Akt/eNOS/NO通路在脓毒症小鼠心肌calpain活化caspase-3和NF-κB中的作用

Cardiac dysfunction is one of the leading causes of death in patients with sepsis, the involving mechnism is unclear and there is no effective way to prevent and treat. Our recent study indicated that myocardial calpain activation triggered activation of caspase-3 and NF-κB, and caused cardiac dysfunction in septic mice, while inhibition of calpain activity blocked the pathological process and improved cardiac function. However, the molecular mechanism under which calpain induced activation of caspase-3 and NF-κB in sepsis remain unknown. Our preliminary data suggested that Akt/eNOS/NO pathway might play a potential role in this pathological process. In this project, we propose to investigate the role of Akt/eNOS/NO signal pathway in the pathologial process of which calpain induced caspase-3 and NF-κB activation in sepsis. Specific inhibition of calpain in the in vitro system will be achieved by using chemical inhibitor of calpain, the overexpression of calpain inhibitor protein calpastatin, and the role of Akt/eNOS signal will be also identified by such ways as chemical drugs and overexpression. For in vivo study, calpastatin transgenic and eNOS transgenic mice will be used. The changes in the activation status of caspase-3 and NF-κB in sepsis will then be assessed.Further understanding of these signaling pathway might lead to novel pharmacological therapy to cardiac dysfunction in sepsis.

心功能障碍是脓毒症患者死亡的主要原因之一，其发生机理不详且缺乏有效的防治措施。申请人近年来研究发现，在脓毒症小鼠中，钙激活中性蛋白酶（calpain）活性增高，并诱导心肌caspase-3和NF-κB活化，导致心功能障碍，抑制calpain活性后可阻断这一病理改变并改善心功能障碍，但具体分子机制尚不清楚。申请人最近研究提示calpain可能通过抑制Akt/eNOS/NO通路来诱导caspase-3和NF-κB活化。本项目拟通过对calpain和Akt/eNOS选择性干预，包括在体外细胞系统中使用药物抑制剂、过表达calpain特异性抑制蛋白-calpastatin和Akt/eNOS，在体内系统中使用calpastatin转基因及eNOS转基因小鼠，从体内体外两个系统中阐明Akt/eNOS/NO是否参与calpain诱导caspase-3和NF-κB活化，为治疗脓毒症心功能障碍提供新的靶点。

心功能障碍是脓毒症患者死亡的主要原因之一，其发生机理不详且缺乏有效的防治措施。在脓毒症小鼠中，钙激活中性蛋白酶（calpain）活性增高，并诱导心肌半胱天冬氨酸蛋白酶-3（caspase-3）和细胞核因子-κB（NF-κB）活化，导致心功能障碍，抑制calpain活性后可阻断这一病理改变并改善心功能障碍。本项目通过对calpain和Akt/eNOS选择性干预，包括在体外细胞系统中使用多种药物抑制剂、过表达calpain特异性抑制蛋白-calpastatin和RNA干扰等，在脓毒症小鼠/大鼠体内系统中使用calpastatin转基因及各种药物抑制剂，从体内体外两个系统中阐明了Akt/eNOS/NO参与calpain诱导caspase-3和NF-κB活化的过程，引起心肌细胞凋亡和炎症反应。在脓毒症体内外模型中，calpain活性增高，导致Akt/eNOS通路活性下降，其抗心肌细胞凋亡和炎症的作用减弱，从而参与介导了calpain诱导脓毒症心功能障碍的发生。我们的研究成果为calpain 抑制剂的临床应用提供实验依据及临床脓毒症所致心功能障碍提供新的治疗靶点。