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Effectiveness of electro-acupuncture for cognitive improvement on Alzheimer's disease quantified via PET imaging of sphingosine-1-phosphate receptor 1
电针对阿尔茨海默病认知改善的有效性通过鞘氨醇-1-磷酸受体 1 的 PET 成像量化

Yongshan Liu 2 , 3 2 , 3 ^(2,3){ }^{2,3} | An Li 1 1 ^(1)^{1} | Xuan Yang 1 ∣ 1 ∣ ^(1)∣{ }^{1} \mid Peining Shen 4 ∣ 4 ∣ ^(4)∣{ }^{4} \mid Junfeng Wang 5 5 ^(5){ }^{5} |
刘永山 2 , 3 2 , 3 ^(2,3){ }^{2,3} | 李安 1 1 ^(1)^{1} | 杨璇 1 ∣ 1 ∣ ^(1)∣{ }^{1} \mid 沈佩宁 4 ∣ 4 ∣ ^(4)∣{ }^{4} \mid 王俊峰 5 5 ^(5){ }^{5} |Qi Zeng 1 ∣ 1 ∣ ^(1)∣{ }^{1} \mid Hongyu Zhang 1 1 ^(1){ }^{1} | Shengqiao Li, 1 , 2 1 , 2 ^(1,2){ }^{1,2} | Hongjun Jin 2 , 3 ∘ 2 , 3 ∘ ^(2,3)^(@){ }^{2,3}{ }^{\circ}
齐增 1 ∣ 1 ∣ ^(1)∣{ }^{1} \mid 张红宇 1 1 ^(1){ }^{1} | 李胜桥 1 , 2 1 , 2 ^(1,2){ }^{1,2} | 金洪军 2 , 3 ∘ 2 , 3 ∘ ^(2,3)^(@){ }^{2,3}{ }^{\circ}

1 1 ^(1){ }^{1} Department of Chinese Medicine Oncology, Cancer Center, The Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai, China
中医肿瘤科，癌症中心，孙中山大学第五附属医院，中国珠海
2 2 ^(2){ }^{2} Guangdong Provincial Engineering Research Center of Molecular Imaging, The Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai, China
2 2 ^(2){ }^{2} 中国珠海，中山大学附属第五医院，分子成像广东省工程研究中心
3 3 ^(3){ }^{3} Guangdong-Hong Kong-Macao University Joint Laboratory of Interventional Medicine, The Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai, China
3 3 ^(3){ }^{3} 广东-香港-澳门大学介入医学联合实验室，中山大学第五附属医院，中国珠海
4 4 ^(4){ }^{4} Pharmaceutical Clinical Trails Office, The Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai, China
4 4 ^(4){ }^{4} 制药临床试验办公室，中山大学第五附属医院，中国珠海
5 5 ^(5){ }^{5} Department of Neurology, The Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai, China
中山大学附属第五医院神经内科，中国珠海

Correspondence 通信

Shengqiao Li, Department of Chinese Medicine Oncology, Cancer Center, The Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai, Guangdong Province 519000, China.
李胜桥，中医肿瘤科，癌症中心，暨南大学第五附属医院，中国广东省珠海市 519000。
Email: lishq26@mail.sysu.edu.cn
电子邮件：lishq26@mail.sysu.edu.cn
Hongjun Jin, Guangdong Provincial Engineering Research Center of Molecular Imaging, Guangdong-Hong Kong-Macao University Joint Laboratory of Interventional Medicine, The Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai, Guangdong Province 519000, China.
金洪俊，广东省分子影像工程研究中心，粤港澳介入医学联合实验室，暨南大学第五附属医院，中国广东省珠海市 519000。
Email: jinhj3@mail.sysu.edu.cn
电子邮件：jinhj3@mail.sysu.edu.cn

Abstract 摘要

INTRODUCTION: Electro-acupuncture (EA) has demonstrated potential in improving mild-to-moderate dementia in clinics, but the underlying scientific target remains unclear. METHODS: EA was administered to APP/PS1 Alzheimer’s disease (AD) mice, with untreated AD, and wild type (WT) mice serving as controls. The efficacy of EA was assessed by the Morris water maze cognitive functional tests. Brain magnetic resonance imaging-positron emission tomography (PET) scans using [ 18 F 18 F ^(18)F{ }^{18} \mathrm{~F} ]TZ4877 targeting sphingosine-1-phosphate receptor 1 (S1PR1) and [ 18 F ] AV 45 18 F AV 45 {:^(18)(F)]AV45\left.{ }^{18} \mathrm{~F}\right] \mathrm{AV} 45 targeting amyloid beta fibrils were conducted. The correlation between regional brain PET quantifications and cognitive functions was analyzed.
引言：电针（EA）在改善轻度至中度痴呆方面在临床上显示出潜力，但其潜在的科学靶点仍不清楚。方法：对 APP/PS1 阿尔茨海默病（AD）小鼠进行电针治疗，未治疗的 AD 小鼠和野生型（WT）小鼠作为对照。通过莫里斯水迷宫认知功能测试评估电针的疗效。使用[ 18 F 18 F ^(18)F{ }^{18} \mathrm{~F} ]TZ4877 靶向鞘氨醇-1-磷酸受体 1（S1PR1）和[ 18 F ] AV 45 18 F AV 45 {:^(18)(F)]AV45\left.{ }^{18} \mathrm{~F}\right] \mathrm{AV} 45 ]靶向淀粉样β纤维进行脑部磁共振成像-正电子发射断层扫描（PET）。分析了区域脑 PET 定量与认知功能之间的相关性。

