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
^(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} Guangdong Provincial Engineering Research Center of Molecular Imaging, The Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai, China ^(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} Guangdong-Hong Kong-Macao University Joint Laboratory of Interventional Medicine, The Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai, China ^(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} Pharmaceutical Clinical Trails Office, The Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai, China ^(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} 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} \mathrm{~F} ]TZ4877 targeting sphingosine-1-phosphate receptor 1 (S1PR1) and [ {:^(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} \mathrm{~F} ]TZ4877 靶向鞘氨醇-1-磷酸受体 1（S1PR1）和[ {:^(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=0.04p=0.04 ) and reduced the uptake of [18 F]TZ4877 in the cortex ( p=0.02p=0.02 ) and hippocampus ( 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 1beta1 \beta and tumor necrosis factor 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=0.04p=0.04 ），并减少了皮层（ p=0.02p=0.02 ）和海马（ 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 1beta1 \beta 和肿瘤坏死因子 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
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)]AV45\left[{ }^{18} \mathrm{~F}\right] \mathrm{AV} 45 was used to detect amyloid beta deposition. An increased uptake of [^(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)]\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)]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)]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)]\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=0.04p=0.04 ). Brain magnetic resonance imaging p(MRI)-\mathrm{p}(\mathrm{MRI})- positron emission tomography (PET) quantifications revealed that significantly reduced the uptake of [^(18)(F)]\left[{ }^{18} \mathrm{~F}\right] TZ4877 in the cortex (p=0.02(p=0.02 ) and hippocampus ( 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=0.04p=0.04 ）。脑部磁共振成像 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)]\left[{ }^{18} \mathrm{~F}\right] TZ4877 的摄取显著减少（ (p=0.02(p=0.02 ）和海马（ 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))\left(\mathrm{V}_{\mathrm{T}}\right) quantifications of [^(18)(F)]\left[{ }^{18} \mathrm{~F}\right] TZ4877. V_(T)\mathrm{V}_{\mathrm{T}} quantifications of [^(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=0.7102p<0.01, r=0.7102; hippocampus 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))\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)\mathrm{V}_{\mathrm{T}} 定量与平台潜伏期（皮层 p < 0.01,r=0.7102p<0.01, r=0.7102 ；海马 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 1beta1 \beta and tumor necrosis factor 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 1beta1 \beta 和肿瘤坏死因子 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)]\left[{ }^{18} \mathrm{~F}\right] TZ4877 showed promise in evaluating the pathological progression of ADA D in clinical settings. S1PR1 靶向 PET 示踪剂 [^(18)(F)]\left[{ }^{18} \mathrm{~F}\right] TZ4877 在临床环境中评估 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\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} 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} 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\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}
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} 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} 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} 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} 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} 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} 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} EA has also shown benefits in attenuating the progression of AD by regulating microglia- and astrocyte-associated neuroinflammation. ^(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} 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} 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} In the central nervous system (CNS), S1PR1 is distributed in astrocytes, microglia, and oligodendrocytes, ^(15){ }^{15} serving as a significant biomarker for neuroinflammation. ^(16){ }^{16} Dysregulation of S1PR1 signaling pathways contributes to the progression of CNS inflammatory diseases, ^(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} 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} Currently, PET probes used in mechanistic research on acupuncture to improve cognitive impairment in AD include [ {:^(18)(F)]AV45\left.{ }^{18} \mathrm{~F}\right] \mathrm{AV} 45 for evaluating amyloid fibers ^(20){ }^{20} and [^(18)(F)]\left[{ }^{18} \mathrm{~F}\right] FDG for measuring glucose metabolism, ^(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)]\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} 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} 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} 作为神经炎症的重要生物标志物。 ^(16){ }^{16} S1PR1 信号通路的失调促进了 CNS 炎症性疾病的进展， ^(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} 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} 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)]AV45\left.{ }^{18} \mathrm{~F}\right] \mathrm{AV} 45 用于评估淀粉样纤维 ^(20){ }^{20} 和 [^(18)(F)]\left[{ }^{18} \mathrm{~F}\right] FDG 用于测量葡萄糖代谢， ^(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)]\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} 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, ~~1.5mm\approx 1.5 \mathrm{~mm} away), according to the Guidance of Experimental Acupuncture and Moxibustion ^(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} ，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， ~~1.5mm\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
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。
