lncRNA NEAT1通过Klotho/mTOR轴调控慢性脑低灌注大鼠的认知障碍  

lncRNA NEAT1 regulates cognitive impairment in chronic cerebral hypoperfusion rats through the Klotho/mTOR axis

作　　者：陈方方[1] 齐会珍 Chen Fangfang;Qi Huizhen(Department of Neurology,the Fifth Affiliated 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6期377-382,共6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 探讨长链非编码RNA (lncRNA)核富集丰度转录物1 (NEAT1)对慢性脑低灌注(CCH)大鼠认知障碍的调控作用和潜在机制。方法双侧颈总动脉闭塞术(BCCAO)诱导CCH模型大鼠。将大鼠分为5组(每组n=10):Ⅰ组(假手术组);Ⅱ组(BCCAO手术组);Ⅲ组[BCCAO后于海马区注射0.3 mg·kg^(-1)·w^(-1)的沉默lncRNA NEAT1的短发夹RNA重组质粒(shNEAT1),持续12 w];Ⅳ组[BCCAO后于海马区每周注射0.3 mg·kg^(-1)·w^(-1)的shNEAT1和50 mg·kg^(-1)·w^(-1)的沉默克洛托蛋白(Klotho)的短发夹RNA重组质粒(shKlotho),持续12 w];V组[BCCAO后于海马区注射0.3 mg·kg^(-1)·w^(-1)的shNEAT1和25mg·kg^(-1)哺乳动物雷帕霉素靶蛋白(mTOR)的抑制剂(AZD-8055),持续12w]。行Morris水迷宫实验记录逃避潜伏期。实时荧光定量PCR (qRT-PCR)测lncRNA NEAT1的表达。Western blot法检测Klotho、mTOR、磷酸化的mTOR (p-mTOR)、神经核抗原(NeuN)、多聚腺苷酸核糖聚合酶(PARP)、半胱天冬酶-3(caspase-3)、切割模式的半胱天冬酶-3(cleaved-caspase-3)的表达。免疫荧光化学检测海马区NeuN阳性细胞数。结果 与Ⅰ组比,Ⅱ组大鼠的逃避潜伏期延长,lncRNA NEAT1、PARP、cleaved-caspase-3的表达均上调,NeuN、Klotho和p-mTOR的表达均下调,NeuN阳性的细胞数减少(~均P<0.05)。与Ⅱ组比,Ⅲ组大鼠的逃避潜伏期缩短,且海马组织中lncRNA NEAT1、PARP、cleaved-caspase-3的表达均下调,NeuN、Klotho和p-mTOR的表达均上调,NeuN阳性的细胞数增加(~均P><0.05)。与Ⅲ组比,Ⅳ组和Ⅴ组中大鼠的逃避潜伏期延长,且海马组织中PARP、cleaved-caspase-3的表达均上调,NeuN、Klotho和p-mTOR的表达均下调,NeuN阳性细胞数都减少(~均P <0.05)。结论 lncRNA NEAT1通过负调控Klotho/mTOR轴促进CCH大鼠的认知障碍,为研究CCH的有效治疗靶点提供理论依据。Objective This study aimed to investigate the regulatory effect and potential mechanism of long non-coding RNA(lncRNA)nuclear-enriched abundant transcript 1(NEAT1)on cognitive impairment in chronic cerebral hypoperfusion(CCH)rats.Methods CCH model rats were induced by bilateral common carotid artery occlusion(BCCAO).The rats were divided into 5 groups(n=10 per group):GroupⅠ(sham group);GroupⅡ(carotid artery ligation);GroupⅢ[injection of short hairpin RNA recombinant plasmid for silencing lncRNA NEAT1(shNEAT1)at a dose of 0.3 mg·kg^(-1)·w^(-1) into the hippocampus after BCCAO,for 12 weeks];GroupⅣ[injection of shNEAT1 at a dose of 0.3mg·kg^(-1)·w^(-1) and short hairpin RNA recombinant plasmid for silencing Klotho(shKlotho)at a dose of 50 mg·kg^(-1)·w^(-1) into the hippocampus after BCCAO,for 12 weeks];Group V(injection of shNEAT1 at a dose of 0.3 mg·kg^(-1)·w^(-1) and mammalian target of rapamycin(mTOR)inhibitor AZD-8055 at a dose of 25 mg·kg^(-1)·w^(-1) into the hippocampus after BCCAO,for 12 weeks).Morris water maze test was conducted to record the escape latency of each group of rats.Real-time quantitative PCR(qRT-PCR)was used to detect the expression of lncRNA NEAT1.Western blot was used to detect the expression of Klotho,mammalian target of rapamycin(mTOR),phosphorylated mTOR(p-mTOR),Neuronuclear antigen(NeuN),polyadenylate ribose polymerase(PARP),caspase-3,and cleaved caspase-3.Immunofluorescence staining was used to detect the number of NeuN-positive cells.Results Compared with GroupⅠ,rats in GroupⅡshowed a prolonged escape latency,and the expression of lncRNA NEAT1,PARP,and cleaved caspase-3 in the hippocampal tissue were upregulated,while the expression of NeuN,Klotho,and p-mTOR were downregulated,and the number of NeuN-positive cells decreased(allP<0.05).Compared with GroupⅡ,rats in GroupⅢshowed a shortened escape latency,and the expression of lncRNA NEAT1,PARP,and cleaved caspase-3 in the hippocampal tissue were downregulated,while the expression of NeuN,Klotho,and p-mTOR were u

关 键 词：lncRNA核富集丰度转录物1 克洛托蛋白 哺乳动物雷帕霉素靶蛋白 慢性脑低灌注 认知功能障碍 

分 类 号：R743.32[医药卫生—神经病学与精神病学]