Klotho通过激活AMPK/mTOR信号通路缓解CCH大鼠海马神经元的铁死亡和氧化应激  

Klotho alleviates ferroptosis and oxidative stress in hippocampal neurons of chronic cerebral hypoperfusion rats by activating the AMPK/mTOR signaling pathway

作　　者：齐会珍 陈方方[1] Qi Huizhen;Chen Fangfang(Department of Neurology,Fifth Affliated Hospital of Xinjiang Medical University,Urumqi 83001l,China)

机构地区：[1]新疆医科大学第五附属医院神经内科,乌鲁木齐830011

出　　处：《脑与神经疾病杂志》2024年第7期438-444,共7页Journal of Brain and Nervous Diseases

基　　金：新疆维吾尔自治区自然科学基金资助项目(2022D01C320)。

摘　　要：目的探讨抗衰老基因Klotho对慢性脑低灌注(CCH)大鼠认知障碍的改善作用及对海马区神经元铁死亡和氧化应激的影响和潜在调控机制。方法使用双侧颈总动脉永久性闭塞术(2-VO)建立CCH大鼠,并将大鼠分为5组(每组n=10),其中包括假手术组、CCH组、重组Klotho蛋白(Klotho)+CCH组(重组Klotho蛋白海马区注射10 mg·kg·w^(-1),x4w)、生理盐水+CCH组(生理盐水海马区注射2mg·kg·w^(-1),x4w)、Klotho+阿卡地新(Acadesine,AICAR)+CCH组(Klotho蛋白10 mg·kg·w^(-1),x4w和AICAR 1.5 mg·kg·w^(-1),x4w海马区联合注射)。4 w后行Morris水迷宫实验检测各组大鼠的逃避潜伏期。随后安乐死大鼠,取海马组织。Western blot法检测海马组织中单磷酸腺苷活化的蛋白激酶(AMPK)、雷帕霉素靶蛋白(mTOR)、磷酸化的AMPK(p-AMPK)、磷酸化的(p-mTOR)、谷胱甘肽(GSH)、谷胱甘肽过氧化物酶4(GPX4)、铁死亡抑制蛋白1(FSP1)、神经元特异性烯醇化酶(NSE)、微管相关蛋白Tau、神经元特异核蛋白(NeuN)的表达水平。ELISA试剂盒检测海马组织中的活性氧(ROS)、丙二醛(MDA)、超氧化物歧化酶(SOD)的含量。结果与假手术组比,CCH组的逃避潜伏期延长(P<0.05),p-AMPK、MDA、NSE、Tau、ROS的水平显著增加(^(均)P<0.05),但p-mTOR、SOD、GSH、GPX4、NeuN、FSP1的水平显著下调(^(均)P<0.05)。与CCH组比,生理盐水+CCH组的逃避潜伏期以及海马区p-AMPK、p-mTOR、MDA、ROS、SOD、GSH、GPX4、NSE、Tau、NeuN、FSP1的水平差异均无统计学意义(^(均)P>0.05)。与CCH组或生理盐水+CCH组比,Klotho+CCH组的逃避潜伏期缩短(^(均)P<0.05),p-AMPK、MDA、ROS、NSE、Tau的水平显著下调(均P<0.05),但p-mTOR、SOD、GSH、GPX4、NeuN、FSP1的水平显著上调(均P<0.05)。与Klotho+CCH组比,Klotho+AICAR+CCH组的逃避潜伏期延长(P<0.05),p-AMPK、MDA、ROS、NSE、Tau的水平显著增加(^(均)P<0.05),但p-mTOR、SOD、GSH、GPX4、NeuN、FSP1的水平显著下调(^(均)P<0.05)。结论Klotho通�Objective This study aimed to explore the therapeutic effect of Klotho,an anti-aging protein,on cognitive impairment,as well as its impact on ferroptosis and oxidative stress in hippocampal neurons of chronic cerebral hypoperfusion(CCH)rats,and the underlying regulatory mechanisms.Methods CCH rats were established by bilateral common carotid arteries ligation(2-VO)and divided into 5 groups(n=10 in each group).These groups included sham operation group,CCH group,recombinant Klotho protein(Klotho)+CCH group(recombinant Klotho protein in the hippocampus injection of 10 mg·kg^(-1)·w^(-1),4w),saline+CCH group(saline injection in the hippocampus of 2 mg·kg·w^(-1),x4w),and Klotho+Acadesine(AICAR)+CCH group(Klotho protein 10 mg·kg·w^(-1),x4w and AICAR 1.5 mg·kg·w^(-1),x4w combined injection).Four weeks later,Morris water maze experiment was performed to detect the escape latency of each group of rats.Then the rats were euthanized and their hippocampal tissue was taken.The expression levels of adenosine monophosphate activated protein kinase(AMPK),rapamycin target protein(mTOR),phosphorylated AMPK(p-AMPK),phosphorylated AMPK(p-mTOR),glutathione(GSH),glutathione peroxidase 4(GPX4),iron death suppressor protein 1(FSP1),neurons in hippocampus specific enolase(NSE),microtuhule-associated protein Tau,and neuron-specific nuclear protein(NeuN)were analyzed by Western blot.The contents of reactive oxygen species(ROS),malondialdehyde(MDA)and superoxide dismutase(SOD)in hippocampus were detected by ELISA.Results Compared with the sham-operated group,the CCH group showed a prolonged escape latency(P<0.05),significant increase in the levels of p-AMPK,MDA,NSE,Tau and ROS(^(all)P<0.05),but significant decrease in the levels of p-mTOR,SOD,GSH,GPX4,NeuN,and FSP1(^(all)P<0.05).There were no significant differences in the escape latency and the levels of p-AMPK,p-mTOR,MDA,ROS,SOD,GSH,GPX4,NSE,Tau,NeuN,and FSP1 between the saline+CCH group and the CCH group(^(all)P>0.05).Compared with the CCH group or the saline+CCH group,the Klotho

关 键 词：KLOTHO 慢性脑低灌注 海马神经元 认知缺陷 氧化应激 细胞铁死亡 AMPK/mTOR通路 

分 类 号：R743.32[医药卫生—神经病学与精神病学]