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]\left.{ }^{[18} \mathrm{F}\right] TZ4877 (targeting S1PR1) and [^(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)]\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)]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]\left.{ }^{[18} \mathrm{F}\right] TZ4877（靶向 S1PR1）和 [^(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)]\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)]TZ 4877\left[{ }^{18} \mathrm{~F}\right] T Z 4877 对评估 AD 治疗效果更为敏感。
Future directions: The first application of the PET probe {:^(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)]\left.{ }^{18} \mathrm{~F}\right] TZ4877 targeting S1PR1 may be a more sensitive probe than [ {:^(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)]\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)]\left.{ }^{18} \mathrm{~F}\right] TZ4877 可能比[ {:^(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 \% 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^{\circ} to 30^(@)30^{\circ}. The needle handles were connected to a HANSLH2O2 针灸，动物使用 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^{\circ} 到 30^(@)30^{\circ} 。针柄连接到 HANSLH2O2。
FIGURE 1 Diagrammatic representation of the experimental procedure (by Figdraw). A 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 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。
The synthesis and quality control of [18 F]TZ4877 were achieved by modifying a previous study. ^(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 \pm 12.7 \% ), and specific activity ( 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)]AV45\left.{ }^{18} \mathrm{~F}\right] \mathrm{AV} 45 were produced from Guangdong Cyclotron Medical Science Co., Ltd. following a previous procedure ^(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 ( 0.389GBq//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} 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 \pm 12.7 \% ）和比活性（ 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)]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} 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（ 0.389GBq//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)\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 =660mT//m=660 \mathrm{mT} / \mathrm{m}, rise time =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)\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 =660mT//m=660 \mathrm{mT} / \mathrm{m} ，上升时间 =4750T//m//s=4750 \mathrm{~T} / \mathrm{m} / \mathrm{s} ）的水平孔 9.4 Biospec 临床前 MRI 系统（Bruker BioSpin MRI GmbH）上进行的。使用 86 mm 四分量体积谐振器进行信号传输，使用四元素阵列冷探头进行信号接收（Cryoprobe，Bruker，BioSpin）。
2.5 | PET imaging 2.5 | PET 成像
PET/computed tomography (CT) imaging studies were performed on the WT (n=5),AD(n=5)(n=5), \mathrm{AD}(n=5), and the EA group (n=5)(n=5) for [^(18)(F)]\left[{ }^{18} \mathrm{~F}\right] TZ4877 and [ {:^(18)(F)]AV 45\left.{ }^{18} \mathrm{~F}\right] A V 45 separately. The interval between [^(18)(F)]\left[{ }^{18} \mathrm{~F}\right] TZ4877 and [18 F]AV45 imaging was 3 days. The mice were anesthetized with 1.5%1.5 \% to 2%2 \% isoflurane inhalation and intravenously injected with 150 muL150 \mu \mathrm{~L} of 8.2+-1.69MBq//mouse[^(18)(F)]8.2 \pm 1.69 \mathrm{MBq} / \mathrm{mouse}\left[{ }^{18} \mathrm{~F}\right] TZ4877 or 8.7+-8.7 \pm 1.47 MBq/mouse [18 F]AV45. A dynamic 0- to 30-minute micro-PET/CT (Mediso nanoScan PET/CT imaging system, Mediso Inc.) scan protocol PET/计算机断层扫描（CT）成像研究在 WT (n=5),AD(n=5)(n=5), \mathrm{AD}(n=5) 和 EA 组 (n=5)(n=5) 上进行，针对 [^(18)(F)]\left[{ }^{18} \mathrm{~F}\right] TZ4877 和[ {:^(18)(F)]AV 45\left.{ }^{18} \mathrm{~F}\right] A V 45 分别进行。 [^(18)(F)]\left[{ }^{18} \mathrm{~F}\right] TZ4877 和[18 F]AV45 成像之间的间隔为 3 天。小鼠在 1.5%1.5 \% 下麻醉，吸入 2%2 \% 异氟烷，并静脉注射 150 muL150 \mu \mathrm{~L} 的 8.2+-1.69MBq//mouse[^(18)(F)]8.2 \pm 1.69 \mathrm{MBq} / \mathrm{mouse}\left[{ }^{18} \mathrm{~F}\right] TZ4877 或 8.7+-8.7 \pm 1.47 MBq/小鼠[18 F]AV45。动态 0 到 30 分钟微型 PET/CT（Mediso nanoScan PET/CT 成像系统，Mediso Inc.）扫描协议。
was performed immediately after [ {:^(18)(F)]\left.{ }^{18} \mathrm{~F}\right] TZ4877 or [ {:^(18)(F)]AV45\left.{ }^{18} \mathrm{~F}\right] \mathrm{AV} 45 injection through vein. Dynamic PET images were reconstructed into 27 frames ( 10 xx310 \times 3 seconds, 3xx103 \times 10 seconds, 4xx604 \times 60 seconds, and 10 xx15010 \times 150 seconds). PET images (voxel size =0.4mm=0.4 \mathrm{~mm} ) were reconstructed using ordered subset expectation maximization (OSEM) algorithm with CT attenuation correction and isotope decay correction. 在[ {:^(18)(F)]\left.{ }^{18} \mathrm{~F}\right] TZ4877 或[ {:^(18)(F)]AV45\left.{ }^{18} \mathrm{~F}\right] \mathrm{AV} 45 静脉注射后立即进行。动态 PET 图像被重建为 27 帧（ 10 xx310 \times 3 秒， 3xx103 \times 10 秒， 4xx604 \times 60 秒和 10 xx15010 \times 150 秒）。PET 图像（体素大小 =0.4mm=0.4 \mathrm{~mm} ）使用有序子集期望最大化（OSEM）算法进行重建，并进行了 CT 衰减校正和同位素衰减校正。
2.6 | PET data analysis 2.6 | PET 数据分析
Through the co-registration of PET and CT, PET data of original [18F]TZ4877 and [18 F]AV45 were quantified using a PMOD Biomedical Image Quantification System (Table S1). The PMOD Fusion Tool was used to obtain the blood input by drawing the region of interest (ROI) of the left ventricle. The volume of interest (VOI) consisting of at least three ROIs on the target area was obtained. The Mouse Brain Atlas of Ma-Benveniste-Mirrione in the PNROD from PMOD was used for mouse brain segmentation. PET data in target region was expressed as standardized uptake value (SUV). SUV was calculated as the activity concentration within the VOI(Bq//mL)\mathrm{VOI}(\mathrm{Bq} / \mathrm{mL}) divided by injected activity ( Bq ) and multiplied by body weight (g). The VOI was plotted on each brain region and the data at 30 minutes post-injection for static PET/CT to obtain SUV values, and over 30 minutes for dynamic PET/CT to obtain the time activity curve (TAC). The TAC curves of different brain regions were recorded and used to calculate volume distribution (V_(T))\left(V_{T}\right) through the Logan plot (LoganAIF). ^(27){ }^{27} TACs of the left ventricle were obtained from the original micro-PET data and used for arterial input function. The goodness of fit was evaluated with r^(2)r^{2}. 通过 PET 和 CT 的共同注册，使用 PMOD 生物医学图像定量系统对原始[18F]TZ4877 和[18F]AV45 的 PET 数据进行了定量（表 S1）。使用 PMOD 融合工具通过绘制左心室的感兴趣区域（ROI）来获取血液输入。获得了由至少三个 ROI 组成的目标区域的感兴趣体积（VOI）。使用 PMOD 中的 Ma-Benveniste-Mirrione 小鼠脑图谱进行小鼠脑分割。目标区域的 PET 数据以标准摄取值（SUV）表示。SUV 的计算方法是将 VOI(Bq//mL)\mathrm{VOI}(\mathrm{Bq} / \mathrm{mL}) 内的活性浓度除以注射活性（Bq），再乘以体重（g）。VOI 在每个脑区上绘制，并在静态 PET/CT 注射后 30 分钟获取 SUV 值，在动态 PET/CT 中超过 30 分钟获取时间活性曲线（TAC）。记录不同脑区的 TAC 曲线，并通过 Logan 图（LoganAIF）计算体积分布 (V_(T))\left(V_{T}\right) 。 ^(27){ }^{27} 左心室的 TAC 从原始微 PET 数据中获得，并用于动脉输入函数。 拟合优度通过 r^(2)r^{2} 进行评估。