RESULTS: EA significantly improved cognitive and memory functions of AD ( p = 0.04 p = 0.04 p=0.04p=0.04 ) and reduced the uptake of [18 F]TZ4877 in the cortex ( p = 0.02 p = 0.02 p=0.02p=0.02 ) and hippocampus ( p = 0.03 p = 0.03 p=0.03p=0.03 ). Immunofluorescence confirmed colocalizations of S1PR1 with glial fibrillary acidic protein and ionized calcium-binding adaptor molecule-1. Furthermore, immunohistochemistry showed a significant reduction of interleukin 1 β 1 β 1beta1 \beta and tumor necrosis factor α α alpha\alpha after EA treatment.
结果：EA 显著改善了阿尔茨海默病（AD）的认知和记忆功能（ p = 0.04 p = 0.04 p=0.04p=0.04 ），并减少了皮层（ p = 0.02 p = 0.02 p=0.02p=0.02 ）和海马（ p = 0.03 p = 0.03 p=0.03p=0.03 ）中[18 F]TZ4877 的摄取。免疫荧光证实了 S1PR1 与胶质纤维酸性蛋白和离子钙结合适配蛋白-1 的共定位。此外，免疫组化显示 EA 治疗后白介素 1 β 1 β 1beta1 \beta 和肿瘤坏死因子 α α alpha\alpha 显著减少。

DISCUSSION: EA may reverse AD by suppressing neuroinflammation, and the PET imaging of S1PR1 seemed potent in evaluating the treatment for AD patients
讨论：EA 可能通过抑制神经炎症来逆转阿尔茨海默病，S1PR1 的 PET 成像在评估阿尔茨海默病患者的治疗方面似乎具有很强的效果

KEYWORDS 关键词

Alzheimer’s disease, astrocyte, electro-acupuncture, microglia, micro-positron emission tomography/computed tomography-magnetic resonance imaging, sphingosine-1-phosphate receptor 1
阿尔茨海默病，星形胶质细胞，电针，微胶质细胞，微正电子发射断层扫描/计算机断层扫描-磁共振成像，鞘氨醇-1-磷酸受体 1

Funding information 资金信息

National Natural Science Foundation of China, Grant/Award Numbers: 82372004, 81871382, 82150610508; Key Realm R&D Program of Guangdong Province, Grant/Award Number: 2018B030337001; Guangdong Provincial Basic and Applied Basic Research Fund Provincial Enterprise Joint Fund, Grant/Award Number: 2021A1515220004; Guangdong Provincial Bureau of Traditional Chinese Medicine Research Project, Grant/Award Number: 20211079
中国国家自然科学基金，资助/奖励编号：82372004，81871382，82150610508；广东省重点领域研发计划，资助/奖励编号：2018B030337001；广东省基础与应用基础研究基金省企业联合基金，资助/奖励编号：2021A1515220004；广东省中医药局研究项目，资助/奖励编号：20211079

