抑制βarrestin2介导受体内吞增强δ阿片受体激动剂在脑缺血再灌注损伤中的保护作用及其机制研究

Ischemic stroke is a leading cause of brain injury worldwide which has high prevalence, high mortality and high morbidity. Ischemia-reperfusion injury (IRI) is the main pathophysiological processes after ischemic stroke. However, there is still no effective therapy available to salvage nerve cell presently. Previous studies have found that the delta opioid receptor (DOR) plays an important role in IRI induced brain injury through two distinct mechanisms. 1) Ischemic/hypoxia preconditioning increases DOR expression, and the exogenous agonists mitigate the damage of nerve cell involving anti-oxidative mechanism. 2) Simulation of DOR by endogenous DOR ligands may play a self-preserving role in IRI. However, exogenous DOR agonists cannot provide further neuroprotective effects due to endocytosis of DOR under IRI. Downregulation DOR is mainly mediated by β arrestin 2. In the present study, we aim to identify whether DOR agonist can provide further neuroprotective effects in IRI by DOR overexpression in both cultured cells and animal models. And also, we will investigate whether decreased DOR expression in IRI is mediated by β arrestin 2, and whether DOR agonist show further neuroprotective effect in IRI after inhibition of endocytosis by knockdown of β arrestin 2 expression. Our study is supposed to further deepen the knowledge of neuroprotective effect of opioid receptor and thus provide new insight to the development of new therapies to relieve neuron damage by ischemic stroke.

缺血性脑卒中具有患病率、死亡率、致残率高的特点，严重威胁人类健康。缺血再灌注损伤（IRI）是缺血性脑卒中的主要病理生理过程，临床尚无有效保护或逆转神经细胞损伤的措施。本课题组研究发现IRI时δ阿片受体（DOR）表达显著下降，内源性DOR配体与受体结合发挥自我保护作用且使DOR功能接近饱和，外源性DOR激动剂仅能产生有限的保护。β arrestin介导的受体内吞是DOR受体下调的主要途径。因此，本项目拟应用过表达DOR的细胞和大鼠模型，查明上调DOR表达是否可增强DOR激动剂对IRI的保护作用；并应用稳定敲减β arrestin 2的细胞模型，应用免疫荧光、免疫共沉淀方法查明IRI所致DOR表达下调是否由β arrestin 2介导，并观察下调β arrestin 2是否可增强DOR激动剂的IRI保护作用。本课题将加深对阿片受体脑保护作用机制的研究，为新型抗缺血性脑卒中药物研究奠定基础。

缺血性脑卒中具有患病率、死亡率、致残率高的特点，严重威胁人类健康。缺血再灌注损伤（IRI）是缺血性脑卒中的主要病理生理过程，临床尚无有效保护或逆转神经细胞损伤的措施。本课题组研究发现IRI时δ阿片受体（DOR）表达显著下降，内源性DOR配体与受体结合发挥自我保护作用且使DOR功能接近饱和，外源性DOR激动剂仅能产生有限的保护。β arrestin介导的受体内吞是DOR受体下调的主要途径。因此，本项目拟应用过表达DOR的细胞，查明上调DOR表达是否可增强DOR激动剂对IRI的保护作用；并应用稳定敲减β arrestin 2的细胞模型，查明IRI所致DOR表达下调是否由β arrestin 2介导，并观察下调β arrestin 2是否可增强DOR激动剂的IRI保护作用。我们发现DOR的细胞保护作用随着DOR受体过表达可以进一步增强，随着DOR受体的敲减而减少；并且这一重要的细胞保护作用机制是通过调控抗氧化应激分子Nrf2，及其下游靶分子NQO1，HO1等发挥作用。β arrestin介导的DOR受体内吞影响阿片受体抗IRI的作用，减少了由DOR受体介导的一系列抗氧化应激作用。本课题发现DOR受体介导的配体受体相互作用可以在神经性细胞中发挥抵抗缺血再灌注损伤的重要功能作用。在今后的实验工作中，针对上述的研究发现，拟采用联合βarrestin和DOR的受体激动剂，联合抵抗缺血再灌注损伤对神经细胞的严重损伤，为新型抗缺血性脑卒中药物研究奠定基础。