2.7 | Immunofluorescence staining and immunohistochemistry 2.7 | 免疫荧光染色和免疫组化
Immunofluorescence (IF) staining for S1PR1 and glial fibrillary acidic proteins (GFAP) or ionized calcium-binding adaptor molecule-1 (IBA1) was conducted on brain tissue collected post-PET experiments. Mice were euthanized and their brains were perfused with 4% paraformaldehyde and phosphate-buffered saline (PBS). Brain tissue was removed to place in a cassette, with optimal cutting temperature compound frozen in liquid nitrogen, and then stored at -80^(@)C-80^{\circ} \mathrm{C}. Brain tissue was sliced into 6mum6 \mu \mathrm{~m} sections by using a frozen microtome and then permeabilized with 0.3% triton-100 for 30 minutes. After washing with PBS, the slices were blocked with 10% bovine serum albumin and incubated overnight at 4^(@)C4^{\circ} \mathrm{C} with S1PR1, GFAP, and IBA-1 primary antibodies (Table S2). After three washes with PBS, the tissues were incubated with a mixture of secondary antibodies against S1PR1, GFAP, IBA-1, and DAPI at room temperature for two hours, and coverslips were added. Also, immunohistochemistry for interleukin (IL)-1 beta\beta and tumor necrosis factor alpha (TNF- alpha\alpha ) was conducted on the paraffin sections of brain tissue. All the above reagent details are described in Table S2. Images were acquired using a ZEISS LSM 880 laser scanning confocal microscope system (Table S1). 对收集的脑组织进行 S1PR1 和胶质纤维酸性蛋白（GFAP）或离子钙结合适配蛋白-1（IBA1）的免疫荧光（IF）染色，样本来自 PET 实验后。小鼠被安乐死，脑部用 4%多聚甲醛和磷酸盐缓冲液（PBS）灌注。脑组织被取出放入盒中，最佳切割温度化合物在液氮中冷冻，然后储存于 -80^(@)C-80^{\circ} \mathrm{C} 。脑组织使用冷冻切片机切成 6mum6 \mu \mathrm{~m} 切片，然后用 0.3% Triton-100 透化 30 分钟。用 PBS 洗涤后，切片用 10%牛血清白蛋白封闭，并在 4^(@)C4^{\circ} \mathrm{C} 与 S1PR1、GFAP 和 IBA-1 一抗孵育过夜（见表 S2）。经过三次 PBS 洗涤后，组织在室温下与针对 S1PR1、GFAP、IBA-1 和 DAPI 的二抗混合物孵育两小时，并添加盖玻片。此外，还对脑组织的石蜡切片进行了白介素（IL）-1 beta\beta 和肿瘤坏死因子α（TNF- alpha\alpha ）的免疫组化。所有上述试剂的详细信息见表 S2。 图像是使用 ZEISS LSM 880 激光扫描共聚焦显微镜系统获取的（表 S1）。
2.8 | Statistical analysis 2.8 | 统计分析
Statistical analyses were performed using GraphPad Prism 9.0 (GraphPad Software, Inc.) and SPSS 26.0 (IBM). Data are expressed as mean and standard deviation ( +-\pm SD). For comparison between multiple groups, one-way analysis of variance (ANOVA) was used for normal distribution. After analysis of homogeneity of variance, the least significant difference (LSD) test was used for pairwise comparison between groups, and the rest were compared by non-parametric test. Differences were considered statistically significant at p < 0.05p<0.05. 统计分析使用 GraphPad Prism 9.0（GraphPad Software, Inc.）和 SPSS 26.0（IBM）进行。数据以均值和标准差（ +-\pm SD）表示。对于多组之间的比较，正态分布使用单因素方差分析（ANOVA）。在方差齐性分析后，使用最小显著差异（LSD）检验进行组间的成对比较，其余使用非参数检验进行比较。差异在 p < 0.05p<0.05 时被认为具有统计学意义。
3 | RESULTS 3 | 结果
3.1 | EA treatment reduced the escape latency of AD mice 3.1 | EA 治疗减少了 AD 小鼠的逃逸潜伏期
In the hidden platform experiment, the escape latency of mice in each group showed a decreasing trend over the training days. Compared to the WT group, the escape latency of mice in the AD group was significantly prolonged ( 46.63+-6.3446.63 \pm 6.34 seconds in AD vs. 25.54+-6.5025.54 \pm 6.50 seconds in WT, n=5,p=0.002n=5, p=0.002 ), confirming that the 8-month-old APP/PS1 mice (genotyped in supplementary materials Figure S2 in supporting information) had obvious impairment in learning and memory. Surprisingly, the escape latency in EA-treated mice ( 30.42+-10.5830.42 \pm 10.58 seconds in EA vs. 46.63+-6.3446.63 \pm 6.34 seconds in AD, n=5,p=0.04n=5, p=0.04 ) was significantly shorter than that of the untreated AD group; and did not significantly differ from the WT group ( 30.42+-10.5830.42 \pm 10.58 seconds in EA vs. 25.54+-6.50sec-25.54 \pm 6.50 \mathrm{sec}- onds in WT, n=5,p=0.44n=5, p=0.44 ), further suggesting that the EA treatment was able to reverse the learning and memory ability of AD mice to close to the WT mice (Figure 2A). 在隐蔽平台实验中，各组小鼠的逃逸潜伏期在训练天数中呈下降趋势。与 WT 组相比，AD 组小鼠的逃逸潜伏期显著延长（AD 组 46.63+-6.3446.63 \pm 6.34 秒 vs. WT 组 25.54+-6.5025.54 \pm 6.50 秒， n=5,p=0.002n=5, p=0.002 ），确认 8 个月大的 APP/PS1 小鼠（基因型见补充材料图 S2）在学习和记忆方面有明显的损伤。令人惊讶的是，EA 处理的小鼠的逃逸潜伏期（EA 组 30.42+-10.5830.42 \pm 10.58 秒 vs. AD 组 46.63+-6.3446.63 \pm 6.34 秒， n=5,p=0.04n=5, p=0.04 ）显著短于未处理的 AD 组；并且与 WT 组没有显著差异（EA 组 30.42+-10.5830.42 \pm 10.58 秒 vs. WT 组 25.54+-6.50sec-25.54 \pm 6.50 \mathrm{sec}- 秒， n=5,p=0.44n=5, p=0.44 ），进一步表明 EA 治疗能够将 AD 小鼠的学习和记忆能力逆转至接近 WT 小鼠的水平（图 2A）。
3.2 | EA treatment improved the spatial exploration time of AD mice 3.2 | EA 治疗改善了 AD 小鼠的空间探索时间
Similarly, a spatial exploration experiment was performed after the hidden platform experiment to test the spatial memory of the mice after 4 days of training. The longer the swimming time in the platform quadrant, the better the memory retention. In this experiment, the exploration time was significantly (p=0.04)(p=0.04) shorter in the AD group ( 10.51+-2.5210.51 \pm 2.52 seconds, n=5n=5 ) than in the WT group (16.85+-1.47(16.85 \pm 1.47 seconds, n=5n=5 ). However, the spatial exploration time was significantly ( p=p= 0.02) longer in the EA group ( 14.88+-4.0314.88 \pm 4.03 seconds, n=5n=5 ) compared to the AD group ( 10.51+-2.5210.51 \pm 2.52 seconds). Despite the longest exploration time in the WT group ( 16.85+-1.4716.85 \pm 1.47 seconds), there was no significant ( p=0.11p=0.11 ) difference compared to the EA group ( 14.88+-4.0314.88 \pm 4.03 seconds; Figure 2B). The map of a typical mouse tour track showed the spatial memory ability of mice in different groups (WT > EA > AD, Figure 2C). 同样，在隐藏平台实验后进行了空间探索实验，以测试小鼠在经过 4 天训练后的空间记忆。小鼠在平台象限中的游泳时间越长，记忆保持得越好。在本实验中，AD 组的探索时间显著 (p=0.04)(p=0.04) 短于 WT 组（ 10.51+-2.5210.51 \pm 2.52 秒， n=5n=5 ）。然而，EA 组的空间探索时间显著（ p=p= 0.02）长于 AD 组（ 10.51+-2.5210.51 \pm 2.52 秒）。尽管 WT 组的探索时间最长（ 16.85+-1.4716.85 \pm 1.47 秒），但与 EA 组（ 14.88+-4.0314.88 \pm 4.03 秒；图 2B）相比没有显著（ p=0.11p=0.11 ）差异。典型小鼠游览轨迹的地图显示了不同组小鼠的空间记忆能力（WT > EA > AD，图 2C）。
