、导致靶基因PTEN表达增加，从而激活PI3K/AKT/mTOR信号通路相关。（5）共有129个mRNAs发生显著变化，其中16个mRNAs经中药复方处理后得到显著调控；这些mRNAs可以通过调节细胞代谢过程、生物粘附、节律过程和细胞自噬等生物学过程发挥治疗作用；参与自噬的细胞信号通路可能是AGE-RAGE/PI3K/Akt /mTOR信号通路。阐明miRNA-214调控细胞自噬的信号转导机制，揭示活血涤痰法干预高血压心室重构是通过miRNA-214靶向PTEN基因介导PI3K/AKT/mTOR信号通路而实现的，有助于阐明活血涤痰法干预高血压心室重构的科学内涵。