Highlights 亮点

  • Electro-acupuncture (EA) was administered to APP/PS1 Alzheimer’s disease (AD) mice, with untreated AD, and wild type (WT) mice serving as controls. The efficacy of EA was assessed by the Morris water maze cognitive functional tests and positron emission tomography (PET) imaging quantifications.
    电针（EA）被施用于 APP/PS1 阿尔茨海默病（AD）小鼠，未治疗的 AD 小鼠和野生型（WT）小鼠作为对照。通过莫里斯水迷宫认知功能测试和正电子发射断层扫描（PET）成像定量评估 EA 的疗效。
  • PET tracer [ 18 F ] AV 45 18 F AV 45 [^(18)(F)]AV45\left[{ }^{18} \mathrm{~F}\right] \mathrm{AV} 45 was used to detect amyloid beta deposition. An increased uptake of [ 18 F ] AV 45 18 F AV 45 [^(18)(F)]AV45\left[{ }^{18} \mathrm{~F}\right] \mathrm{AV} 45 was found in AD compared to WT mice, with significance observed only in the cortex and not in the hippocampus. EA treatment exhibited a trend toward reduced 18 F ] 18 F {:^(18)(F)]\left.{ }^{18} \mathrm{~F}\right] AV45 uptake in AD mouse brains post-treatment. However, statistical difference was not attained in most brain regions.
    PET 示踪剂 [ 18 F ] AV 45 18 F AV 45 [^(18)(F)]AV45\left[{ }^{18} \mathrm{~F}\right] \mathrm{AV} 45 用于检测淀粉样β沉积。与野生型小鼠相比，阿尔茨海默病小鼠中发现 [ 18 F ] AV 45 18 F AV 45 [^(18)(F)]AV45\left[{ }^{18} \mathrm{~F}\right] \mathrm{AV} 45 的摄取增加，且仅在皮层观察到显著性，而在海马中未观察到。EA 治疗显示出阿尔茨海默病小鼠大脑中 18 F ] 18 F {:^(18)(F)]\left.{ }^{18} \mathrm{~F}\right] AV45 摄取减少的趋势。然而，在大多数脑区未达到统计学差异。
  • EA “Baihui (DU20) and Sishencong (EX-HN1)” significantly improved cognitive and memory functions of AD ( p = 0.04 p = 0.04 p=0.04p=0.04 ). Brain magnetic resonance imaging p ( MRI ) − p ( MRI ) − p(MRI)-\mathrm{p}(\mathrm{MRI})- positron emission tomography (PET) quantifications revealed that significantly reduced the uptake of [ 18 F ] 18 F [^(18)(F)]\left[{ }^{18} \mathrm{~F}\right] TZ4877 in the cortex ( p = 0.02 ( p = 0.02 (p=0.02(p=0.02 ) and hippocampus ( p = p = p=p= 0.03) after EA treatment.
    耳针“百会（DU20）和四神聪（EX-HN1）”显著改善了阿尔茨海默病（AD）的认知和记忆功能（ p = 0.04 p = 0.04 p=0.04p=0.04 ）。脑部磁共振成像 p ( MRI ) − p ( MRI ) − p(MRI)-\mathrm{p}(\mathrm{MRI})- 正电子发射断层扫描（PET）定量显示，耳针治疗后皮层 [ 18 F ] 18 F [^(18)(F)]\left[{ }^{18} \mathrm{~F}\right] TZ4877 的摄取显著减少（ ( p = 0.02 ( p = 0.02 (p=0.02(p=0.02 ）和海马（ p = p = p=p= 0.03）。
  • The correlation between PET quantifications and cognitive functions was analyzed and the most notable correlations were found between escape latency (reaction cognitive and memory behavior) and volume distribution ( V T ) V T (V_(T))\left(\mathrm{V}_{\mathrm{T}}\right) quantifications of [ 18 F ] 18 F [^(18)(F)]\left[{ }^{18} \mathrm{~F}\right] TZ4877. V T V T V_(T)\mathrm{V}_{\mathrm{T}} quantifications of [ 18 F ] 18 F [^(18)(F)]\left[{ }^{18} \mathrm{~F}\right] TZ4877 in key brain regions for cognitive and memory ability, such as the cortex and hippocampus, positively correlated with platform latency (cortex p < 0.01 , r = 0.7102 p < 0.01 , r = 0.7102 p < 0.01,r=0.7102p<0.01, r=0.7102; hippocampus p < 0.01 , r = 0.6891 p < 0.01 , r = 0.6891 p < 0.01,r=0.6891p<0.01, r=0.6891 ).
    PET 定量与认知功能之间的相关性进行了分析，发现逃逸潜伏期（反应认知和记忆行为）与 TZ4877 的体积分布 ( V T ) V T (V_(T))\left(\mathrm{V}_{\mathrm{T}}\right) 定量之间的相关性最为显著。TZ4877 在关键脑区（如皮层和海马）对认知和记忆能力的 V T V T V_(T)\mathrm{V}_{\mathrm{T}} 定量与平台潜伏期（皮层 p < 0.01 , r = 0.7102 p < 0.01 , r = 0.7102 p < 0.01,r=0.7102p<0.01, r=0.7102 ；海马 p < 0.01 , r = 0.6891 p < 0.01 , r = 0.6891 p < 0.01,r=0.6891p<0.01, r=0.6891 ）呈正相关。
  • Immunofluorescence confirmed colocalizations of S1PR1 with glial fibrillary acidic protein and ionized calcium-binding adaptor molecule-1 in the AD brain. And the EA treatment significantly reduced the signals in the cortex and hippocampus.
    免疫荧光确认了 S1PR1 与胶质纤维酸性蛋白和离子钙结合适配蛋白-1 在阿尔茨海默病大脑中的共定位。并且 EA 治疗显著减少了皮层和海马中的信号。
  • Immunohistochemistry showed a significant reduction of interleukin 1 β 1 β 1beta1 \beta and tumor necrosis factor α α alpha\alpha after EA treatment. EA reversed AD by suppressing neuroinflammation in the cortex and hippocampus.
    免疫组化显示，经过 EA 治疗后，白介素 1 β 1 β 1beta1 \beta 和肿瘤坏死因子 α α alpha\alpha 显著减少。EA 通过抑制皮层和海马体的神经炎症逆转了 AD。
  • The S1PR1 targeting PET tracer [ 18 F ] 18 F [^(18)(F)]\left[{ }^{18} \mathrm{~F}\right] TZ4877 showed promise in evaluating the pathological progression of A D A D ADA D in clinical settings.
    S1PR1 靶向 PET 示踪剂 [ 18 F ] 18 F [^(18)(F)]\left[{ }^{18} \mathrm{~F}\right] TZ4877 在临床环境中评估 A D A D ADA D 的病理进展方面显示出前景。