Swimming distance in the platform quadrant also served as an indicator of spatial cognitive and memory abilities. The longer the swimming distance in the platform, the better the memory retention. The longest swimming distance was in the WT group ( 40.50+-4.28cm40.50 \pm 4.28 \mathrm{~cm} ), followed by the EA group ( 33.70+-9.07cm33.70 \pm 9.07 \mathrm{~cm} ), and the shortest was in the 平台象限中的游泳距离也作为空间认知和记忆能力的指标。游泳距离越长，记忆保持越好。最长的游泳距离在 WT 组（ 40.50+-4.28cm40.50 \pm 4.28 \mathrm{~cm} ），其次是 EA 组（ 33.70+-9.07cm33.70 \pm 9.07 \mathrm{~cm} ），最短的是在
AD group (20.07 +-6.62cm\pm 6.62 \mathrm{~cm}; WT vs. AD: p=0.0008;EAp=0.0008 ; \mathrm{EA} vs. AD: p=0.04p=0.04; WT vs. EA: p=0.21,n=5p=0.21, n=5 ) as shown in Figure 2D. Similarly, the greater the number of platform crossings, the better the memory retention (2 +-0.63\pm 0.63 times in the WT group, 1.2+-0.751.2 \pm 0.75 times in the EA group, and 0.8 +-0.4\pm 0.4 times in the AD group, WT vs. AD: p=0.01p=0.01; EA vs. AD: p=0.37p=0.37; WT vs. EA: p=0.14,n=5p=0.14, n=5, respectively; Figure 2 E ). AD 组（20.07 +-6.62cm\pm 6.62 \mathrm{~cm} ；WT 与 AD： p=0.0008;EAp=0.0008 ; \mathrm{EA} 与 AD： p=0.04p=0.04 ；WT 与 EA： p=0.21,n=5p=0.21, n=5 ），如图 2D 所示。同样，平台穿越次数越多，记忆保持越好（WT 组为 2 +-0.63\pm 0.63 次，EA 组为 1.2+-0.751.2 \pm 0.75 次，AD 组为 0.8 +-0.4\pm 0.4 次，WT 与 AD： p=0.01p=0.01 ；EA 与 AD： p=0.37p=0.37 ；WT 与 EA： p=0.14,n=5p=0.14, n=5 ，分别；图 2E）。
3.3 | EA treatment reduced uptake of {:^([18)F]\left.{ }^{[18} \mathrm{F}\right] TZ4877 in the cortex and hippocampus of AD mice 3.3 | EA 治疗减少了阿尔茨海默病小鼠皮层和海马中 {:^([18)F]\left.{ }^{[18} \mathrm{F}\right] TZ4877 的摄取
To assess the potential impact of EA treatment on neuroinflammation in these mice in vivo, PET/CT/MRI brain imaging and quantifications were performed. As shown in Figure 3 and Figure S3 in supporting information, compared to the WT group ( n=5n=5 ), a notable increase of [^(18):}\left[{ }^{18}\right. F]TZ4877 uptake was found in AD mice (n=5)(n=5) at 30 minutes postinjection (Table 1). Furthermore, the increased uptake in the AD brain can be visibly reduced especially in the cortex and hippocampus by EA (n=5)(n=5) treatment as illustrated in Figure 3A, and the quantification of SUV is presented in Table 1. Although there was a slightly higher SUV in EA mice than in WT mice, the disparity was not statistically significant. 为了评估 EA 治疗对这些小鼠体内神经炎症的潜在影响，进行了 PET/CT/MRI 脑成像和定量分析。如图 3 和支持信息中的图 S3 所示，与 WT 组（ n=5n=5 ）相比，AD 小鼠 (n=5)(n=5) 在注射后 30 分钟发现了显著增加的 [^(18):}\left[{ }^{18}\right. F]TZ4877 摄取（表 1）。此外，EA (n=5)(n=5) 治疗可以明显减少 AD 脑中，特别是在皮层和海马体的摄取，如图 3A 所示，SUV 的定量结果见表 1。尽管 EA 小鼠的 SUV 略高于 WT 小鼠，但差异没有统计学意义。
3.4 | EA treatment declines amyloid beta expression in brain regions of AD mice 3.4 | EA 治疗降低阿尔茨海默病小鼠脑区的淀粉样β表达
To further investigate the efficacy of EA in AD, PET tracer [18 F]AV45 was used to detect amyloid beta ( A betaA \beta ) deposition in mouse brains. As shown in Figure 4 and Figure S4 in supporting information, an increased uptake of [^(18)(F)]\left[{ }^{18} \mathrm{~F}\right] AV45 was found in AD mice (n=5)(n=5) compared to WT mice (n=3)(n=3), with significance observed only in the cortex and not in the hippocampus. Similarly, EA treatment (n=5)(n=5) exhibited a trend toward reduced [18 F]AV45 uptake in AD mouse brains post-treatment. However, statistical difference was not attained in most brain regions (Table 1). 为了进一步研究 EA 在阿尔茨海默病（AD）中的疗效，使用 PET 示踪剂[18 F]AV45 检测小鼠大脑中的淀粉样β（ A betaA \beta ）沉积。如图 4 和支持信息中的图 S4 所示，与野生型小鼠（WT） (n=3)(n=3) 相比，AD 小鼠 (n=5)(n=5) 中发现 [^(18)(F)]\left[{ }^{18} \mathrm{~F}\right] AV45 的摄取增加，且仅在皮层观察到显著性，而在海马体中未观察到类似情况。同样，EA 治疗 (n=5)(n=5) 在治疗后表现出 AD 小鼠大脑中[18 F]AV45 摄取减少的趋势。然而，在大多数脑区未达到统计学差异（表 1）。
3.5 | The V_(T)V_{\mathrm{T}} quantifications 3.5 | V_(T)V_{\mathrm{T}} 的量化
FIGURE 2 EA treatment improved the cognitive learning and memory ability of AD mice. A, The trend of escape latency of mice in the hidden platform experiment. B, The exploration time of mice in the platform quadrant experiment. C, The typical trajectory of three groups in a spatial exploration experiment. D , The exploration distance in the platform quadrant of each group in the space exploration trial (WT vs. AD, p < 0.01p<0.01; EA vs. AD, p < 0.05p<0.05; WT vs. EA, p > 0.05p>0.05 ). E, The platform crossing numbers of each group in the space exploration trial (WT vs. AD, p < 0.05p<0.05; EA vs. AD and WT vs. EA, p > 0.05;n=5).^(**)p < 0.05,^(****)p < 0.01,^(******)p < 0.001p>0.05 ; n=5) .{ }^{*} p<0.05,{ }^{* *} p<0.01,{ }^{* * *} p<0.001, ns: p > 0.05p>0.05. AD, Alzheimer’s disease; EA, electro-acupuncture; WT, wild type. 图 2 EA 治疗改善了 AD 小鼠的认知学习和记忆能力。A、隐蔽平台实验中小鼠逃逸潜伏期的趋势。B、小鼠在平台象限实验中的探索时间。C、三组在空间探索实验中的典型轨迹。D、各组在空间探索试验中平台象限的探索距离（WT 与 AD， p < 0.01p<0.01 ；EA 与 AD， p < 0.05p<0.05 ；WT 与 EA， p > 0.05p>0.05 ）。E、各组在空间探索试验中的平台穿越次数（WT 与 AD， p < 0.05p<0.05 ；EA 与 AD 和 WT 与 EA， p > 0.05;n=5).^(**)p < 0.05,^(****)p < 0.01,^(******)p < 0.001p>0.05 ; n=5) .{ }^{*} p<0.05,{ }^{* *} p<0.01,{ }^{* * *} p<0.001 ，ns： p > 0.05p>0.05 。AD，阿尔茨海默病；EA，电针；WT，野生型。
suggested that the binding of these two tracers was reduced in EA versus AD mice; significant statistical reductions were observed only in the binding of [^(18)(F)]\left[{ }^{18} \mathrm{~F}\right] TZ4877, especially in the cortex and hippocampus, whereas the binding of [^(18)(F)]AV45\left[{ }^{18} \mathrm{~F}\right] \mathrm{AV} 45 was not. 建议在 EA 小鼠与 AD 小鼠中，这两种示踪剂的结合减少；仅在 [^(18)(F)]\left[{ }^{18} \mathrm{~F}\right] TZ4877 的结合中观察到显著的统计减少，特别是在皮层和海马中，而 [^(18)(F)]AV45\left[{ }^{18} \mathrm{~F}\right] \mathrm{AV} 45 的结合则没有。
