AMPK信号通路对缺血性脑卒中后小胶质细胞吞噬作用调节与认知功能恢复的影响
The effect of AMPK signaling pathway on the phagocytic regulation and cognitive function recovery of micro-glia after ischemic stroke
白康 1闫研 1姚丽娜1
作者信息
- 1. 072750 河北省保定市第二中心医院老年病科
- 折叠
摘要
目的 观察腺苷酸活化蛋白激酶(Adenosine monophosphate activated protein kinase,AMPK)信号通路对小鼠脑缺血性脑卒中后小胶质细胞吞噬作用调节与认知功能恢复的影响.方法 取成年雄性C57小鼠,随机分为假手术+溶媒(Sham+vehicle)组＼大脑中动脉短暂性闭塞模型+溶媒(Transient middle cerebral artery occlusion,tMCAO+vehicle)组、大脑中动脉闭塞模型(Middle cerebral artery occlusion,MCAO+com)组,每组各27只;构造短暂性大脑中动脉闭塞(Transient middle cerebral artery occlusion,tMCAO)小鼠模型,MCAO+com组、tMCAO+vehicle组和Sham+vehicle组分别于拔出线栓后以20 mg/kg剂量腹腔注射AMPK抑制剂复合物C(Compound C)和等体积生理盐水;再灌注15 min前、后观察各组小鼠脑血流情况,MAP2免疫荧光染色观察脑梗死体积,TUNEL染色观察神经元凋亡情况,用离子钙结合适配器分子1(Ionic calcium binding adapter molecule 1,Iba1)、神经元特异性 RNA 剪接蛋白(Neuron specific RNA splicing pro-tein,NeuN)、脱氧核苷酸末端转移酶介导的缺口末端标记技术(Notch end labeling technique mediated by de-oxyribonucleotide terminal transferase,TUNEL)标记各组小鼠梗死周围区小胶质细胞的吞噬情况;Western blot 法观察 AMPK、磷酸化的腺苷酸活化蛋白激酶(Phosphorylation-Adenosine monophosphate activated pro-tein kinase,p-AMPK)、糖原合酶激酶-3β(Glycogen synthase kinase-3β,GSK-3β)、磷酸化的糖原合酶激酶-3β(Phosphorylation-glycogen synthase kinase-3β,p-GSK-3β)、白细胞分化抗原 36(Cell differentiation antigen 36,CD36)、引发髓性细胞受体表达 2(Triggering receptor expressed on myeloid cells 2,TREM2)等蛋白表达情况;检测各组小鼠神经功能评分并用水迷宫实验评估认知功能恢复.结果 再灌注损伤7 d后MCAO+com组较tMCAO+vehicle组脑梗死体积和神经元凋亡细胞数显著降低(P＜0.05);免疫荧光显示MCAO+com组较tMCAO+vehicle组小胶质细胞吞噬凋亡神经元效率显著提高,对存活神经元的"误吞"比率显著降低,且残留凋亡神经元显著减少(P＜0.05);Western blot显示tMCAO+vehicle组小鼠梗死周围区p-AMPK/AMPK,p-GSK-3β/GSK-3β 比例、CD36 和 TREM2 蛋白表达水平较 Sham+vehicle 组显著增高,而 MCAO+com 组表达 p-AMPK/AMPK,p-GSK-3β/GSK-3β 比例较 tMCAO+vehicle 组显著降低,同时 CD36 和TREM2蛋白表达水平显著增高(P＜0.05);MCAO+com组小鼠较tMCAO+vehicle组神经功能评分显著改善,通过水迷宫观察MCAO+com组小鼠目标象限停留时间显著提高,逃离潜伏期显著降低(P＜0.05),但游泳速度无明显差异(P=0.16).结论 抑制AMPK信号通路促进小鼠缺血性脑卒中后认知功能恢复,此过程可能通过增强小胶质细胞吞噬作用来完成.
Abstract
Objective To observe the effect of AMPK signaling pathway on the phagocytic regulation and cognitive function recovery of microglia after ischemic stroke in rats.Methods Adult male C57 mice were taken and randomly divided into sham operation+vehicle(Sham+vehicle)group\transient middle cerebral ar-tery occlusion model+vehicle(tMCAO+vehicle)group,middle cerebral artery occlusion model(MCAO+com)group,with 27 members in each group,constructing a mouse model of transient middle cerebral artery occlusion(tMCAO).After removal of the wire bolus,the MCAO+com,tMCAO+vehicle,and Sham+vehi-cle groups were injected intraperitoneally with AMPK inhibitor complex C(Compound C)and an equal volume of saline at a dose of 20 mg/kg,respectively.Before and after 15 minutes of reperfusion,the cerebral blood flow of rats in each group was observed.The volume of cerebral infarction was observed by MAP2 immunoflu-orescence staining,and neuronal apoptosis was observed by TUNEL staining.Ionic calcium binding adapter molecule 1(Iba1),Neuron specific RNA splicing protein(NeuN),Notch end labeling technique mediated by deoxyribonucleotide terminal transferase(TUNEL)were used to label microglia in the peri-infarct area of each group.Notch end labeling technique mediated by deoxyribonucleotide terminal transferase(TUNEL)labeled the phagocytosis of microglia in the peri-infarct area of mice in each group.Western blot was performed to ob-serve AMPK,phosphorylation-Adenosine monophosphate activated protein kinase,p-AMPK,glycogen syn-thase kinase-3β,GSK-3β,phosphorylation-glycogen synthase kinase-3β,p-GSK-3βcell differentiation antigen 36,CD36,triggering receptor expressed on myeloid cells 2,TREM2 and other protein expression.The neuro-logical function scores of mice in each group were detected and cognitive function recovery was assessed by wa-ter maze experiment.Results After 7 days of reperfusion injury,the volume of cerebral infarction and the number of apoptotic neurons were significantly reduced in MCAO+com group compared with tMCAO+vehi-cle group(P＜0.05);Immunofluorescence showed that microglial cells phagocytosis of apoptotic neurons was significantly more efficient and the rate of"misphagocytosis"was significantly reduced in MCAO+com group compared with tMCAO+vehicle group,the"misphagocytosis"ratio was significantly lower,and the residual apoptotic neurons were significantly reduced(P＜0.05).Western blot showed that p-AMPK/AMPK,p-GSK-3β/GSK-3β ratio,CD36 and TREM2 protein expression levels in peri-infarct area of mice in tMCAO+vehicle group were significantly higher than those in Sham+vehicle group,while the expression levels of p-AMPK/AMPK,p-GSK-3β/GSK-3β ratio were significantly lower in the MCAO+com group compared to the tMCAO+vehicle group(P＜0.05),and CD36 and TREM2 protein expression levels were increasedly significantly.The neurological function scores of the mice in the MCAO+com group were significantly improved compared to the tMCAO+vehicle group.The mice in the MCAO+com group had a significantly higher residence time in the destination quadrant and a significantly lower escape latency as observed by the water maze(P＜0.05),but there was no significant difference in the swimming speed(P=0.16).Conclusion Inhibition of AMPK signaling pathway can promote the recovery of cognitive function after cerebral ischemic stroke in mice,which may be accomplished by enhancing microglial phagocytosis.
关键词
腺苷酸活化蛋白激酶/小胶质细胞/吞噬作用/脑卒中Key words
AMPK/Microglia/Phagocytosis/Cerebral infarction引用本文复制引用
基金项目
河北省卫生健康委科研项目(2021547)
出版年
2024
NETL
NSTL
万方数据