1 | BACKGROUND 1 | 背景

Alzheimer’s disease (AD) represents a pressing global health challenge that is yet to be effectively addressed. Characterized by progressive cognitive dysfunction and behavioral impairment, AD poses a significant social and economic burden on health-care systems worldwide. It is reported that ≈ 50 ≈ 50 ~~50\approx 50 million individuals are currently suffering from AD, a number projected to triple by 2050.1,2 Despite decades of research, significant advances in AD treatment remain elusive. Although recent antibody drugs, such as aducanumab and lecanemab, have shown promise in delaying the progression of AD, concerns persist regarding their long-term efficacy and potential adverse effects. 3 3 ^(3){ }^{3} Therefore, there is a pressing need for a comprehensive treatment approach that encompasses not only pharmacological interventions but also alternative non-drug therapies. 4 4 ^(4){ }^{4}
阿尔茨海默病（AD）代表了一个亟待解决的全球健康挑战。其特征是逐渐加重的认知功能障碍和行为损害，AD 对全球医疗系统造成了显著的社会和经济负担。据报道，目前有 ≈ 50 ≈ 50 ~~50\approx 50 百万个体正在遭受 AD 的困扰，预计到 2050 年这一数字将增加三倍。尽管经过数十年的研究，AD 治疗的重大进展仍然难以实现。尽管最近的抗体药物，如阿杜卡奴单抗和利卡单抗，已显示出延缓 AD 进展的希望，但人们对其长期疗效和潜在不良反应仍然存在担忧。因此，迫切需要一种综合治疗方法，不仅包括药物干预，还应涵盖替代的非药物疗法。 4 4 ^(4){ }^{4}
Acupuncture, as a traditional Chinese medicine (TCM) therapy, involves the insertion of needles into specific acupoints to alleviate various diseases and has been practiced for thousands of years with beneficial interactions and few side effects. 5 5 ^(5){ }^{5} A 2002 report by the World Health Organization reviewed controlled clinical trials and confirmed the effectiveness of acupuncture in treating various diseases. In 2017, McDonald et al. reviewed the effectiveness of acupuncture in 122 treatments in 14 clinical fields and found that it was effective in 117 situations. 6 6 ^(6){ }^{6} The external structure of the body can reflect internal pathophysiological changes, by stimulating specific acupoints on the body through which it is possible to acquire therapeutic effects of certain symptoms. Although the precise mechanisms of meridians and acupoints are not fully understood, relevant research suggests that acupuncture can regulate body function through the nerve-endocrine-immune network. 7 , 8 7 , 8 ^(7,8){ }^{7,8} Research has demonstrated
针灸作为一种传统中医（TCM）疗法，涉及将针插入特定的腧穴以缓解各种疾病，已经有数千年的历史，具有良好的相互作用和较少的副作用。 5 5 ^(5){ }^{5} 2002 年，世界卫生组织发布了一份报告，回顾了受控临床试验，并确认针灸在治疗各种疾病方面的有效性。2017 年，McDonald 等人回顾了针灸在 14 个临床领域的 122 种治疗中的有效性，发现其在 117 种情况下有效。 6 6 ^(6){ }^{6} 身体的外部结构可以反映内部病理生理变化，通过刺激身体上的特定腧穴，可以获得某些症状的治疗效果。尽管经络和腧穴的精确机制尚未完全理解，但相关研究表明，针灸可以通过神经-内分泌-免疫网络调节身体功能。 7 , 8 7 , 8 ^(7,8){ }^{7,8} 研究已证明
the anti-inflammatory effects of electro-acupuncture (EA) in neurological disorders, such as brain injury, Parkinson’s disease, dementia, post-traumatic stress disorder, and so forth; 9 , 10 9 , 10 ^(9,10){ }^{9,10} EA has also shown benefits in attenuating the progression of AD by regulating microglia- and astrocyte-associated neuroinflammation. 11 , 12 11 , 12 ^(11,12){ }^{11,12} However, further scientific evidence is needed to elucidate the underlying mechanisms and support EA as a viable alternative treatment strategy for AD.
电针（EA）在神经系统疾病中的抗炎作用，例如脑损伤、帕金森病、痴呆、创伤后应激障碍等； 9 , 10 9 , 10 ^(9,10){ }^{9,10} EA 还显示出通过调节小胶质细胞和星形胶质细胞相关的神经炎症来减缓阿尔茨海默病（AD）的进展的好处。 11 , 12 11 , 12 ^(11,12){ }^{11,12} 然而，需要进一步的科学证据来阐明其潜在机制，并支持 EA 作为阿尔茨海默病的可行替代治疗策略。
Sphingosine-1-phosphate receptors (S1PRs) signaling plays a crucial role in the neuroimmune system. Among the S1P receptors, S1PR1 is the predominant receptor expressed in the brain, involved in inflammation, immunity, and brain function regulation. 13 , 14 13 , 14 ^(13,14){ }^{13,14} In the central nervous system (CNS), S1PR1 is distributed in astrocytes, microglia, and oligodendrocytes, 15 15 ^(15){ }^{15} serving as a significant biomarker for neuroinflammation. 16 16 ^(16){ }^{16} Dysregulation of S1PR1 signaling pathways contributes to the progression of CNS inflammatory diseases, 17 17 ^(17){ }^{17} especially in AD. A previous study reported that S1PR1 expression is increased in the brains of patients with AD, and that inhibition of S1PR1 can alleviate AD-related pathological progression. 18 18 ^(18){ }^{18} Positron emission tomography (PET) is a non-invasive, dynamic, and sensitive method used to study pathophysiological neuroimmune systems in live subjects. In recent years, various imaging systems, including PET, have been used in acupuncture studies, which enables quantitative and imaging data collection and comparison at different time points before, after, and during acupuncture interventions. Such use enhances the design of high-level acupuncture research schemes and facilitates the generation of high-quality evidence. 19 19 ^(19){ }^{19} Currently, PET probes used in mechanistic research on acupuncture to improve cognitive impairment in AD include [ 18 F ] AV 45 18 F AV 45 {:^(18)(F)]AV45\left.{ }^{18} \mathrm{~F}\right] \mathrm{AV} 45 for evaluating amyloid fibers 20 20 ^(20){ }^{20} and [ 18 F ] 18 F [^(18)(F)]\left[{ }^{18} \mathrm{~F}\right] FDG for measuring glucose metabolism, 21 21 ^(21){ }^{21} which may not fully meet the expectations for feasibly evaluating anti-inflammation therapies for AD. Building upon our previous work with the S1PR1 targeting probe 18 F ] 18 F {:^(18)(F)]\left.{ }^{18} \mathrm{~F}\right] TZ4877, which was used to evaluate neuroinflammation in various physiological states and the effects of different therapeutic interventions, 22 − 24 22 − 24 ^(22-24){ }^{22-24} the present study aims to evaluate the effectiveness of EA therapy in APP/PS1 transgenic mice as an AD model; it also seeks to elucidate the mechanisms underlying EA therapy’s ability in delaying the progression of AD, particularly regarding its impact on neuroinflammation.
鞘氨醇-1-磷酸受体（S1PRs）信号在神经免疫系统中发挥着关键作用。在 S1P 受体中，S1PR1 是大脑中主要表达的受体，参与炎症、免疫和大脑功能调节。 13 , 14 13 , 14 ^(13,14){ }^{13,14} 在中枢神经系统（CNS）中，S1PR1 分布在星形胶质细胞、小胶质细胞和少突胶质细胞中， 15 15 ^(15){ }^{15} 作为神经炎症的重要生物标志物。 16 16 ^(16){ }^{16} S1PR1 信号通路的失调促进了 CNS 炎症性疾病的进展， 17 17 ^(17){ }^{17} 尤其是在阿尔茨海默病（AD）中。先前的研究报告显示，AD 患者的大脑中 S1PR1 的表达增加，抑制 S1PR1 可以减轻与 AD 相关的病理进展。 18 18 ^(18){ }^{18} 正电子发射断层扫描（PET）是一种非侵入性、动态和敏感的方法，用于研究活体对象中的病理生理神经免疫系统。近年来，包括 PET 在内的各种成像系统已被用于针灸研究，这使得在针灸干预前、后和期间在不同时间点进行定量和成像数据的收集与比较成为可能。 这种使用增强了高水平针灸研究方案的设计，并促进了高质量证据的生成。 19 19 ^(19){ }^{19} 目前，用于研究针灸改善阿尔茨海默病（AD）认知障碍的机制研究的 PET 探针包括[ 18 F ] AV 45 18 F AV 45 {:^(18)(F)]AV45\left.{ }^{18} \mathrm{~F}\right] \mathrm{AV} 45 用于评估淀粉样纤维 20 20 ^(20){ }^{20} 和 [ 18 F ] 18 F [^(18)(F)]\left[{ }^{18} \mathrm{~F}\right] FDG 用于测量葡萄糖代谢， 21 21 ^(21){ }^{21} 这可能无法完全满足对可行评估 AD 抗炎疗法的期望。在我们之前的工作基础上，使用 S1PR1 靶向探针 18 F ] 18 F {:^(18)(F)]\left.{ }^{18} \mathrm{~F}\right] TZ4877，评估了不同生理状态下的神经炎症及不同治疗干预的效果， 22 − 24 22 − 24 ^(22-24){ }^{22-24} 本研究旨在评估电针（EA）疗法在 APP/PS1 转基因小鼠作为 AD 模型中的有效性；同时探讨 EA 疗法延缓 AD 进展的机制，特别是其对神经炎症的影响。