3.6 | Positive correlation between 3.6 | 正相关
cognitive-memory behavior and V_(T)V_{\mathrm{T}} quantifications of [^(18):}\left[{ }^{18}\right. F]TZ4877 认知-记忆行为和 V_(T)V_{\mathrm{T}} 对 [^(18):}\left[{ }^{18}\right. F]TZ4877 的量化
To further clarify the association between the neuroinflammatory target S1PR1 and cognitive-memory behavior across various brain 进一步阐明神经炎症靶点 S1PR1 与各种大脑中认知记忆行为之间的关联
regions by examining the correlation between the cognitive-memory behavior and V_(T)V_{T} quantifications of [^(18)(F)]\left[{ }^{18} \mathrm{~F}\right] TZ4877 was investigated. We explored all behavioral correlations and found the most notable correlations between escape latency (reaction cognitive and memory behavior) and V_(T)V_{T} quantifications of [^(18)(F)]\left[{ }^{18} \mathrm{~F}\right] TZ4877. The results (Figure 5 and Figure S 6 in supporting information) showed the V_(T)V_{T} quantifications of [^(18)(F)]\left[{ }^{18} \mathrm{~F}\right] TZ4877 in key brain regions for cognitive and memory ability, such as the cortex and hippocampus positively correlating with platform latency (cortex p < 0.01p<0.01, r=0.7102r=0.7102; hippocampus p < 0.01,r=0.6891p<0.01, r=0.6891 ), indicating that higher V_(T)V_{T} values were associated with longer periods to find the platform. 通过检查认知记忆行为与 [^(18)(F)]\left[{ }^{18} \mathrm{~F}\right] TZ4877 的 V_(T)V_{T} 量化之间的相关性，研究了不同区域。我们探索了所有行为相关性，并发现逃逸潜伏期（反应认知和记忆行为）与 [^(18)(F)]\left[{ }^{18} \mathrm{~F}\right] TZ4877 的 V_(T)V_{T} 量化之间的最显著相关性。结果（支持信息中的图 5 和图 S6）显示，关键脑区（如皮层和海马体）中 [^(18)(F)]\left[{ }^{18} \mathrm{~F}\right] TZ4877 的 V_(T)V_{T} 量化与平台潜伏期（皮层 p < 0.01p<0.01 ， r=0.7102r=0.7102 ；海马体 p < 0.01,r=0.6891p<0.01, r=0.6891 ）呈正相关，表明更高的 V_(T)V_{T} 值与寻找平台的时间更长相关。
FIGURE 3 The PET-MRI imaging and uptake comparisons of [18 F]TZ4877 among WT, AD, and EA mice. A, The representative PET-MRI images of [^(18)(F)]\left[{ }^{18} \mathrm{~F}\right] TZ4877 in the brain regions of WT, AD, and EA mice. B, The averaged TACs of [ {:^(18)(F)]\left.{ }^{18} \mathrm{~F}\right] TZ4877 in the brain regions from the WT (black lines), AD (red lines), and EA (green lines) groups, n=5.^(**)p < 0.05,^(****)p < 0.01,^(******)p < 0.001,ns:p > 0.05n=5 .{ }^{*} p<0.05,{ }^{* *} p<0.01,{ }^{* * *} p<0.001, \mathrm{~ns}: p>0.05. AD, Alzheimer’s disease; EA, electro-acupuncture; MRI, magnetic resonance imaging; PET, positron emission tomography; SUV, standardized uptake values; TACs, time activity curves; WT, wild type. 图 3 [18 F]TZ4877 在 WT、AD 和 EA 小鼠中的 PET-MRI 成像和摄取比较。A，WT、AD 和 EA 小鼠脑区的代表性 PET-MRI 图像。B，来自 WT（黑线）、AD（红线）和 EA（绿线）组的脑区[ {:^(18)(F)]\left.{ }^{18} \mathrm{~F}\right] TZ4877 的平均 TACs， n=5.^(**)p < 0.05,^(****)p < 0.01,^(******)p < 0.001,ns:p > 0.05n=5 .{ }^{*} p<0.05,{ }^{* *} p<0.01,{ }^{* * *} p<0.001, \mathrm{~ns}: p>0.05 。AD，阿尔茨海默病；EA，电针；MRI，磁共振成像；PET，正电子发射断层扫描；SUV，标准化摄取值；TACs，时间活性曲线；WT，野生型。
3.7 | The IF staining showed a colocalization of S1PR1 with neuroinflammatory markers 3.7 | IF 染色显示 S1PR1 与神经炎症标志物的共定位
The post-PET immunofluorescence staining confirmed S1PR1 expression and explored its relationship with neuroinflammation markers. Cortex and hippocampus tissues of AD mice showed colocalization of S1PR1 (red) with neuroinflammation marker GFAP for astrocytes and IBA-1 for microglia (Figure 6 and Figure S7 in supporting information). Colocalization in EA mice was significantly reduced (Figure 6A,B), suggesting suppression of astrocytes or microglia activation, which PET 后免疫荧光染色确认了 S1PR1 的表达，并探讨了其与神经炎症标志物的关系。阿尔茨海默病小鼠的皮层和海马组织显示 S1PR1（红色）与星形胶质细胞的神经炎症标志物 GFAP 和小胶质细胞的 IBA-1 的共定位（图 6 和支持信息中的图 S7）。EA 小鼠中的共定位显著减少（图 6A,B），这表明星形胶质细胞或小胶质细胞活化的抑制。
thereby can be assessed by PET imaging via [18 F]TZ4877 targeting to S1PR1 receptor. 因此可以通过 PET 成像评估[18 F]TZ4877 对 S1PR1 受体的靶向。
3.8 | The immunohistochemistry showed a reduction of IL-1 beta\beta and TNF- alpha\alpha after EA treatment 3.8 | 免疫组化显示 EA 治疗后 IL-1 beta\beta 和 TNF- alpha\alpha 的减少
The post-PET immunohistochemistry confirmed a significant reduction of IL-1 beta\beta and TNF- alpha\alpha in the AD brain, especially in the cortex and hippocampus after EA treatment (Figure S8 in supporting information). PET 后免疫组化确认在 AD 大脑中，尤其是在皮层和海马中，EA 治疗后 IL-1 beta\beta 和 TNF- alpha\alpha 显著减少（支持信息中的图 S8）。
TABLE 1 The SUV comparisons of [^(18)(F)]\left[{ }^{18} \mathrm{~F}\right] TZ4877 (S1PR1:AD, n=5n=5; EA, n=5n=5; WT, n=5n=5 ) and [ {:^(18)(F)]AV45\left.{ }^{18} \mathrm{~F}\right] \mathrm{AV} 45 (A : AD, n=5n=5; EA, n=5n=5; WT, n=3n=3 ) in AD,WTA D, W T, and EAE A groups at 30 minutes post-injection. 表 1 [^(18)(F)]\left[{ }^{18} \mathrm{~F}\right] TZ4877 (S1PR1:AD, n=5n=5 ; EA, n=5n=5 ; WT, n=5n=5 ) 和 [ {:^(18)(F)]AV45\left.{ }^{18} \mathrm{~F}\right] \mathrm{AV} 45 (A : AD, n=5n=5 ; EA, n=5n=5 ; WT, n=3n=3 ) 在 AD,WTA D, W T 和 EAE A 组在注射后 30 分钟的 SUV 比较。
Paraffin sections of brain cortex and hippocampus tissue from AD mice showed expression of IL-1 beta\beta (Figure S8A,B) and TNF- alpha\alpha (Figure S8C,D). However, in EA-treated mice, expression was significantly reduced (Figure S8A-D), suggesting EA’s suppression of inflammation in AD brain tissue. 阿尔茨海默病小鼠的脑皮层和海马组织的石蜡切片显示出 IL-1 beta\beta （图 S8A,B）和 TNF- alpha\alpha （图 S8C,D）的表达。然而，在接受电针治疗的小鼠中，表达显著减少（图 S8A-D），这表明电针抑制了阿尔茨海默病脑组织中的炎症。
4 | DISCUSSION 4 | 讨论
Neuroinflammation is an important factor in accelerating the pathological process of AD. As a neuroinflammation mediator, S1PR1 has been reported to be involved in the development of AD, although direct evidence from on-site imaging is lacking. In this study, the {:^([18)F]\left.{ }^{[18} \mathrm{F}\right] TZ4877 神经炎症是加速阿尔茨海默病（AD）病理过程的重要因素。作为神经炎症介导因子，S1PR1 已被报道参与 AD 的发展，尽管缺乏现场成像的直接证据。在本研究中， {:^([18)F]\left.{ }^{[18} \mathrm{F}\right] TZ4877
