【作者】 刘倩；

【导师】 何俐；

【作者基本信息】 四川大学 ， 神经病学， 2021， 博士

【摘要】 目的:慢性脑血流低灌注(Chronic cerebral hypoperfusion,CCH)是老年人脑白质损伤(White matter lesions,WMLs)和血管因素导致的认知障碍和痴呆(Vascular contributions to cognitive impairment and dementia,VCID)的核心原因。脑白质损伤、胶质细胞激活、氧化应激和慢性炎性反应被认为是VCID的关键病理过程,但目前仍缺乏有效的治疗手段调控该病理过程以延缓VCID的疾病进展。钠氢交换体1(Sodium hydrogen exchanger 1,NHE1)是一种关键离子转运蛋白,其通过排出细胞内H~+交换细胞外Na~+来调节细胞内p H平衡以维持细胞形态和功能。研究显示NHE1的激活与中枢神经系统胶质细胞反应性活化和炎性反应密切相关,抑制NHE1的活性或者减少NHE1蛋白表达均可减轻急性缺血或缺氧后脑损伤并改善神经功能缺损。然而,NHE1蛋白在VCID中的作用尚不明确。因此,本研究探索了NHE1在双侧颈动脉狭窄(Bilateral carotid artery stenosis,BCAS)小鼠的CCH模型中脑白质损伤和认知功能减退的作用及其机制,旨在为VCID寻找潜在治疗靶点。材料和方法:采用0.18mm内径规格的微弹簧构建小鼠BCAS的CCH模型。用激光散斑血流灌注成像仪监测小鼠造模(BCAS或假手术Sham)前、造模后即刻、术后第7天和术后第30天的脑血流灌注。此外,BCAS小鼠造模后第三天随机分为安慰剂(Veh)组、HOE642以0.3mg/kg/d每日一次腹腔注射(HOE i.p.)组和术后植入微型渗透泵以0.3mg/kg/d的HOE642持续泵入(HOE pump)组治疗至术后第30天。采用旷场实验(OF,open field)检测小鼠局部运动功能和情绪紧张度变化。采用Y迷宫(Y maze)自主交替试验检测小鼠空间工作记忆变化。术后第30天取小鼠脑送检完善ex vivo MRI DTI检查明确脑组织结构变化。采用免疫荧光染色及western blot技术观察BCAS术后30天轴索(NF200)、髓鞘(MBP)蛋白表达变化。采用免疫荧光染色检测神经元(Neu N)、星形胶质细胞(GFAP)、小胶质细胞(Iba1)的标记物表达水平和与NHE1蛋白共染细胞计数。此外,提取了假手术(Sham)组、安慰剂(Veh)组和0.3mg/kg/天HOE642腹腔注射(HOE)组的星形胶质细胞RNA送检进行RNA-seq以探索BCAS术后和NHE1高选择性抑制剂HOE642治疗后星形胶质细胞m RNA的基因表达情况,进而探索NHE1蛋白在CCH后对星形胶质细胞的作用及机制。最后通过q PCR和免疫荧光染色对相关机制进行进一步验证。结果:BCAS诱导小鼠出现脑白质损伤和空间工作记忆减退:(1)小鼠BCAS术后导致小鼠脑血流灌注相对于基线下降约45%,随后脑血流逐渐恢复,术后第30天脑血流灌注下降约25%;(2)BCAS术后第28天的Y迷宫自主轮替实验提示小鼠BCAS术后存在明显的空间工作记忆减退,但无明显运动功能和情绪紧张度改变;(3)BCAS术后第30天小鼠脑白质的胼胝体(CC)和外囊(EC)的神经髓鞘标记物髓鞘碱性蛋白MBP和神经轴索标记物神经纤维丝蛋白NF200表达显著下降;(4)BCAS术后第30天脑白质CC和EC存在显著星形胶质细胞和小胶质细胞激活增殖,但脑灰质皮层和海马(CA1、CA3和齿状回)无神经元缺损,并且各神经细胞均有NHE1蛋白表达。NHE1高选择性抑制剂(HOE642)治疗可改善BCAS术后小鼠脑白质损伤病理及认知功能减退:(1)给予HOE642每日腹腔注射(HOE i.p.组)和植入性微量泵(HOE pump组)治疗后小鼠脑血流无明显改变,且对小鼠自发运动功能和情绪紧张度无明显影响,但均可显著改善BCAS小鼠的空间工作记忆减退;(2)给予HOE642每日腹腔注射(HOE i.p.组)和植入性微量泵(HOE pump组)可显著减轻脑白质CC和EC脱髓鞘和轴索损伤,以及降低了星形胶质细胞和小胶质细胞的激活增殖;(3)HOE i.p.组相对于Veh组显著抑制了海马星形胶质细胞和小胶质细胞活化增殖;(4)Pearson相关性分析显示CC和EC星形胶质细胞和小胶质细胞数与脑白质微结构损伤和Y迷宫成功自主轮替率呈显著线性相关。NHE1抑制剂治疗抑制了BCAS小鼠星形胶质细胞氧化应激反应及炎性激活:(1)星形胶质细胞的RNA-seq分析结果显示Veh组相对于Sham组共有1278个差异表达基因,包括833个上调基因和445个下调基因。GO富集分析显示这些上调差异表达基因主要与细胞周期和促进炎性反应相关;(2)HOE组和Veh组相比共有1130个差异表达基因,包括340个上调基因和790个下调基因。GO富集分析显示这些下调的差异表达基因也主要与细胞周期和炎性反应相关,而上调的差异表达基因的GO功能注释显示这些基因参与突触递质释放和促进神经元和神经系统发育;(3)IPA经典通路分析也表明Veh组相对于Sham组的差异表达基因激活了细胞周期和炎性反应相关通路,而HOE组相对于Veh组的差异表达基因抑制了细胞周期和炎性反应相关通路的激活;(4)此外,与Sham组相比Veh组存在ROS产生的关键基因cybb和ptgs2基因表达显著升高,而HOE组相对于Veh组这些基因表达显著下调;(5)同时,Veh组的炎性因子相关基因表达较Sham组显著上调,包括Lcn2、Tnfsf8、Tnfrsf9、Itgal、Itga4、Mmp9、Mmp8、Ccl5等,而HOE组相对于Veh组这些炎性基因表达显著下调;(6)最后,免疫荧光染色从蛋白水平揭示了Veh组相对于Sham组星形胶质细胞的NOX2活性显著增强且促炎蛋白Lcn2表达显著升高,而HOE642治疗显著抑制了BCAS小鼠星形胶质细胞的NOX2活性并降低了Lcn2的表达。结论:NHE1蛋白在CCH诱导的WMLs和VCID的核心病理过程中起重要作用,其激活促进了星形胶质细胞反应性活化增殖,并加重了其氧化应激和慢性炎症反应过程。因此,NHE1蛋白是VCID药物治疗的潜在关键治疗靶点,也为未来VCID的治疗研究提供了新的研究方向。更多还原

【Abstract】 Objective:Chronic cerebral hypoperfusion(CCH)is the key contributor for white matter lesions(WML)and vascular contributions to cognitive impairment and dementia(VCID)in the elderly.White matter damage,glial cell activation,oxidative stress and chronic inflammatory responses are the major pathological processes of VCID.However,there is still a lack of effective treatment methods to control these pathological processes to delay the progression of VCID disease.Sodium-hydrogen exchanger 1(NHE1)is an important ion transporter,which