2 | METHODS 2 | 方法

2.1 | Animal grouping and intervention
2.1 | 动物分组与干预

Ten APP/PS1 mice were randomly divided into the AD group and the EA group. Additionally, five age- and sex-matched wild-type (WT) C57BL/6 mice were used as controls. Acupuncture was performed at five acupoints on the head of each mouse, including Baihui (GV20, also DU20, located at the midpoint between the auricular apices) and four Sishencong EX-HN1 points (located at the four sides of DU20, ≈ 1.5 mm ≈ 1.5 mm ~~1.5mm\approx 1.5 \mathrm{~mm} away), according to the Guidance of Experimental Acupuncture and Moxibustion 25 25 ^(25){ }^{25} (Figure 1; Table S1 in supporting information). Prior to
十只 APP/PS1 小鼠被随机分为 AD 组和 EA 组。此外，五只年龄和性别匹配的野生型（WT）C57BL/6 小鼠作为对照。根据《实验针灸与艾灸指导》 25 25 ^(25){ }^{25} ，在每只小鼠的头部进行五个腧穴的针灸，包括百会（GV20，也称 DU20，位于耳尖之间的中点）和四个四神聪 EX-HN1 点（位于 DU20 的四侧， ≈ 1.5 mm ≈ 1.5 mm ~~1.5mm\approx 1.5 \mathrm{~mm} 远），如图 1 所示；支持信息中的表 S1。

