激活星形胶质细胞Kappa阿片受体缓解缺血再灌注后脑水肿的作用及机制研究

The clinical outcome of perioperative cerebral ischemia-reperfusion injury is closely related to the severity of cerebral edema. At present, the treatment of cerebral edema is limited. Our previous studies have shown that activation of kappa opioid receptor (KOR) in astrocytes can alleviate brain edema, protect hippocampal neurons and improve neural function in mice after ischemia-reperfusion injury, but the specific mechanism remains unclear. The astrocyte can alleviate the brain edema by regulating the balance of water, ion and glutamic acid. We speculate that activation of KOR in astrocytes may confer neuroprotection by regulating the expression of functional molecules. On the basis of our previous work, we plan to use rat global cerebral ischemia-reperfusion model, oxygen and glucose deprivation model, co-immunoprecipitation and immunofluorescence to detect the regulation of activated KOR on the expression of GFAP, a marker of activation of astrocytes, and the expression of functional molecule, GLT-1 and AQP4 in astrocytes. In order to clarify the molecular mechanism of activation of KOR in astrocytes, we also plan to investigate the role of β-arrestin2/p38 MAPK and NF-κB involved in modulating the expression of GFAP, GLT-1 and AQP4. The results will enrich our understanding of the molecular mechanism of KOR neuroprotection, and provide a scientific basis for clinical treatment of ischemic brain edema.

围术期脑缺氧损伤的转归与脑水肿严重程度密切相关，目前脑水肿的治疗方法有限。我们前期研究发现，激活星形胶质细胞kappa阿片受体(KOR) 参与缓解小鼠脑缺血再灌注后脑水肿，保护海马区神经元，提高神经功能，但具体的机制尚不明确。星形胶质细胞可通过调节水、离子及谷氨酸平衡缓解脑水肿损伤。我们推测激活星形胶质细胞KOR可通过调节其功能分子表达产生脑保护效应。本课题拟在前期工作的基础上利用大鼠全脑缺血再灌注模型、细胞缺氧及复氧模型，运用免疫共沉淀及荧光免疫标记法检测激活KOR对星形胶质细胞活化标志物GFAP和功能分子GLT-1、AQP4表达的调节，探讨KOR激活后下游β-arrestin2/p38 MAPK信号通路及NF-κB参与调控GFAP、GLT-1和AQP4表达方面的作用，以明确激活KOR调节星形胶质细胞功能产生脑保护作用的分子机制。所获成果将为临床治疗缺氧性脑水肿提供科学依据。

脑缺血再通后临床转归与脑水肿严重程度密切相关，因而临床上仍急需提供神经保护及降低再灌注后脑水肿的药物，提高患者恢复脑灌注后的神经功能。本研究应用四血管阻断法建立大鼠全脑缺血模型，评价再灌注后给予kappa阿片受体（KOR）激动剂，KOR激活在再灌注不同时间点对脑水肿的影响及机制。方法：选用雄性SD大鼠（270g-290g）建立全脑缺血模型，分为对照组、缺血组、溶剂组、KOR激动组及拮抗组。通过神经行为学评价（18分制评分）、脑水含量测定、尼氏染色、Tunel染色评价kappa阿片受体激动剂Salvinorin A（SA）在再灌注24h及48h对海马CA1区和颞侧皮层脑水肿及神经元受损情况的影响；通过免疫荧光染色及Western blot免疫印迹探索KOR激活在全脑缺血再灌注模型中对GFAP、GLT-1、AQP-4、CD31时空表达的影响。结果：再灌注后24h及48h后，SA处理组mNSS评分及脑水含量显著低于缺血组、海马CA1区和颞侧皮层神经元损失及凋亡情况低于缺血组。免疫荧光染色显示SA处理组KOR激活在再灌注24h及48h后与海马CA1及颞侧皮层神经元区GFAP、GLT-1、CD31增殖共存。免疫印迹结果显示再灌注24h及48h后海马CA1及颞侧皮层神经元区KOR、p-KOR、GFAP、GLT-1、CD31表达高于缺血组，AQP-4的增多低于低于缺血组。结论：全脑缺血再灌注损伤中，KOR 可通过调节星形胶质细胞形态及功能产生减少脑水肿的脑保护作用，机制涉及激活星形胶质细胞内调节水离子平衡的 GLT-1、AQP4 的表达及血管重塑。