probe targeting S1PR1 and [^(18)(F)]AV45\left[{ }^{18} \mathrm{~F}\right] \mathrm{AV} 45 probe targeting Abeta\mathrm{A} \beta were used to evaluate the mechanisms by which EA therapy improves AD. Onsite imaging (Figures 3 and 4) revealed increased tracer uptake both for [^(18)(F)]\left[{ }^{18} \mathrm{~F}\right] TZ4877 and [18 F]AV45 in AD mouse brain regions compared to WT, while [ {:^(18)(F)]\left.{ }^{18} \mathrm{~F}\right] TZ4877 was more pronounced. After EA treatment, uptakes of [18F]TZ4877 and [18 F]AV45 were both reduced with concomitant improvement in AD symptoms (Tables 1 and 2). Similarly, the reduction in {:^(18)(F)]\left.{ }^{18} \mathrm{~F}\right] TZ4877 was also more notable, which may suggest that the {:^(18)(F)]\left.{ }^{18} \mathrm{~F}\right] TZ4877 may be a more potentially sensitive probe in evaluating the pathological progression of AD. 用于评估 EA 疗法改善 AD 机制的探针包括针对 S1PR1 的探针和针对 Abeta\mathrm{A} \beta 的 [^(18)(F)]AV45\left[{ }^{18} \mathrm{~F}\right] \mathrm{AV} 45 探针。现场成像（图 3 和图 4）显示，与 WT 相比，AD 小鼠脑区对 [^(18)(F)]\left[{ }^{18} \mathrm{~F}\right] TZ4877 和[18 F]AV45 的示踪剂摄取均有所增加，而[ {:^(18)(F)]\left.{ }^{18} \mathrm{~F}\right] TZ4877 的增加更为明显。经过 EA 治疗后，[18F]TZ4877 和[18 F]AV45 的摄取均减少，同时 AD 症状有所改善（表 1 和表 2）。同样， {:^(18)(F)]\left.{ }^{18} \mathrm{~F}\right] TZ4877 的减少也更为显著，这可能表明 {:^(18)(F)]\left.{ }^{18} \mathrm{~F}\right] TZ4877 在评估 AD 的病理进展方面可能是一个更具潜在敏感性的探针。
Glial cells, including microglia and astrocytes, significantly influence impact on the pathology of AD. Previous knowledge ^(28){ }^{28} has generally attributed CNS immunosurveillance only to resident microglia as 胶质细胞，包括小胶质细胞和星形胶质细胞，显著影响阿尔茨海默病的病理。先前的知识 ^(28){ }^{28} 通常将中枢神经系统的免疫监视仅归因于常驻的小胶质细胞。
FIG URE 4 The PET-MRI imaging and uptake comparisons of [ {:^(18)(F)]AV45\left.{ }^{18} \mathrm{~F}\right] \mathrm{AV} 45 among WT, AD, and EA mice. A, The representative PET images showed the uptake of [18 F]AV45 in brain regions of WT, AD, and EA mice. The images of different brain regions are indicated by white dotted lines. B, TAC shows the uptake of [18F]AV45 in WT (black lines), AD (red lines), and EA (blue lines) mice. n=3n=3 in WT, and n=5n=5 in AD and EA, respectively. ^(**)p < 0.05,^(****)p < 0.01,^(******)p < 0.001,ns:p > 0.05{ }^{*} p<0.05,{ }^{* *} p<0.01,{ }^{* * *} p<0.001, \mathrm{~ns}: p>0.05. AD, Alzheimer’s disease; EA, electro-acupuncture; MRI, magnetic resonance imaging; PET, positron emission tomography; SUV, standardized uptake values; TAC, time activity curve; WT, wild type. 图 4 PET-MRI 成像和 [ {:^(18)(F)]AV45\left.{ }^{18} \mathrm{~F}\right] \mathrm{AV} 45 在 WT、AD 和 EA 小鼠中的摄取比较。A，代表性的 PET 图像显示了 [18 F]AV45 在 WT、AD 和 EA 小鼠大脑区域的摄取。不同大脑区域的图像由白色虚线表示。B，TAC 显示了 WT（黑线）、AD（红线）和 EA（蓝线）小鼠中 [18F]AV45 的摄取。 n=3n=3 在 WT 中， n=5n=5 在 AD 和 EA 中，分别为 ^(**)p < 0.05,^(****)p < 0.01,^(******)p < 0.001,ns:p > 0.05{ }^{*} p<0.05,{ }^{* *} p<0.01,{ }^{* * *} p<0.001, \mathrm{~ns}: p>0.05 。AD，阿尔茨海默病；EA，电针；MRI，磁共振成像；PET，正电子发射断层扫描；SUV，标准化摄取值；TAC，时间活动曲线；WT，野生型。
resident macrophages of the CNS; they are involved in the process of maintaining brain homeostasis. The concept of microglia is crucial in the early stage of the disease research and can even be used as a potential therapeutic target. Astrocytes have been identified as key cortical regulators in the arousal state ^(29){ }^{29} and are considered indispensable players in cognitive function. ^(30){ }^{30} Our IF results (Figure 6 and S7) indicated the co-localization of S1PR1 with GFAP (astrocyte) or IBA-1 (microglia), further suggesting EA may improve cognitive and memory functions of AD by modulating astrocyte or microgliarelated functions. Additionally, the results of immunohistochemistry 中枢神经系统的常驻巨噬细胞；它们参与维持大脑稳态的过程。小胶质细胞的概念在疾病研究的早期阶段至关重要，甚至可以作为潜在的治疗靶点。星形胶质细胞被确定为觉醒状态的关键皮层调节因子 ^(29){ }^{29} ，并被认为是认知功能中不可或缺的参与者。 ^(30){ }^{30} 我们的免疫荧光结果（图 6 和 S7）表明 S1PR1 与 GFAP（星形胶质细胞）或 IBA-1（小胶质细胞）的共定位，进一步表明 EA 可能通过调节星形胶质细胞或小胶质细胞相关功能来改善阿尔茨海默病的认知和记忆功能。此外，免疫组化的结果
for IL-1 beta\beta and TNF- alpha\alpha (Figure S8) suggest that EA may alleviate AD pathology by suppressing astrocyte or microglia-related inflammation. To further explore the association between S1PR1 and cognitivememory behavior, correlation analysis was conducted between V_(T)V_{T} quantifications across various brain regions of [^(18)(F)]\left[{ }^{18} \mathrm{~F}\right] TZ4877 and the escape latency (Figure S6). The result revealed that, as a widespread neuroinflammatory target, S1PR1 in brain areas was positively associated with AD cognitive and memory behavior impairment; the higher the S1PR1 expressed the longer the platform latency period (the time taken to find the platform), especially in the cortex and 对于 IL-1 beta\beta 和 TNF- alpha\alpha (图 S8) 的结果表明，EA 可能通过抑制星形胶质细胞或小胶质细胞相关的炎症来减轻 AD 病理。为了进一步探讨 S1PR1 与认知记忆行为之间的关联，对 [^(18)(F)]\left[{ }^{18} \mathrm{~F}\right] TZ4877 的各个脑区的 V_(T)V_{T} 定量结果与逃逸潜伏期进行了相关性分析 (图 S6)。结果显示，作为广泛的神经炎症靶点，脑区中的 S1PR1 与 AD 的认知和记忆行为损害呈正相关；S1PR1 表达越高，平台潜伏期越长（找到平台所需的时间），尤其是在皮层中。
TABLE 2 The V_(T)V_{T} comparisons of [^(18)(F)]\left[{ }^{18} \mathrm{~F}\right] TZ4877 (S1PR1:AD, n=5;EA,n=5;WT,n=5n=5 ; \mathrm{EA}, n=5 ; \mathrm{WT}, n=5 ) and [^(18)(F)]AV45\left[{ }^{18} \mathrm{~F}\right] \mathrm{AV} 45 (AB:AD, n=5;EA,n=5n=5 ; \mathrm{EA}, n=5; WT, n=3n=3 ) in AD, WT , and EA groups at 30 minutes post-injection. 表 2 在注射后 30 分钟， [^(18)(F)]\left[{ }^{18} \mathrm{~F}\right] TZ4877 (S1PR1:AD, n=5;EA,n=5;WT,n=5n=5 ; \mathrm{EA}, n=5 ; \mathrm{WT}, n=5 ) 和 [^(18)(F)]AV45\left[{ }^{18} \mathrm{~F}\right] \mathrm{AV} 45 (AB:AD, n=5;EA,n=5n=5 ; \mathrm{EA}, n=5 ; WT, n=3n=3 ) 在 AD、WT 和 EA 组的 V_(T)V_{T} 比较。
Brain areas 脑区
AD
EA
WT
AD 与 WT 的 p 值
AD vs. WT
p value
AD vs. WT
p value| AD vs. WT |
| :--- |
| p value |
ADvs. EA pp 值
ADvs. EA
pp value
ADvs. EA
p value| ADvs. EA |
| :--- |
| $p$ value |
EA 与 WT pp 值
EA vs. WT
pp value
EA vs. WT
p value| EA vs. WT |
| :--- |
| $p$ value |
Abbreviations: A betaA \beta, amyloid beta; ADA D, Alzheimer’s disease; EAE A, electro-acupuncture; S1PR1, sphingosine-1-phosphate receptor 1; V_(T)V_{T}, volume distribution; WTW T, wild type. 缩写： A betaA \beta ，淀粉样β； ADA D ，阿尔茨海默病； EAE A ，电针；S1PR1，鞘氨醇-1-磷酸受体 1； V_(T)V_{T} ，体积分布； WTW T ，野生型。
*p < 0.05. *p < 0.05。 ^(****)p < 0.01{ }^{* *} p<0.01.