RESEARCH IN CONTEXT 研究背景

  1. Systematic review: Given the continued increasing demand for complementary treatment strategies to enhance cognitive functions for Alzheimer’s disease (AD), acupuncture-as a non-pharmacologic therapy- holds considerable potential for its effectiveness, minimal side effects, and low cost for long-term medical management. Electro-acupuncture (EA) has shown promise in clinical treatment and animal studies of various neurological conditions, including AD. Unfortunately, scientific targeting of EA for neuroinflammation has remained explored.
    系统评价：鉴于对增强阿尔茨海默病（AD）认知功能的辅助治疗策略的需求持续增加，针灸作为一种非药物疗法，因其有效性、最小的副作用和低成本在长期医疗管理中具有相当大的潜力。电针（EA）在阿尔茨海默病等各种神经系统疾病的临床治疗和动物研究中显示出希望。不幸的是，针对神经炎症的电针科学研究仍未得到充分探索。
  2. Interpretation: The EA “Baihui (DU2O, also DV20)Sishencong (EX-HN1)” therapy significantly improved the cognitive and memory behavioral representations in the transgenic (APP/PS1) mouse model of AD, while reducing the expression of sphingosine-1-phosphate receptor 1 (S1PR1) in the cortex and hippocampus. Both [ 18 F ] [ 18 F {:^([18)F]\left.{ }^{[18} \mathrm{F}\right] TZ4877 (targeting S1PR1) and [ 18 F ] 18 F [^(18)(F)]\left[{ }^{18} \mathrm{~F}\right] AV45 (targeting amyloid composition) showed increased uptake in the AD group; the increase of which can be reduced by EA treatment significantly only indicated by [ 18 F ] 18 F {:^(18)(F)]\left.{ }^{18} \mathrm{~F}\right] TZ4877 positron emission tomography (PET). There is a negative correlation between PET quantifications (S1PR1) in different brain regions and cognitive-memory behavior. Immunofluorescence results confirmed that S1PR1 colocalized with the astrocyte and microglial activations, which proved that S1PR1 is an important target associated with glial activations for AD. Targeting of the S1PR1 PET probe [ 18 F ] T Z 4877 18 F T Z 4877 [^(18)(F)]TZ 4877\left[{ }^{18} \mathrm{~F}\right] T Z 4877 is more sensitive for evaluation of the effectiveness of AD treatments.
    解释：EA“百会（DU20，也称 DV20）四神聪（EX-HN1）”疗法显著改善了转基因（APP/PS1）阿尔茨海默病（AD）小鼠模型中的认知和记忆行为表现，同时减少了皮层和海马中鞘氨醇-1-磷酸受体 1（S1PR1）的表达。 [ 18 F ] [ 18 F {:^([18)F]\left.{ }^{[18} \mathrm{F}\right] TZ4877（靶向 S1PR1）和 [ 18 F ] 18 F [^(18)(F)]\left[{ }^{18} \mathrm{~F}\right] AV45（靶向淀粉样成分）在 AD 组中显示出增加的摄取；这种增加可以通过 EA 治疗显著减少，仅通过[ 18 F ] 18 F {:^(18)(F)]\left.{ }^{18} \mathrm{~F}\right] TZ4877 正电子发射断层扫描（PET）指示。不同脑区的 PET 定量（S1PR1）与认知-记忆行为之间存在负相关。免疫荧光结果证实 S1PR1 与星形胶质细胞和小胶质细胞的激活共定位，这证明 S1PR1 是与 AD 胶质细胞激活相关的重要靶点。靶向 S1PR1 的 PET 探针 [ 18 F ] T Z 4877 18 F T Z 4877 [^(18)(F)]TZ 4877\left[{ }^{18} \mathrm{~F}\right] T Z 4877 对评估 AD 治疗效果更为敏感。
  3. Future directions: The first application of the PET probe 18 F ] 18 F {:^(18)(F)]\left.{ }^{18} \mathrm{~F}\right] TZ4877 targeting S1PR1 demonstrated that EA “Baihui (DU2O) and Sishencong (EX-HN1)” therapy significantly improved the cognitive and memory behavior in a mouse model of AD by reducing the expression of S1PR1 in the cortex and hippocampus. From the perspective of non-pharmacological alternative treatment, this proves that S1PR1 is an important neuroinflammation biomarker for evaluating the treatment effectiveness in AD. The study suggests that the probe 18 F ] 18 F {:^(18)(F)]\left.{ }^{18} \mathrm{~F}\right] TZ4877 targeting S1PR1 may be a more sensitive probe than [ 18 F ] AV 45 18 F AV 45 {:^(18)(F)]AV45\left.{ }^{18} \mathrm{~F}\right] \mathrm{AV} 45 with potential AD clinical application regarding prediction, efficacy evaluation, or prognostic evaluation.
    未来方向：首次应用 PET 探针 18 F ] 18 F {:^(18)(F)]\left.{ }^{18} \mathrm{~F}\right] TZ4877 靶向 S1PR1 的研究表明，EA“百会（DU20）和四神聪（EX-HN1）”疗法通过减少皮层和海马体中 S1PR1 的表达，显著改善了阿尔茨海默病小鼠模型的认知和记忆行为。从非药物替代治疗的角度来看，这证明了 S1PR1 是评估阿尔茨海默病治疗效果的重要神经炎症生物标志物。研究建议，靶向 S1PR1 的探针 18 F ] 18 F {:^(18)(F)]\left.{ }^{18} \mathrm{~F}\right] TZ4877 可能比[ 18 F ] AV 45 18 F AV 45 {:^(18)(F)]AV45\left.{ }^{18} \mathrm{~F}\right] \mathrm{AV} 45 更敏感，具有潜在的阿尔茨海默病临床应用，涉及预测、疗效评估或预后评估。
    acupuncture, animals were anesthetized with 1.5 % 1.5 % 1.5%1.5 \% isoflurane, and the area of operation was disinfected using 75% alcohol. Disposable sterile acupuncture needles were then inserted to a depth of 4 mm at an angle of 15 ∘ 15 ∘ 15^(@)15^{\circ} to 30 ∘ 30 ∘ 30^(@)30^{\circ}. The needle handles were connected to a HANSLH2O2
    针灸，动物使用 1.5 % 1.5 % 1.5%1.5 \% 异氟烷麻醉，手术区域使用 75%酒精消毒。然后插入一次性无菌针灸针，深度为 4 毫米，角度为 15 ∘ 15 ∘ 15^(@)15^{\circ} 到 30 ∘ 30 ∘ 30^(@)30^{\circ} 。针柄连接到 HANSLH2O2。
FIGURE 1 Diagrammatic representation of the experimental procedure (by Figdraw). A β β beta\beta, amyloid beta; EA, electro-acupuncture; MRI, magnetic resonance imaging; PET, positron emission tomography; S1PR1, sphingosine-1-phosphate receptor 1.
图 1 实验程序的示意图（由 Figdraw 提供）。A β β beta\beta ，淀粉样β；EA，电针；MRI，磁共振成像；PET，正电子发射断层扫描；S1PR1，鞘氨醇-1-磷酸受体 1。
Electro-acupuncture instrument (Table S1), and acupuncture was performed with the following parameters: continuous wave, frequency of 2 Hz , voltage of 2 V , and current intensity of 2 mA . Mice in the EA group received EA treatment for 20 minutes once daily for 15 days, while mice in the AD and WT group were subjected to the same anesthesia conditions without EA treatment.
电针仪器（表 S1），针灸的参数如下：连续波，频率为 2 Hz，电压为 2 V，电流强度为 2 mA。EA 组的小鼠每天接受 20 分钟的电针治疗，持续 15 天，而 AD 组和 WT 组的小鼠在相同的麻醉条件下未接受电针治疗。

