BDNF减轻糖尿病心肌缺血再灌注损伤的新机制：Caveolin-3/Trk介导的抗细胞程序性坏死

Patients with diabetes are susceptible to myocardial ischemia reperfusion injury (IRI) and the underlying mechanism is not clear. Recent studies showed that brain derived neurotrophic factor (BDNF) can protect hearts against myocardial IRI, while our preliminary studies showed that BDNF cardioprotection is lost in hearts from diabetes that was associated with increased myocardial necroptosis, a new form of programmed cell death. We further found that the association of caveolin-3 and BDNF receptor Trk was significantly reduced in hearts from diabetes after myocardial IRI. In cardiomyocytes exposed to high glucose, supplementation of caveolin-3 in combination with BDNF conferred cellular protection accompanied with reduced necroptosis. Thus, we hypothesized that BDNF confers cardioprotection by reducing necroptosis through caveolin-3/Trk signaling, while in diabetes, the impairment of caveolin-3/Trk leads to the loss of BDNF. In the proposed study, we will induce in vivo myocardial IRI and diabetes in C57BL/6 mice or Caveolin-3-/- mice in cooperation with BDNF and Caveolin-3 adenovirus to overexpress BDNF and caveolin-3 in diabetes. We will also use in vitro cell model with hypoxic reoxygenation to confirm our in vivo model and to determine the role of necroptosis in caveolin-3/Trk-mediated BDNF cardioprotection. The proposed studies will improve our understanding of the pathophysiology of this phenomenon and may help facilitate the development of novel and optimal therapies in diabetic heart disease.

糖尿病心肌缺血再灌注损伤（IRI）加重，但机制未明。新近研究表明脑源性神经营养因子（BDNF）具有心肌保护作用，然而我们预实验发现糖尿病小鼠心肌中BDNF保护效果被取消且心肌细胞程序性坏死增加，同时，糖尿病心肌IRI过程中BDNF受体Trk与小窝蛋白Caveolin-3结合降低。体外高糖培养心肌细胞中，过表达心肌Caveolin-3并给予外源性BDNF，缺氧复氧后心肌细胞程序性坏死减少。我们推测糖尿病心肌中Caveolin-3/Trk信号通路受损是导致BDNF保护效果消失的关键原因。本课题拟在此基础上，在野生型及Caveolin-3基因敲除小鼠上建立糖尿病模型和心肌I/R损伤模型，结合Caveolin-3腺病毒及BDNF腺病毒，同时建立离体细胞缺氧复氧模型，确认在体实验结果。从在体及细胞水平阐明BDNF减轻糖尿病心肌IRI的作用机制。本研究将为寻找新的围术期心肌保护靶点提供线索及依据。

糖尿病心肌缺血再灌注损伤（IRI）加重，但机制未明。新近研究表明脑源性神经营养因子（BDNF）具有心肌保护作用，然而我们研究发现糖尿病心肌中BDNF保护效果被取消且necroptosis（一种新型的细胞程序性死亡）升高，同时糖尿病心肌IRI过程中BDNF受体TrkB与Caveolin-3结合降低。给予外源性necroptosis抑制剂Nec-1处理后，糖尿病心肌IRI梗死面积明显较小。且体外高糖培养心肌细胞中，过表达心肌Caveolin-3并给予外源性BDNF，能减少缺氧复氧后心肌细胞necroptosis。因此，我们推测糖尿病心肌中Caveolin-3/TrkB信号通路受损是导致BDNF保护效果消失的关键原因。.为探讨其可能机制，我们研究了cAMP/PKA、β2AR及BDNF/TrkB信号通路在Caveolin-3改善糖尿病心肌IRI中的作用。正常及糖尿病大鼠进行心肌缺血再灌注，与假手术组相比，非糖尿病I/R组ADRB2 (β2AR) 和cAMP/PKA 信号通路受损，ADRB mRNA表达下调、ADRB2和cAMP蛋白表达下调及p-PKA磷酸化下调；与I/R组相比，糖尿病I/R组受损更加严重。在H9C2细胞系中，进行高糖培养能进一步加重细胞缺氧复氧（H/R）损伤：增加细胞凋亡、降低细胞活性以及抑制TrkB和Akt信号通路。然而，给予ADRB2激动剂ISO能促进BDNF/TrkB和cAMP/PKA信号通路的活性，从而明显减轻上述损伤。此外，结果表明，Caveolin-3过表达能促进ADRB2在高糖培养H9C2细胞系的细胞膜定位，从而抑制凋亡，促进细胞的活性。而在高糖刺激下，Caveolin-3过表达可增强cAMP/PKA和BDNF/TrkB信号通路的活性，cAMP、TrkB 蛋白表达和PKA、Akt磷酸化增加，而ADRB2沉默能逆转Caveolin-3 过表达引起的这些改变。.综上所述，necroptosis在Caveolin-3/TrkB介导的 BDNF心肌保护中起到重要作用。Caveolin-3可通过β2AR、cAMP/PKA和BDNF/TrkB信号通路发挥糖尿病心肌缺血再灌注损伤的保护作用。.探索在非糖尿病机体上有明确保护作用的措施在糖尿病机体上失效的原因。将为寻找新的围术期心肌保护靶点提供线索，并为制定相关有效的防治措施提供依据。