regulates intracellular p H balance and maintains cell volume and function by exchanging intracellular H~+for extracellular Na~+.Studies have shown that the activation of NHE1 facilitates glial activation and pro-inflammatory responses in the central nervous system.Inhibition of NHE1 either by selective blockade or gene knockout can improve brain damage and neurological function deficits after acute ischemic stroke and hypoxia injury.However,the role of NHE1 protein in brain injury of CCH is still unclear.Therefore,this study investigated the role and mechanisms of NHE1 in CCH induced WMLs and VCID in mice with bilateral carotid artery stenosis(BCAS)to help find potential therapeutic targets for VCID.Materials and Methods:Mice BCAS were created by constriction of bilateral carotid commom artery using micro coils with an inner diameter of 0.18mm to induce CCH.The Laser speckle contrast imaging of cerebral blood flow was used to monitor the regional cerebral blood flow(r CBF)at baseline,onset of BCAS or Sham,the 7 days and the30 days after surgery.In addition,BCAS mice were randomly divided into vehicle control(Veh)group,HOE642 0.3mg/kg/d daily intraperitoneal injection for 28 days(HOE i.p.)group and HOE642 0.3mg/kg/d constant delivery(HOE pump)group with implanted 4 week’s osmotic mini pump.The open field test(OF)was used to detect changes in locomotor function and emotion of mice.The spontaneous alternation in Y maze test was used to assess spatial working memory in mice.At 30days post surgery,mice were sacrificed for ex vivo MRI-DTI examination to detect the cerebral structural changes.Immunofluorescence staining and western blot were used to measure the level of axonal marker NF200 and myelin marker MBP at 30days after BCAS.Immunofluorescence double staining was used to detect the changes of NHE1 and neuronal marker Neu N,astrocytic marker GFAP or microglia marker Iba1,respectively.Additionally,RNA of astrocyte from Sham group,Veh group,and HOE642 0.3 mg/kg/day intraperitoneal injection(HOE)group was extracted for RNA-seq to explore the transcriptome profiling of the astrocyte after BCAS surgery and with HOE642 treatment.At last,we validated the possible mechanisms from transcriptome analysis by using q PCR and immunofluorescence staining methods.Results:BCAS induced WML and spatial working memory decline in mice:(1)The r CBF of the mice reduced～45%(relative to the baseline)after BCAS surgery,and the r CBF gradually recovered during experiment,which reduced～25%at 30 days after surgery;(2)The BCAS mice showed spatial working memory deficits in Y maze at 28 days after surgery,whereas no locomotor function and anxiety were detected in those BCAS mice;(3)The expression of the myelin marker MBP and the axonal marker NF were significantly decreased in the corpus callosum(CC)and the external capsule(EC)of the white matter at 30 days after BCAS;(4)Besides,there was significantly increased reactive astrogliosis and microglia activation in CC and EC at 30 days after BCAS.There was no neuronal loss either in cortex or hippocampus(CA1,CA3 and dentate gyrus)of cerebral gray matter at 30 days after BCAS.Almost all cell types in mice brain colabeled with NHE1 protein more or less.Administration of NHE1 selective inhibitor improved pathologic changes and cognitive decline in BCAS mice:(1)The administration of HOE642 either by i.p.