2.2 | Morris water maze experiment
2.2 | 莫里斯水迷宫实验

The Morris water maze (MWM) experiment was based on a wellestablished model, providing valuable insights into spatial learning and memory capabilities. The data were collected and analyzed using MWM system software and a Smart V3.0 camera system (Table S1).
莫里斯水迷宫（MWM）实验基于一个成熟的模型，为空间学习和记忆能力提供了宝贵的见解。数据通过 MWM 系统软件和 Smart V3.0 摄像系统收集和分析（表 S1）。

2.2.1 | Visual platform experiment
2.2.1 | 视觉平台实验

The mice were trained for 1 day to eliminate visual interference and assess swimming ability. During the experiment, visual platforms were randomly placed in four quadrants, with two entry points on opposite sides of the platform quadrant. The swimming distance and time were recorded based on whether the animal was able to find the platform within 60 seconds and remained on it for 5 seconds. If the animal
小鼠经过 1 天的训练，以消除视觉干扰并评估游泳能力。在实验过程中，视觉平台随机放置在四个象限中，平台象限的对面有两个入口点。根据动物是否能够在 60 秒内找到平台并在上面停留 5 秒，记录游泳距离和时间。如果动物
failed to find the platform within 60 seconds, it was then removed from the water and placed on the platform for 15 seconds to observe and remember the position, with an escape incubation period of 60 seconds.
未能在 60 秒内找到平台，随后将其从水中取出并放置在平台上观察和记住位置 15 秒，逃逸孵化期为 60 秒。

2.2.2 | Hidden platform experiment
2.2.2 | 隐藏平台实验

The mice underwent a hidden platform experiment for 4 days to assess changes in learning and memory. The hidden platform remained at a fixed position in a specific quadrant throughout the experiment. Subsequently, four fixed entry points were set, the daily training launching points were sorted randomly, and the distance between the entry point and the center of the sink was equal. The experimenter oriented the mouse with its head facing the tank wall and placed it in the water.
小鼠进行了为期 4 天的隐蔽平台实验，以评估学习和记忆的变化。隐蔽平台在整个实验过程中保持在特定象限的固定位置。随后设置了四个固定的入口点，日常训练的发射点随机排序，入口点与水槽中心之间的距离相等。实验者将小鼠的头部朝向水箱壁，并将其放入水中。

2.2.3 | Space exploration experiment
2.2.3 | 太空探索实验

In this experiment, the platform was removed and mice were placed at the midpoint opposite to the targeted quadrant where it had been previously located, then each mouse was also observed for 60 seconds after entering the water, recording and analyzing the spatial memory ability.
在这个实验中，平台被移除，老鼠被放置在与之前所在的目标象限相对的中点，然后每只老鼠在进入水中后也被观察 60 秒，记录和分析空间记忆能力。

2.3 | Radiosynthesis of [ 18 F ] 18 F [^(18)(F)]\left[{ }^{18} \mathrm{~F}\right] TZ4877 and [ 18 F ] 18 F [^(18)(F)]\left[{ }^{18} \mathrm{~F}\right] AV45
2.3 | [ 18 F ] 18 F [^(18)(F)]\left[{ }^{18} \mathrm{~F}\right] TZ4877 和 [ 18 F ] 18 F [^(18)(F)]\left[{ }^{18} \mathrm{~F}\right] AV45 的放射合成

