[Mechanism of electroacupuncture at "Neiguan" (PC6) and "Jianshi" (PC5) in ameliorating blood-brain barrier damage in APP/PS1 mice based on the nucleus tractus solitarius-locus coeruleus neural circuit].

摘要

OBJECTIVES: To observe the role of tyrosine hydroxylase (TH)-positive neurons in the nucleus tractus solitarius (NTS) and locus coeruleus (LC) in electroacupuncture (EA)-mediated improvement of blood-brain barrier (BBB) damage in APP/PS1 mice, so as to explore the mechanism of the NTSTH-LC neural circuit underlying the effect of EA on prevention and treatment of Alzheimer's disease (AD). METHODS: (1) Eight 4-month-old male C57BL/6 mice served as the control group, and 16 age-matched male APP/PS1 mice were randomly divided into the model and EA groups (n=8). The EA group received EA stimulation at "Neiguan" (PC6) and "Jianshi" (PC5) once every other day for 4 weeks. After intervention, Morris water maze and novel object recognition tests were used to evaluate learning and memory abilities. Western blot was performed to detect the expression levels of hippocampal tight junction proteins, including Occludin, Claudin-5, and zonula occludens-1 (ZO-1). Immunofluorescence was used to assess the co-localization of TH/c-Fos in NTS and LC, as well as TH/norepinephrine (NE) co-localization in LC. (2) Five TH-cre mice received retrograde tracing virus injection into LC to observe whether TH-positive neurons in NTS project to LC. (3) Twenty-one 7-month-old APP/PS1 mice were injected with chemogenetic activation virus (AAV2/9-hSyn-DIO-hM3D-mCherry-WPRE-hGH-pA) or empty virus (AAV2/9-Ef1α - DIO-mCherry-WPRE-hGH-pA) into the NTS, and AAVretro-TH-CRE-WPRE-hGH-pA virus into the LC. After 21 d of virus expression, 6 mice injected with empty virus were taken as mCherry+CNO+EA group;3 mice from the activation virus group were randomly selected for brain slice patch-clamp to verify virus functionality. The remaining mice were randomly divided into 2 groups (n=6):hM3D+CNO+EA, and hM3D+saline+EA groups. EA was applied to PC6 and PC5 once daily for 15 d. After intervention, learning and memory abilities were evaluated by Morris water maze and novel object recognition tests. BBB permeability was detected by Evans blue (EB) staining. Immunofluorescence was used to measure TH/c-Fos and TH/NE co-localization in the LC. RESULTS: (1) Compared with the control group, the model group showed significantly impaired learning and memory abilities (P<0.01), decreased expressions of hippocampal Occludin, Claudin-5, and ZO-1 (P<0.01), and increased TH/c-Fos co-localization (c-Fos expression in TH-positive neurons) in NTS and LC, as well as TH/NE co-localization in LC (P<0.01). Compared with the model group, the EA group exhibited improved learning and memory abilities (P<0.05, P<0.01), increased expressions of hippocampal tight junction proteins (P<0.01), and reduced TH/c-Fos and TH/NE co-localization (P<0.01, P<0.05). (2) Retrograde tracing confirmed that TH-positive neurons in NTS project to LC. (3) Compared with the mCherry+CNO+EA group and hM3D+saline+EA group, the hM3D+CNO+EA group showed significantly impaired learning and memory (P<0.01), increased EB content in brain tissue, and elevated TH/c-Fos and TH/NE co-localization in the LC (P<0.01). CONCLUSIONS: EA at PC6 and PC5 can ameliorate BBB damage and learning/memory deficits in AD mice, and its mechanism may be related to inhibiting the activation of the NTSTH-LC neural circuit. 目的: 观察孤束核（NTS）中酪氨酸羟化酶（TH）神经元及蓝斑（LC）在电针改善APP/PS1小鼠血脑屏障（BBB）损伤中的作用，探讨电针防治阿尔茨海默病（AD）的NTSTH-LC神经环路机制。方法: （1）8只4月龄雄性C57BL/6小鼠为对照组，16只同月龄雄性APP/PS1小鼠随机分为模型组、电针组（n=8）。电针组予电针刺激“内关”“间使”，隔日1次，共4周。干预结束后，以Morris水迷宫、新物体识别实验评估各组小鼠学习记忆能力，Western blot法检测海马闭锁蛋白（Occludin）、紧密连接蛋白-5（Claudin-5）、闭锁小带蛋白-1（ZO-1）表达水平，免疫荧光染色法检测NTS和LC中TH/c-Fos共定位、LC中TH/去甲肾上腺素（NE）共定位表达情况。（2）5只TH-cre小鼠，LC注射逆行示踪病毒，观察NTS中的TH神经元是否投射到LC。（3）21只7月龄APP/PS1小鼠，NTS注射化学遗传激活病毒（AAV2/9-hSyn-DIO-hM3D-mCherry-WPRE-hGH-pA）或空载病毒（AAV2/9-Ef1α-DIO-mCherry-WPRE-hGH-pA），LC注射AAVretro-TH-CRE-WPRE-hGH-pA病毒，待病毒表达21 d后，注射空载病毒小鼠（n=6）为空载病毒+氯氮平-N-氧化物（CNO）+电针组（mCherry+CNO+EA），随机选3只注射激活病毒小鼠用于脑片膜片钳验证病毒功能，剩余小鼠随机分为2组（n=6）:激活病毒+CNO+电针组（hM3D+CNO+EA）、激活病毒+盐水+电针组（hM3D+saline+EA），各组均电针“内关”“间使”，每日1次，共15 d。干预结束后，以Morris水迷宫、新物体识别实验评估小鼠学习记忆能力，EB染色法检测小鼠脑组织BBB通透性，免疫荧光染色法检测小鼠LC中TH/c-Fos、TH/NE共定位表达情况。结果: （1）与对照组相比，模型组小鼠学习记忆能力显著降低（P<0.01），海马Occludin、Claudin-5和ZO-1蛋白表达明显减少（P<0.01），NTS与LC中TH/c-Fos共定位表达、LC中TH/NE共定位表达明显增多（P<0.01）。与模型组相比，电针组小鼠学习记忆能力提高（P<0.05，P<0.01），海马Occludin、Claudin-5和ZO-1蛋白表达明显增加（P<0.01），NTS与LC中TH/c-Fos共定位表达、LC中TH/NE共定位表达减少（P<0.01，P<0.05）。（2）通过逆行示踪病毒证明，NTS中TH神经元可以投射到LC。（3）与mCherry+CNO+EA组和hM3D+saline+EA组相比，hM3D+CNO+EA组小鼠学习记忆能力显著降低（P<0.01），脑组织EB含量，LC中TH/c-Fos共定位表达、TH/NE共定位表达明显增多（P<0.01）。结论: 电针“内关”“间使”能改善AD小鼠BBB损伤及学习记忆功能障碍，其作用机制可能与抑制NTSTH-LC神经环路的激活有关。.