Delta阿片受体激动剂调控脑缺血/再灌注后星形胶质细胞反应及其机制研究

Ischemic injury is traditionally viewed from an axiomatic perspective of neuronal loss. Yet the ischemic infarct encompasses all cell types, including astrocytes. Astrocytes provide structural, trophic, and metabolic support to neurons and can critically influence neuron survival. Thus,regulation of astrocytes is important to neuroprotective effect under ischemia.In our previous studies, we discovered that DADLE (a delta opioid receptor agonist) postconditioning protect against cerebral ischemia/reperfusion injury that not only promotes beneficial astrocytes activation but reduced damaged astrocytes as well, suggesting that functional modulation of the astrocytes may involve in reducing their harmful effects to and hereby contribute to neuronal survival against ischemia. Current understanding on how DOR activation affects astrocytic functions under ischemia remains incomplete. The present study we will investigated the morphological and functional changes with time of the astrocytic reaction following OGD or cerebral ischemia.Further, recent studies indicate that autophagy, a double-edged sword，function as a cytoprotective mechanism under stress or mediates cell death under specific circumstances is needed to allow its manipulation for therapeutic purposes. AMPK, as a critical positive regulatory molecule in promoting autophagy,is upregulated by DOR activation.All of them are involved in controlling neuronal survival and energy metabolic homeostasis.We then evaluated autophagy activation in astrocytes in response to DADLE in the condition of OGD or global ischemia. Alterations of astrocytes were evaluated with astroglia markers glial fibrillary acidic protein (GFAP). The formation of autophagosomes in astrocytes was examined with transmission electron microscopy (TEM). The expression of autophagy-related proteins were examined with immunoblotting or western blotting. The role of autophagy or AMPK in OGD or global ischemia was assessed by pharmacological intervention with 3-methyladenine (3-MA), Rapamycin and compound C. The study will provide a new experimental evidence for the protective mechanisms of DADLE, and expect to find out a novel strategy for future neuroprotection.

既往脑缺血研究常常集中于神经元，而缺血损伤涉及所有的神经细胞，也包括星形胶质细胞。脑缺血损伤条件下星形胶质细胞的功能调控对神经元的保护有至关重要的影响。我们先前的研究发现delta阿片受体(DOR)激动剂DADLE减轻全脑缺血/再灌注损伤，促进星形胶质细胞活化，同时减少受损的星形胶质细胞。而DOR如何影响缺血后星形胶质细胞的功能及其作用机制还不清楚。因此,本课题提出假说即激动DOR可以调控星形胶质细胞的功能状态,进而影响神经元的存活，并通过调控AMPK依赖的自噬途径发挥脑保护作用。本课题拟观察DADLE对氧糖剥夺（OGD）或全脑缺血后不同时间星形胶质细胞形态和功能的影响；并进一步借助多种分子生物学手段来研究OGD或全脑缺血后DADLE对星形胶质细胞自噬的调控机制。这一研究为DADLE脑保护作用提供新的实验证据，有助于寻找神经保护功能的新途径和药物作用的新靶标。

越来越多的研究结果显示，星形胶质细胞在大多数中枢神经系统重要功能中起着举足轻重的作用，这些作用不仅体现在正常大脑运转，还包含许多病理情况。在缺血性脑损伤发生后星形胶质细胞做出的应激反应是极其复杂的。Delta阿片受体(DOR)是G蛋白耦联受体，除了参与镇痛之外，激动DOR可以诱导动物进入冬眠，还具有器官保护作用。因此，我们对缺血后星形胶质细胞反应做了一系列研究。我们的研究发现DOR激动剂DADLE促进缺血后星形胶质细胞活化，减少受损的星形胶质细胞，这些作用可能与降低能量代谢有关。我们通过建立体外缺血/再灌注原代细胞模型，发现DADLE可以减轻氧糖剥夺(OGD)的损伤，而自噬抑制剂3-MA则取消了DADLE的保护作用，在荧光显微镜下，点状聚集在细胞浆的LC3在OGD组较对照组明显增多，而DADLE组较OGD组进一步增加，自噬相关蛋白LC3，Beclin 1和P62蛋白表达也有相似表现。在自噬和凋亡相互作用研究中，DADLE可以使OGD后星形胶质细胞的cytomchrome c 释放减少，取消缺血缺氧诱导Bcl-2的表达增加，线粒体凋亡相关蛋白caspase 3,9, cleaved PARP-1表达减少。而抑制自噬后，DADLE的这些作用消失，说明DADLE抑制缺血缺氧导致的凋亡发生。这说明DADLE通过诱导自噬和抑制凋亡促进氧糖剥夺后星形胶质细胞的存活。同时，我们也建立全脑缺血大鼠模型，研究发现在全脑缺血大鼠，通过诱导自噬调控星形胶质细胞反应促进神经元的存活。并且AMPK是缺血后神经元存活的一个关键分子。此外，神经功能恢复最终表现不仅在促进神经元的存活，还包括突触功能及神经网络的建立，通过对大鼠行为学实验考察，发现DADLE可改善全脑缺血后一个月大鼠的神经认知功能，可能与调控缺血后神经发生有一定的关系。