抑制p16保护心肌缺血再灌注损伤的作用和机制研究

The apoptosis of cardiomyocytes is an essential pathological process in myocardial ischemia/reperfusion (I/R) injury. p16 is a cyclin kinase inhibitor, while inhibition of p16 can prolong cell lifespan and reduce cell apoptosis. We previously found that p16 was upregulated in a mouse model of myocardial I/R injury, while inhibition of p16 reduced cardiomyocyte apoptosis in vitro and reduced infarct size after I/R injury in vivo, indicating that p16 might be involved in the regulation of myocardial I/R injury. In the present project, we will use neonatal rat cardiomyocytes transfected with p16 overexpression plasmid or siRNA, as well as myocardial specific p16 overexpression or knockout mouse, and establish oxygen glucose deprivation/reperfusion (OGD/R) cell model and surgical I/R injury animal model respectively, to observe the impact of p16 on cardiomyocyte apoptosis and I/R injury. Further, we will use Akt activator and inhibitor in vitro, as well as intramyocardial injection of Akt overexpression lentivirus and shRNA lentivirus in vivo, to perform function-rescue experiments to clarify the role of Akt mediating the regulatory effect of p16 in myocardial I/R injury. Our project will propose a novel therapeutic strategy for myocardial I/R injury based on inhibition of p16.

心肌细胞凋亡是心肌缺血再灌注（I/R）损伤的重要病理改变。p16是一种细胞周期抑制蛋白，抑制p16可以延长细胞寿命、降低细胞凋亡。我们在前期初步发现，p16在小鼠心肌I/R损伤模型中的表达上调，抑制p16可以在体外降低心肌细胞凋亡，在体内降低心肌I/R损伤后的梗死面积，这提示p16可能参与调控心肌I/R损伤。在本项目中，我们将在新生大鼠心肌细胞水平转染p16的过表达质粒和小干扰RNA，或者采用心肌特异性p16过表达和敲除小鼠，分别在氧葡萄糖剥夺/恢复（OGD/R）模型和手术诱导心肌I/R损伤模型中，观察p16对心肌细胞凋亡和I/R损伤的影响。进一步，运用Akt的激动剂和抑制剂（细胞水平），或者运用Akt过表达慢病毒和shRNA慢病毒的心肌层注射（动物水平），通过功能逆转实验，明确Akt在p16调控心肌I/R损伤中的分子机制。本项目将基于抑制p16提出防治心肌I/R损伤的新思路。

心肌凋亡是心肌缺血再灌注（I/R）损伤的重要病理改变。p16是一种细胞周期抑制蛋白，抑制p16可以延长细胞寿命、降低细胞凋亡。我们在前期初步发现，p16在小鼠心肌I/R损伤模型中的表达上调，但p16是否参与调控心肌I/R损伤过程是未知的。本项目首先在新生大鼠心肌细胞水平构建了氧葡萄糖剥夺恢复（OGDR）诱导的细胞凋亡模型，明确p16在OGDR模型中表达上调。接着，在新生大鼠心肌细胞水平，明确了下调p16可以抑制OGDR诱导的心肌细胞凋亡，但不影响心肌细胞增殖，过表达p16可以加剧OGDR诱导的心肌细胞凋亡，抑制心肌细胞的增殖。接着，我们通过细胞水平功能逆转实验明确了AKT/mTOR信号通路参与介导了p16调控心肌细胞凋亡过程，鉴定出AKT和mTOR是p16的下游分子。进一步，基于心肌特异性p16敲除小鼠，构建心肌I/R模型，明确心肌特异性敲除p16可显著改善I/R手术后的心肌梗死面积和心肌凋亡，且心肌特异性敲除p16能够在I/R损伤重构期改善心功能。通过上述研究，本项目明确了p16 与心肌缺血再灌注损伤的关系，鉴定出下调p16通过激活AKT/mTOR信号通路抑制心肌凋亡的分子机制，提出抑制 p16 防治心肌缺血再灌注损伤的新思路。综上，下调p16在心肌保护中发挥重要作用，抑制p16有望为抑制心肌凋亡和心肌缺血再灌注损伤提供新的治疗靶点。