hippocampus. Additionally, a stronger correlation was observed in the right hippocampal than in the left, possibly due to its greater contribution to spatial memory. ^(31,32){ }^{31,32} EA treatment also mainly functions by decreasing S1PR1 expression in the cortex and hippocampus (a more pronounced effect observed on the right hippocampus), which provides evidence for EA’s efficacy in improving cognitive and memory behavior in AD mice by acting on S1PR1 in the cortex and hippocampus (Tables 1 and 2). 海马。此外，右侧海马的相关性比左侧更强，这可能是由于其对空间记忆的更大贡献。 ^(31,32){ }^{31,32} EA 治疗主要通过降低皮层和海马中 S1PR1 的表达来发挥作用（在右侧海马观察到更明显的效果），这为 EA 通过作用于皮层和海马中的 S1PR1 改善 AD 小鼠的认知和记忆行为的有效性提供了证据（表 1 和表 2）。
EA O wT
r
(D) The p\mathbf{p} value of the correlation analysis (D) 相关分析的 p\mathbf{p} 值
p
FIGURE 5 The correlations between cognitive-memory behavior and V_(T)V_{T} quantifications of [ {:^(18)(F)]\left.{ }^{18} \mathrm{~F}\right] TZ4877. A, The schematic representation in the correlation of different brain regions. B, The pp-value in the correlation of V_(T)([^(18)(F)]TZ 4877)V_{T}\left(\left[{ }^{18} \mathrm{~F}\right] T Z 4877\right) in different brain areas and escape latency in the hidden platform experiment. C, The positive linear correlation between V_(T)([^(18)(F)]TZ 4877)V_{T}\left(\left[{ }^{18} \mathrm{~F}\right] T Z 4877\right) and escape latency. D, The Pearson rr in the correlation of {:V_(T)(^(18)(F)]TZ4877)\left.V_{\mathrm{T}}\left({ }^{18} \mathrm{~F}\right] \mathrm{TZ} 4877\right) in different brain regions and escape latency in the hidden platform experiment. AD, Alzheimer’s disease; EA, electro-acupuncture; V_(T)V_{\mathrm{T}}, volume distribution; WT, wild type. 图 5 认知记忆行为与 V_(T)V_{T} 对[ {:^(18)(F)]\left.{ }^{18} \mathrm{~F}\right] TZ4877 的量化之间的相关性。A，不同脑区相关性的示意图。B，不同脑区 V_(T)([^(18)(F)]TZ 4877)V_{T}\left(\left[{ }^{18} \mathrm{~F}\right] T Z 4877\right) 的相关性中的 pp 值和隐蔽平台实验中的逃逸潜伏期。C， V_(T)([^(18)(F)]TZ 4877)V_{T}\left(\left[{ }^{18} \mathrm{~F}\right] T Z 4877\right) 与逃逸潜伏期之间的正线性相关性。D，隐蔽平台实验中不同脑区 {:V_(T)(^(18)(F)]TZ4877)\left.V_{\mathrm{T}}\left({ }^{18} \mathrm{~F}\right] \mathrm{TZ} 4877\right) 与逃逸潜伏期的相关性中的 Pearson rr 。AD，阿尔茨海默病；EA，电针； V_(T)V_{\mathrm{T}} ，体积分布；WT，野生型。
effectiveness in AD therapy (Figure 3). It is challenging to study TCM because its clinical practices defy the dominant biomedical paradigm. However, such challenges are pivotal in fostering continuous scientific innovation and breakthroughs. It is imperative to identify useful biomarkers for discerning the varying efficacies of acupuncture treatments. Liu et al. ^(8){ }^{8} demonstrated that PROKR2 ^("Cre-marked sen- "){ }^{\text {Cre-marked sen- }} sory neurons are the neuroanatomical basis of EA. In this study, we focused on S1PR1, an important biomarker for neuroinflammation. Our findings suggest that S1PR1 holds promise as a biomarker for evaluating acupuncture efficacy in diseases. The study’s innovation lies in its pioneering use of S1PR1 PET probe {:^(18)(F)]\left.{ }^{18} \mathrm{~F}\right] TZ4877 to evaluate the efficacy of EA treatment in AD, underscoring the potential clinical utility of targeting S1PR1 PET in the diagnosis and treatment of AD. 在阿尔茨海默病（AD）治疗中的有效性（图 3）。研究中医（TCM）具有挑战性，因为其临床实践违背了主流生物医学范式。然而，这些挑战在促进持续的科学创新和突破方面至关重要。识别有用的生物标志物以区分针灸治疗的不同疗效是至关重要的。刘等人 ^(8){ }^{8} 证明了 PROKR2 ^("Cre-marked sen- "){ }^{\text {Cre-marked sen- }} 感觉神经元是电针（EA）的神经解剖基础。在本研究中，我们关注 S1PR1，这是一个重要的神经炎症生物标志物。我们的研究结果表明，S1PR1 作为评估疾病中针灸疗效的生物标志物具有潜力。该研究的创新在于首次使用 S1PR1 PET 探针 {:^(18)(F)]\left.{ }^{18} \mathrm{~F}\right] TZ4877 评估电针治疗阿尔茨海默病的疗效，强调了靶向 S1PR1 PET 在阿尔茨海默病诊断和治疗中的潜在临床应用。
There are still limits to this study, for instance, the sample size of AD animals is relatively small. However, despite this constraint, the identification of statistical discrepancies in V_(T)V_{T} of PET tracer [^(18)(F)]TZ 4877\left[{ }^{18} \mathrm{~F}\right] T Z 4877 这项研究仍然存在局限性，例如，AD 动物的样本量相对较小。然而，尽管有这一限制，PET 示踪剂 [^(18)(F)]TZ 4877\left[{ }^{18} \mathrm{~F}\right] T Z 4877 在 V_(T)V_{T} 中的统计差异的识别仍然存在。
for EA provides compelling evidence that suggests that, within the confines of our study, values of V_(T)V_{T} from tracer kinetics analysis can provide more robust evidence for assessing the efficacy of acupuncture. Also, clinical AD samples and human translation research on more specimens may need to be collected in the future to verify the preliminary animal study. We believe that if applied to clinical research, the precise acupuncture treatment protocols and quantitative assessment of AD could significantly promote nuclear medicine’s role in AD therapies. In the future, our research group will actively promote the clinical translation of [^(18)(F)]\left[{ }^{18} \mathrm{~F}\right] TZ4877 and strive to further verify our conclusions on patients with AD. Establishing this hypothesis will hopefully provide novel approaches to AD treatment. We believe that S1PR1 bridges acupuncture and CNS diseases providing a new perspective in both fields for clinical practice. 对于 EA 提供了有力的证据，表明在我们研究的范围内，来自示踪动力学分析的 V_(T)V_{T} 值可以为评估针灸的疗效提供更可靠的证据。此外，未来可能需要收集临床 AD 样本和更多标本的人类转化研究，以验证初步的动物研究。我们相信，如果应用于临床研究，精确的针灸治疗方案和 AD 的定量评估可以显著促进核医学在 AD 治疗中的作用。未来，我们的研究小组将积极推动 [^(18)(F)]\left[{ }^{18} \mathrm{~F}\right] TZ4877 的临床转化，并努力进一步验证我们对 AD 患者的结论。建立这一假设希望能为 AD 治疗提供新的方法。我们相信 S1PR1 在针灸和中枢神经系统疾病之间架起了桥梁，为临床实践提供了两个领域的新视角。