(HOE i.p.)or mini-pump pump(HOE pump)caused no changes in r CBF during experiment compared with Veh mice.However,both HOE i.p.and HOE pump groups significantly improved the spatial working memory function in BCAS mice,but had little influence on locomotor function and level of anxiety;(2)HOE i.p.and HOE pump treatments significantly reduced the demyelination and axonal damage of CC and EC white matter,as well as the reactive astrogliosis and microglia activation;(3)In addition,HOE i.p.or HOE pump showed less reactive astrogliosis and microglia activation in hippocampus as well;(4)Pearson correlation analysis showed that the number of astrocytes and microglia in CC and EC white matter was linearly correlated with microstructure damage in CC and EC and spontaneous alternation rate in Y maze test.Administration of NHE1 selective inhibitor suppressed the oxidative stress and pro-inflammatory response from reactive astrocytes after hypoperfusion:(1)The RNA-seq analysis of astrocytic m RNA showed that the Veh group had a total of1278 differentially expressed genes compared to the Sham group,including 833 up-regulated genes and 445 down-regulated genes.Gene ontology(GO)enrichment analysis revealed that these differentially expressed genes are mainly related to the activation of cell cycle and the pro-inflammatory response;(2)Compared with the Veh group,the HOE group had 1130 differentially expressed genes,including 340up-regulated genes and 790 down-regulated genes.GO enrichment analysis demonstrated that these down-regulated differentially expressed genes are mainly related to cell cycle and inflammatory response,while those up-regulated differentially expressed genes were mainly involved in synaptic transmitters release and helping neurons or brain development;(3)Cononical pathway analysis of differentially expressed genes in IPA further verified that the Veh group activated the cell cycle and inflammatory response related pathways compared with the Sham group,while the HOE group inhibited the activation of the cell cycle and inflammatory response related pathways compared to the Veh group;(4)In addition,the up-regulated expression of key genes in ROS production in Veh group,like cybb and ptgs2,were significantly down-regulated in HOE group;(5)In addition,up-regulated genes of pro-inflammatory cytoines in the Veh group were significantly reduced in HOE group,including Lcn2,Tnfsf8,Tnfrsf9,Itgal,Itga4,Mmp9,Mmp8,Ccl5,etc;(6)Moreover,the immunofluorescence staining showed the BCAS triggered the higher NOX2 activity and Lcn2 expression,while those changes were significantly surpressed by HOE642 treatment.Conclusion:NHE1 protein play a pivotal role in the basic pathological processes of CCH induced WMLs and VCID.Activating NHE1 protein could increase the number of reactive astrocytes,and promote the oxidative stress and inflammatory responses in those reactive astrocytes.Therefore,this study suggests that NHE1 protein is a potential therapeutic target for VCID and provides a new reaserch direction for future VCID treatment.更多还原