β-arrestin2在δ阿片受体激动剂抑制TLR4炎症通路减轻脑缺血再灌注损伤中的作用及机制研究

Due to the surgical and other reasons, cerebral ischemia-reperfusion injury in the perioperative period is often difficult to avoid, but the treatment is limited, causing the patient disability or death. Our previous study found that delta opioid receptor (DOR) agonist DADLE can significantly reduce cerebral ischemia-reperfusion injury, improve neurological function, but its mechanism is unclear. Neurological inflammation is an important cause and crucial point for brain injury after ischemia and reperfusion. Our Prior study suggests that DOR agonists can inhibit Toll like receptor 4 up-regulation in ischemic penumbra and decrease NF-κB production. While β-arrestin2 is known as a key molecule that mediates G protein-coupled receptors and TLR4. According to these, our hypothesis is DOR agonists can negatively control TLR4 pathway through β-arrestin2, inhibitingTLR4 downstream TRAF6 and NF-κB signal, reducing the production of downstream inflammatory factors and so play a role in ameliorating crebral ischemia-reperfusion injury. This present study is to use TLR4, β-arrestin2 knockout mice setting up middle cerebral artery occlusion model and use primary microglia, BV2 and neuron with hypoxia and sugar-free model to confirm the hypothesis. This study is expected to reveal the mechanism of DOR agonist brain protection, and provide new ideas for prevention and treatment of cerebral ischemia-reperfusion injury.

由于手术操作等原因，脑缺血再灌注损伤在围术期往往难以避免，但治疗手段有限，病人残疾或死亡，预后极差。课题组先前的研究发现，δ阿片受体（DOR）激动剂DADLE可明显减轻脑缺血再灌注损伤，改善神经功能，但其机制尚不明确。神经炎症是缺血再灌注过程中加重脑损伤的重要原因。预实验结果提示，DOR激动剂可抑制缺血半暗带区TLR4上调，减少NF-κB产生。而β-arrestin2是介导DOR和TLR4的关键分子。据此提出假说：DOR激动剂通过β-arrestin2负调控TLR4通路，抑制其下游TRAF6 和NF-κB信号转导，减少下游炎症因子产生，稳定血脑屏障，从而发挥减轻脑缺血再灌注损伤作用。本研究拟采用TLR4、β-arrestin2基因敲除鼠，应用大脑中动脉栓塞模型和原代小胶质细胞、神经元等缺氧复氧模型证实上述假说。本研究有望揭示DOR激动剂脑保护作用的机制，为防治脑缺血再灌注损伤提供新切入点。

本研究成功建立SD大鼠、C57BL/6J小鼠脑缺血再灌注损伤在体模型和SH-SY5Y缺氧复氧体外模型。体外研究发现，缺氧过程中给予δ阿片受体激动剂DADLE能够提高细胞生存率，且其保护作用与剂量相关；体内研究证实，侧脑室注射和尾静脉注射δ阿片受体激动剂DADLE均对脑缺血再灌注损伤具有保护作用。进一步探索相关机制发现，神经元和小胶质细胞的TLR4在脑缺血再灌注后表达上调；而δ阿片受体激动剂可抑制脑缺血再灌注损伤后TLR4的上调并抑制受体后炎症调控基因NF-κB的表达，同时降低下游炎症因子TNF-α和IL-6的水平；δ阿片受体激动剂还可抑制脑缺血再灌注损伤后TLR4信号通路下游分子Myd88的上调。据此我们得出结论：δ阿片受体激动剂DADLE可能通过抑制依赖Myd88的TLR4信号通路抑制NF-κB的表达水平，从而降低中枢炎症反应，减轻脑缺血再灌注损伤。此外，我们还对调控去乙酰化酶（HDAC）的表达能否减轻脑损伤做了一定探索，发现抑制HDAC3可减轻脑缺血再灌注损伤。综上所述，本项目提示δ阿片受体有望成为防治脑缺血再灌注损伤的新靶点，为防治脑损伤提供了重要的实验室依据。如今后能拓展到临床，将极大减轻多种原因导致的脑缺血再灌注损伤，具有广阔应用前景。