proBDNF通过P75NTR/sortilin受体促进心肌缺血再灌注损伤的作用及机制研究

The cardiopulmonary bypass (CPB) is the essential method of cardiac surgery. As a result of many years of clinical and experimental research, the cardiac protection and skills of operation have been improved, but the mortality of heart operation with CPB still remains 2-5 %. The myocardial ischemia-reperfusion (I/R) injury during the CPB, could be one of the main reasons contributing to cardiac dysfunction, which is influenced by many risk factors and various molecular mechanisms. Pro-brain derived neurotrophic factor (BDNF), the precursor of BDNF, is a well characterized cytokine in the brain induced by injury. ProBDNF activates p75 neurotrophin receptor (p75(NTR)) and sortilin receptors to mediate proapoptotic responses. In our previous research, we found that Ab-proBDNF can significantly improve cardiac function and reduce infarct size after myocardial I/R in mouse. Further studies found that rapid upregulation of proBDNF and its receptors, P75NTR and sortilin, was observed in myocardium after reperfusion. This implied that proBDNF may be an important factor to regulate myocardial ischemia-reperfusion injury. In this study, firstly, we will isolate primary cardiomyocytes from neonatal mouse and established an in vitro model of hypoxia/reoxygenation (H/R) which resembles I/R in vivo. Together with the classic in vivo model of myocardial I/R in mouse, we will investigate the effects of proBDNF on myocardial I/R injury. Next, we will clarify the molecular mechanism of signaling pathway of P75NTR/sortilin -JNK-caspase-3 in myocardial I/R injury induced by proBDNF. Our findings will suggest an important role of cardiac proBDNF in exacerbation of myocardial I/R injury and serve as a target for pharmacotherapeutic intervention in I/R-induced myocardial injury.

尽管心肌保护策略得到了迅速发展，如何减轻体外循环下心血管大手术因缺血/再灌注(ischemia/reperfusion, I/R)导致的心肌损伤仍是心血管麻醉医生面临的重大问题。课题组前期实验发现抗-脑源性神经营养因子前体抗体(Ab-proBDNF)可显著改善心肌I/R损伤后的心功能并减少梗死面积；并且proBDNF及其受体在I/R后心肌内表达上调，提示proBDNF可能是调控心肌I/R损伤的重要因子。本课题拟在此基础上，采用心肌细胞缺氧复氧模型和小鼠心肌I/R损伤模型，通过干预proBDNF 的表达，分别从体外和体内水平证实proBDNF促进心肌I/R损伤；进一步采用受体缺失的基因敲除小鼠模型探讨P75NTR/sortilin-JNK- caspase-3信号通路在proBDNF促进心肌I/R损伤中的作用机制。本研究将为探索I/R损伤新机制及寻找新的围术期心肌保护靶点提供线索及依据。

缺血再灌注损伤是指器官在缺血的基础上恢复血供后，损伤反而加重的现象。体外循环下心血管大手术往往对心脏、脑等重要器官造成了缺血再灌注损伤，而这些器官的损伤程度则往往决定了病人的预后。因此，围术期重要器官的保护是心血管大手术麻醉医师非常关注的问题，而心肌保护则是核心。有研究表明：脑源性神经营养因子除了在神经系统中起着重要作用之外，还在血管内皮细胞的存活、促进缺血部位内皮细胞的增殖、移行中扮演着重要角色。BDNF在胚胎发育过程中调控了心肌细胞的增殖、迁移和分化，在心脏发育、血管生成，心肌肥厚及动脉粥样硬化发生发展过程中发挥了重要作用。课题组前期研究发现，proBDNF及其受体P75在正常心肌组织中几乎不表达，然而在受到缺血再灌注等伤害刺激的时候会被激活。因此，课题组研究内容主要为明确proBDNF是促进心肌的缺氧复氧损伤的重要因子，并进一步探讨其促凋亡的机制。免疫荧光及western blot结果初步表明大鼠心肌经历缺血再灌注损伤后的2h,1d，梗死区proBDNF及其受体、相关凋亡因子表达显著上调，心肌注射proBDNF腺病毒转染成功后，一定程度上加重了大鼠心肌缺血再灌注损伤，TTC检测梗死面积扩大，TUNEL检测凋亡增加，其可能作用的通路为JNK-caspase-3，心肌注射proBDNF的中和抗体之后，对心肌的损伤作用减轻。离体心肌细胞培养及缺氧复氧模型的结果同在体实验一致。上述结果在一定程度上揭示了proBDNF的生物学新功能，为基础理论做补充，初步表明ab-proBDNF作为心肌保护的新药物将具有很好的临床应用价值，可以作为临床心肌保护的新靶点。