α7 烟碱型乙酰胆碱受体激动剂通过抑制 GSK-3β在脑出血小鼠模型中发挥神经保护作用。
α7 nicotinic acetylcholine receptor agonism confers neuroprotection through GSK-3β inhibition in a mouse model of intracerebral hemorrhage.
机构信息
Department of Physiology and Pharmacology, Loma Linda University, Loma Linda, CA 92354, USA.
出版信息
Stroke. 2012 Mar;43(3):844-50. doi: 10.1161/STROKEAHA.111.639989. Epub 2011 Dec 29.
BACKGROUND AND PURPOSE
Perihematomal edema formation and consequent cell death contribute to the delayed brain injury evoked by intracerebral hemorrhage (ICH). We aimed to evaluate the effect of α7 nicotinic acetylcholine receptor (α7nAChR) stimulation on behavior, brain edema, and neuronal apoptosis. Furthermore, we aimed to determine the role of the proapoptotic glycogen synthase kinase-3β (GSK-3β) after experimental ICH.
METHODS
Male CD-1 mice (n=109) were subjected to intracerebral infusion of autologous blood (n=88) or sham surgery (n=21). ICH animals received vehicle administration, 4 or 12 mg/kg of α7nAChR agonist PHA-543613, 12 mg/kg of α7nAChR agonist PNU-282987, 6 mg/kg of α7nAChR antagonist methyllycaconitine (MLA), 15 μg/kg of phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin, or PHA-543613 combined with MLA or wortmannin. Behavioral deficits and brain water content were evaluated at 24 and 72 hours after surgery. Western blotting and immunofluorescence staining were used for the quantification and localization of activated Akt (p-Akt), GSK-3β (p-GSK-3β), and cleaved caspase-3 (CC3). Neuronal cell death was quantified through terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL).
RESULTS
α7nAChR stimulation improved neurological outcome and reduced brain edema at 24 and 72 hours after surgery (P<0.05 compared with vehicle). Furthermore, PHA-543613 treatment increased p-Akt and decreased p-GSK-3β and CC3 expressions in the ipsilateral hemisphere (P<0.05, respectively), which was reversed by MLA and wortmannin. P-Akt, p-GSK-3β, and CC3 were generally localized in neurons. PHA-543613 reduced neuronal cell death in the perihematomal area (P<0.05).
CONCLUSIONS
α7nAChR stimulation improved functional and morphological outcomes after experimental ICH in mice. PHA-543613 reduced the expression of proapoptotic GSK-3β through the PI3K-Akt signaling pathway.
背景与目的
血肿周围水肿的形成和随之而来的细胞死亡导致了脑出血（ICH）引起的迟发性脑损伤。我们旨在评估α7 烟碱型乙酰胆碱受体（α7nAChR）刺激对行为、脑水肿和神经元凋亡的影响。此外，我们旨在确定实验性 ICH 后促凋亡糖原合酶激酶-3β（GSK-3β）的作用。
方法
雄性 CD-1 小鼠（n=109）接受自体血脑内输注（n=88）或假手术（n=21）。ICH 动物给予载体给药，4 或 12 mg/kg α7nAChR 激动剂 PHA-543613、12 mg/kg α7nAChR 激动剂 PNU-282987、6 mg/kg α7nAChR 拮抗剂甲基lycaconitine（MLA）、15 μg/kg 磷脂酰肌醇 3-激酶（PI3K）抑制剂wortmannin 或 PHA-543613 联合 MLA 或 wortmannin。术后 24 和 72 小时评估行为缺陷和脑水含量。Western blot 和免疫荧光染色用于定量和定位激活的 Akt（p-Akt）、GSK-3β（p-GSK-3β）和 cleaved caspase-3（CC3）。通过末端脱氧核苷酸转移酶介导的 dUTP 缺口末端标记（TUNEL）定量神经元细胞死亡。
结果
α7nAChR 刺激可改善术后 24 和 72 小时的神经功能和减轻脑水肿（与载体相比，P<0.05）。此外，PHA-543613 治疗增加了同侧半球的 p-Akt，并降低了 p-GSK-3β 和 CC3 的表达（分别为 P<0.05），这被 MLA 和 wortmannin 逆转。p-Akt、p-GSK-3β 和 CC3 通常定位于神经元中。PHA-543613 减少了血肿周围区域的神经元细胞死亡（P<0.05）。
结论
α7nAChR 刺激可改善实验性 ICH 后小鼠的功能和形态学结果。PHA-543613 通过 PI3K-Akt 信号通路降低促凋亡 GSK-3β 的表达。
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