In conclusion, the function and mechanism of S1PR1 in the EA treatment of AD have not been previously documented. We initially confirmed that EA at “Baihui (GV20)-Sishencong (EX-HN1)” effectively 总之，S1PR1 在耳针治疗阿尔茨海默病中的功能和机制尚未被记录。我们最初确认，在“百会（GV20）-四神聪（EX-HN1）”进行耳针治疗有效。
FIGURE 6 Colocalization of S1PR1 with GFAP and IBA-1. A, Multiple IF staining showed colocalizations (white arrows) of S1PR1 (red), GFAP (green for astrocytes), and DAPI (blue) in hippocampus of AD and EA mice; (B) multiple IF staining showed colocalization (white arrows) of S1PR1 (red), IBA-1 (green for microglia), and DAPI (blue) in cortex of AD and EA mice. The EA-treated mice showed a significant reduction of the colocalization (white arrows) (A,B). AD, Alzheimer’s disease; EA, electro-acupuncture; GFAP, glial fibrillary acidic protein; IBA-1, ionized calcium-binding adaptor molecule-1; IF, immunofluorescence; S1PR1, sphingosine-1-phosphate receptor 1. 图 6 S1PR1 与 GFAP 和 IBA-1 的共定位。A，多重免疫荧光染色显示 S1PR1（红色）、GFAP（绿色，星形胶质细胞）和 DAPI（蓝色）在 AD 和 EA 小鼠的海马中的共定位（白色箭头）；（B）多重免疫荧光染色显示 S1PR1（红色）、IBA-1（绿色，小胶质细胞）和 DAPI（蓝色）在 AD 和 EA 小鼠的皮层中的共定位（白色箭头）。EA 处理的小鼠显示出共定位（白色箭头）的显著减少（A，B）。AD，阿尔茨海默病；EA，电针；GFAP，胶质纤维酸性蛋白；IBA-1，离子钙结合适配蛋白-1；IF，免疫荧光；S1PR1，鞘氨醇-1-磷酸受体 1。
improved cognitive function in AD mice by decreasing S1PR1 in the cortex and hippocampus. The co-localization of S1PR1 with GFAP or IBA-1 and the reduction of IL-1 beta\beta and TNF- alpha\alpha suggested that EA may exert beneficial effects via an anti-inflammatory effect as an alternative therapy for AD. Additionally, our study marked the first use of [^(18)(F)]\left[{ }^{18} \mathrm{~F}\right] TZ4877 to assess the capacity of EA in the brains of AD mice. This study further promotes the exploratory application of PET quantifications for evaluating the efficacy of AD. 通过降低皮层和海马中的 S1PR1，改善了阿尔茨海默病小鼠的认知功能。S1PR1 与 GFAP 或 IBA-1 的共定位以及 IL-1 beta\beta 和 TNF- alpha\alpha 的减少表明，EA 可能通过抗炎作用作为阿尔茨海默病的替代疗法发挥有益效果。此外，我们的研究标志着首次使用 [^(18)(F)]\left[{ }^{18} \mathrm{~F}\right] TZ4877 评估 EA 在阿尔茨海默病小鼠大脑中的能力。这项研究进一步推动了 PET 定量在评估阿尔茨海默病疗效中的探索性应用。
ACKNOWLEDGMENTS 致谢
The authors would like to thank Dr. Miao Liu from the Department of Geriatrics of our hospital and Guangdong Cyclotron Medical Science Co., Ltd. for their cooperation and technical support. The authors also thank Dr. Chunlei Han (Turku PET Center, Turku University Hospital, Turku, Finland) for providing technical support with the Carimas software. This work was funded by the National Natural Science Foundation of China (82372004, 81871382, 82150610508), the Key Realm R&D Program of Guangdong Province (2018B030337001), the 作者感谢我院老年医学科的刘苗博士和广东环形加速器医学科学有限公司的合作与技术支持。作者还感谢汉春雷博士（芬兰图尔库大学医院图尔库 PET 中心）提供 Carimas 软件的技术支持。本研究得到了中国国家自然科学基金（82372004, 81871382, 82150610508）、广东省重点领域研发计划（2018B030337001）的资助。
Guangdong Provincial Basic and Applied Basic Research Fund Provincial Enterprise Joint Fund (2021A1515220004) and Talent Program of Sun Yat-Sen University, and the Guangdong Provincial Bureau of Traditional Chinese Medicine Research Project (20211079). 广东省基础与应用基础研究基金省企业联合基金（2021A1515220004）和中山大学人才计划，以及广东省中医药局研究项目（20211079）。
CONFLICT OF INTEREST STATEMENT 利益冲突声明
All authors declare no financial interests or potential competing interests. A patent is pending regarding this new method for evaluation of the EA treatment (No. 202311832723.3). No other potential conflicts of interest relevant to this article exist. Author disclosures are available in the supporting information. 所有作者声明没有财务利益或潜在的竞争利益。关于这种新的评估 EA 治疗的方法，专利正在申请中（编号：202311832723.3）。与本文相关的其他潜在利益冲突不存在。作者披露信息可在支持信息中获得。
ETHICS STATEMENT 伦理声明
All rodent study was conducted under the administration of the IACUC of the Guangdong Molecular Imaging Engineering Research Center in the Fifth Affiliated Hospital of Sun Yat-sen University (ethical protocol number # 00274.1.2). Eight-month-old male APP/PS1 mice (C57BL/6) and age- and sex-matched C57BL/6 mice were purchased from Cavens 所有啮齿动物研究是在中山大学附属第五医院广东分子影像工程研究中心 IACUC 的管理下进行的（伦理协议编号# 00274.1.2）。八个月大的雄性 APP/PS1 小鼠（C57BL/6）和年龄及性别匹配的 C57BL/6 小鼠是从 Cavens 购买的。
(Changzhou) Laboratory Animals, Ltd. (license No. SCXK [Su] 20210013). All the animal experiments were performed under the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. The animals were housed in standard cages in the laboratory animal center of the Fifth Affiliated Hospital of Sun Yat-sen University. （常州）实验动物有限公司（许可证编号：SCXK [苏] 20210013）。所有动物实验均在国家卫生研究院实验动物护理和使用指南下进行。动物被安置在中山大学附属第五医院的实验动物中心的标准笼中。
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How to cite this article: Wang L, Bi L, Qiu Y, et al. Effectiveness of electro-acupuncture for cognitive improvement on Alzheimer’s disease quantified via PET imaging of sphingosine-1-phosphate receptor 1. Alzheimer’s Dement. 2024;1-15. https://doi.org/10.1002/alz. 14260 如何引用本文：Wang L, Bi L, Qiu Y, 等. 电针对阿尔茨海默病认知改善的有效性，通过 PET 成像量化鞘氨醇-1-磷酸受体 1. Alzheimer’s Dement. 2024;1-15. https://doi.org/10.1002/alz.14260
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