摘要
目的 旨在明确二甲双胍(Met)能否通过Sirt1抑制神经元凋亡以减轻蛛网膜下腔出血(SAH)后早期脑损伤(EBI).方法 将小鼠使用简单随机抽样分为假手术组、SAH24h组、SAH+Met 24h组、SAH 48 h组、SAH+Met 48 h组、SAH 72 h组、SAH+Met 72 h组,每组10只.使用视交叉前池注血法建立SAH小鼠模型.各组小鼠进行改良Garcia评分法评分、脑水含量测定、血脑屏障破坏评估及Morris水迷宫实验.检测各组Sirt1、Bcl-2、Bax、Cleaved Caspase-3蛋白水平并结合Tunel实验与Nissl染色分析Met是否抑制EBI过程中神经元凋亡.体外采用胎鼠原代神经元SAH模型,分为对照组、OxyHb组、OxyHb+Met组、OxyHb+Vehicle 组及 OxyHb+Met+EX527 组,每组 12 只,检测各组 Sirt 1、Bcl-2、Bax、Cleaved Caspase-3 蛋白水平并结合 Tunel实验验证动物实验的结果.加入EX527抑制Sirt1研究Met抑制神经元凋亡的可能机制.结果 与SAH各组相比,SAH+Met各组小鼠的神经功能损伤显著改善.与SAH 48 h组相比,SAH+Met 48 h组Sirt1与Bcl-2水平显著升高(P＜0.01),Bax和Cleaved Caspase-3水平显著降低(P＜0.01),Tunel与Nissl染色结果显示SAH+Met组凋亡的细胞显著减少(P＜0.01).在体外实验中,与OxyHb组相比,OxyHb+Met组Sirt1与Bcl-2水平显著升高(P＜0.01),Bax和Cleaved Caspase-3水平显著降低(P＜0.01),且此作用在加入EX527后受抑制.结论 Met可通过激活Sirt1通路抑制EBI过程的神经元凋亡,改善SAH预后,为SAH的临床治疗提供了新的药物靶点和治疗策略.
Abstract
Objective To determine whether metformin(Met)can attenuate early brain injury(EBI)after subarachnoid hemorrhage(SAH)by inhibiting neuronal apoptosis through Sirt1.Methods Mice were randomly divided into sham operation group,SAH 24 h group,SAH+Met 24 h group,SAH 48 h group,SAH+Met 48 h group,SAH 72 h group and SAH+Met 72 h group(n=10 per group).The mouse model of SAH was established by blood injection of the anterior cisterna optic chiasma.The modified Garcia score,brain water content,blood-brain barrier damage assessment and Morris water maze test were performed in each group.Sirt1,Bcl-2,Bax,Cleaved Caspase-3 protein levels were detected in each group,and Tunel assay and Nissl staining were combined to determine whether Met inhibited neuronal apoptosis during EBI.Fetal rat prima-ry neuron SAH model was used in vitro and divided into control group,OxyHb group,OxyHb+Met group,OxyHb+Vehicle group and OxyHb+Met+EX527 group(n=12 per group).The protein levels of Sirt1,Bcl-2,Bax,Cleaved Caspase-3 were detected in each group,and the results of animal experiments were verified by Tunel assay.EX527 was added to inhibit Sirt1 to investigate the potential mecha-nism by which Met inhibits neuronal apoptosis.Results Compared with SAH group,neurological dysfunction was significantly improved in the SAH+Met group.Compared with SAH 48h group,Sirt1 and Bcl-2 levels in SAH+Met 48h group were significantly increased(P＜0.01),while Bax and Cleaved Caspase-3 levels were significantly decreased(P＜0.01).Tunel and Nissl staining showed that the apoptotic cells in SAH+Met group were significantly reduced(P＜0.01).In vitro experiments,compared with the OxyHb group,the levels of Sirt1 and Bcl-2 in the OxyHb+Met group were significantly in-creased(P＜0.01),and the levels of Bax and Cleaved Caspase-3 were significantly decreased(P＜0.01),and this effect was inhibited after the addition of EX527.Conclusion Met can inhibit neuronal apoptosis during the process of EBI by activating the Sirt1 path-way,improve the prognosis of SAH,and provide a new therapeutic target and treatment strategy for clinical treatment of SAH.
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