The synthesis and quality control of [18 F]TZ4877 were achieved by modifying a previous study. 24 24 ^(24){ }^{24} The nucleophilic reaction between the tosylate precursor and [18 F]KF in acetonitrile with Kryptofifix 222 was followed by deprotection of the methoxymethyl group using hydrochloric acid (6 M). After purification using semi-preparative high-performance liquid chromatography (HPLC) combined with solidphase extraction, [18F]TZ4877 was formulated using 10% ethanol in saline with high radiochemical purity (> 95%), radiochemical yields ( 58.5 ± 12.7 % 58.5 ± 12.7 % 58.5+-12.7%58.5 \pm 12.7 \% ), and specific activity ( 17.6 ± 5.3 GBq / μ mol , n = 18 17.6 ± 5.3 GBq / μ mol , n = 18 17.6+-5.3GBq//mumol,n=1817.6 \pm 5.3 \mathrm{GBq} / \mu \mathrm{mol}, n=18, decay corrected to the end of synthesis; Figure S1 in supporting information). The synthesis and quality control of [ 18 F ] AV 45 18 F AV 45 {:^(18)(F)]AV45\left.{ }^{18} \mathrm{~F}\right] \mathrm{AV} 45 were produced from Guangdong Cyclotron Medical Science Co., Ltd. following a previous procedure 26 26 ^(26){ }^{26} and formulated using 10 mL ascorbic acid aqueous solution (0.15%) with high radiochemical purity (91.60%), radiochemical yields (62%), and specific activity ( 0.389 GBq / μ mol 0.389 GBq / μ mol 0.389GBq//mumol0.389 \mathrm{GBq} / \mu \mathrm{mol}, decay corrected to the end of synthesis; Table S2 in supporting information).
[18 F]TZ4877 的合成和质量控制是通过修改之前的研究实现的。 24 24 ^(24){ }^{24} 在含有 Kryptofifix 222 的乙腈中，tosylate 前体与[18 F]KF 之间的亲核反应后，使用盐酸（6 M）去保护甲氧基甲基基团。经过半制备高效液相色谱（HPLC）结合固相萃取的纯化后，使用 10%乙醇在生理盐水中配制[18F]TZ4877，具有高放射化学纯度（> 95%）、放射化学产率（ 58.5 ± 12.7 % 58.5 ± 12.7 % 58.5+-12.7%58.5 \pm 12.7 \% ）和比活性（ 17.6 ± 5.3 GBq / μ mol , n = 18 17.6 ± 5.3 GBq / μ mol , n = 18 17.6+-5.3GBq//mumol,n=1817.6 \pm 5.3 \mathrm{GBq} / \mu \mathrm{mol}, n=18 ，衰变校正至合成结束；支持信息中的图 S1）。[ 18 F ] AV 45 18 F AV 45 {:^(18)(F)]AV45\left.{ }^{18} \mathrm{~F}\right] \mathrm{AV} 45 的合成和质量控制是由广东环流医学科学有限公司按照之前的程序 26 26 ^(26){ }^{26} 生产的，并使用 10 mL 抗坏血酸水溶液（0.15%）配制，具有高放射化学纯度（91.60%）、放射化学产率（62%）和比活性（ 0.389 GBq / μ mol 0.389 GBq / μ mol 0.389GBq//mumol0.389 \mathrm{GBq} / \mu \mathrm{mol} ，衰变校正至合成结束；支持信息中的表 S2）。

2.4 | Magnetic resonance imaging acquisition
2.4 | 磁共振成像获取

For magnetic resonance imaging (MRI) experiments, T2-weighted brain images of mice were obtained using a DOTY 400 MHz 1H Rx surface coil on a 9.4 Tesla Bruker BioSpec small-animal MRI system (Bruker BioSpin MRI, Ettlingen, Germany). Animals from each group were imaged individually. Initially, animals were anesthetized under 2% isoflurane in an induction chamber. The anesthetized mice were transferred to an MR-compatible cradle and positioned in an MRIcompatible head holder to minimize head motion. Anesthesia was then maintained at 1.5% isoflurane in the air throughout imaging. The respiration rate was monitored using a pressure pad placed under the animal’s abdomen and the animal’s body temperature was maintained by a warming pad ( 37 ∘ C ) 37 ∘ C (37^(@)C)\left(37^{\circ} \mathrm{C}\right) placed under the animal. The imaging was conducted on a horizontal bore 9.4 Biospec pre-clinical MRI system (Bruker BioSpin MRI GmbH) equipped with shielded gradients (maximum gradient strength = 660 mT / m = 660 mT / m =660mT//m=660 \mathrm{mT} / \mathrm{m}, rise time = 4750 T / m / s = 4750 T / m / s =4750T//m//s=4750 \mathrm{~T} / \mathrm{m} / \mathrm{s} ). An 86 mm quadrature volume resonator was used for transmission and a four-element array cryocoil was used for signal reception (Cryoprobe, Bruker, BioSpin).
对于磁共振成像（MRI）实验，使用 DOTY 400 MHz 1H Rx 表面线圈在 9.4 特斯拉 Bruker BioSpec 小动物 MRI 系统（Bruker BioSpin MRI，德国埃特林根）上获得小鼠的 T2 加权脑图像。每组动物单独成像。最初，动物在诱导舱中接受 2%异氟烷麻醉。麻醉后的小鼠被转移到 MR 兼容的摇篮中，并放置在 MRI 兼容的头部固定器中，以最小化头部运动。麻醉在成像过程中保持在 1.5%异氟烷空气中。通过放置在动物腹部下方的压力垫监测呼吸频率，并通过放置在动物下方的加热垫 ( 37 ∘ C ) 37 ∘ C (37^(@)C)\left(37^{\circ} \mathrm{C}\right) 维持动物的体温。成像是在配备有屏蔽梯度（最大梯度强度 = 660 mT / m = 660 mT / m =660mT//m=660 \mathrm{mT} / \mathrm{m}