异功散通过调控脑水液代谢改善APP/PS1转基因小鼠的学习记忆能力

doi:10.12122/j.issn.1673-4254.2024.10.20

南方医科大学学报 ›› 2024, Vol. 44 ›› Issue (10): 2015-2023.doi: 10.12122/j.issn.1673-4254.2024.10.20

• • 上一篇

异功散通过调控脑水液代谢改善APP/PS1转基因小鼠的学习记忆能力

曾静¹^,²^,³(), 花雷¹^,², 阳勇¹^,², 张小梅¹^,², 魏江平¹^,²(), 李利生³()

^1.中药新药创制川渝共建重点实验室
^2.国家中医药管理局中药化学三级实验室，重庆市中药研究院，重庆 400065；遵义医科大学3. 基础药理教育部重点实验室暨特色民族药教育部国际合作联合实验室
^4.贵州省基础药理重点实验室//药学院药理学教研室，贵州遵义 563000

收稿日期:2024-04-11 出版日期:2024-10-20 发布日期:2024-10-31
通讯作者: 魏江平,李利生 E-mail:1589905936@qq.com;sichuanwjp@163.com;medlls@qq.com
作者简介:曾静，在读硕士研究生，E-mail: 1589905936@qq.com
基金资助:
重庆市技术创新与应用发展专项重点项目(cstc2021jscx-dxwtBX0012);重庆市自然科学基金项目博士后项目(cstc2021jscx-bshX0054);重庆市中药研究院基本科研业务费项目(jbky20210003)

Yigong San improves learning and memory functions of APP/PS1 transgenic mice by regulating brain fluid metabolism

Jing ZENG¹^,²^,³(), Lei HUA¹^,², Yong YANG¹^,², Xiaomei ZHANG¹^,², Jiangping WEI¹^,²(), Lisheng LI³()

^1.Sichuan -Chongqing Joint Key Laboratory of Innovation of New Drugs of Traditional Chinese Medicine
^2.Third Level Laboratory of Traditional Chinese Medicine Chemistry of the National Administration of Traditional Chinese Medicine, Chongqing Academy of Chinese Materia Medica, Chongqing 400065, China
^3.Key Laboratory of Basic Pharmacology of Ministry of Education & Ministry of Education International Cooperation Joint Laboratory of Characteristic Ethnic Medicine
^4.Department of Pharmacology & Guizhou Provincial Key Laboratory of Basic Pharmacology, Zunyi Medical University, Zunyi 563000, China

Received:2024-04-11 Online:2024-10-20 Published:2024-10-31
Contact: Jiangping WEI, Lisheng LI E-mail:1589905936@qq.com;sichuanwjp@163.com;medlls@qq.com

摘要/Abstract

摘要：

目的观察异功散对APP/PS1小鼠学习记忆能力的影响，并从调控脑水液代谢系统探讨异功散抗痴呆的作用机制。方法将3月龄雄性APP/PS1转基因小鼠及同龄同背景野生型C57BL/6小鼠随机分为4组（8只/组）：对照组、模型组、多奈哌齐（1.67 mg/kg）组、异功散（7.5 g/kg）组。灌胃给予各组对应药物1次/d，1月后采用Morris水迷宫实验考察小鼠的学习记忆能力； HE染色观察海马皮层组织病理形态学变化；免疫组化染色、硫磺素S染色观察脑内淀粉样蛋白斑块数量变化；ELISA法检测脑组织和血清Aβ_1-40和Aβ_1-42含量；伊文思蓝法检测血脑屏障（BBB）通透性变化；免疫荧光共定位考察AQP4在星型胶质细胞上极化情况；Western blotting观察血管内皮钙黏蛋白（VE-cadherin）、闭锁连接蛋白1（ZO-1）、闭合蛋白（Occludin）、β-淀粉样前体蛋白（APP）、β-分泌酶（BACE1）、胰岛素降解酶（IDE）、低密度脂蛋白受体相关蛋白1（LRP1）、晚期糖基化终末产物受体（RAGE）、水通道蛋白4 （AQP4）蛋白表达情况。结果与对照组比较， APP/PS1小鼠第 5 天的逃避潜伏期显著延长（P<0.001），平台象限停留时间降低（P<0.05）；海马和皮层神经细胞变性或坏死细胞数量增加且病理评分升高（P<0.001）；Aβ阳性斑块明显升高以及硫磺素S荧光强度明显增强（P<0.05）；脑组织和血清Aβ_1-40、Aβ_1-42含量升高（P<0.05）；BBB通透性增加（P<0.01），RAGE蛋白表达上调（P<0.01）而VE-cadherin、LRP1、ZO-1、Occludin、AQP4蛋白表达下调（P<0.05），且AQP4在GFAP阳性细胞上表达的数量减少（P<0.05）。与模型组比较，异功散组小鼠第 5 天的逃避潜伏期缩短（P<0.001），平台象限停留时间增加（P<0.05）且平均游泳速度加快（P>0.05）；海马和皮层神经细胞变性或坏死细胞数量减少且病理评分降低（P<0.01）；Aβ阳性斑块减少以及硫磺素S荧光强度减弱（P<0.05）；脑组织和血清Aβ_1-40、Aβ_1-42含量降低（P<0.05）；维持了BBB通透性（P<0.01），RAGE蛋白表达下调（P<0.05），而VE-cadherin、LRP1、ZO-1、Occludin、AQP4蛋白表达上调（P<0.05），且AQP4在GFAP阳性细胞上表达的数量明显增加（P<0.01）。结论异功散能够通过改善APP/PS1小鼠脑内神经细胞损伤、Aβ病变，调控脑水液代谢系统相关蛋白表达而改善学习记忆变化。

关键词: 异功散, 痴呆症, 脑水液代谢系统, APP/PS1小鼠, AQP4

Abstract:

Objective To explore the mechanism by which Yigong San (YGS) improves learning and memory abilities of APP/PS1 transgenic mice in light of cerebral fluid metabolism regulation. Methods Three-month-old male APP/PS1 transgenic mice and wild-type C57BL/6 mice were both randomized into control group, model group, donepezil (1.67 mg/kg) group, and YGS (7.5 g/kg) group and received the corresponding treatments via gavage once daily for one month. After the treatments, the mice were assessed for learning and memory functions using Morris water maze test and examined for hippocampal and cortical pathologies and amyloid plaques using HE, immunohistochemical and thioflavin S staining; ELISA and Evans blue method were used for detecting Aβ_1-40 and Aβ_1-42 levels in the brain tissue and serum and assessing blood-brain barrier (BBB) integrity. Immunofluorescence colocalization was used to investigate AQP4 polarization on astrocytes. Western blotting was performed to detect the expressions of VE-cadherin, ZO-1, occludin, β‑amyloid precursor protein (APP), BACE1, insulin-degrading enzyme (IDE), LRP1, RAGE, and AQP4 proteins. Results Compared with the control mice, APP/PS1 mice showed significant impairment of learning and memory abilities, increased degeneration or necrosis of hippocampal and cortical neurons, pathological scores, Aβ‑positive plaques, elevated Aβ_1-40 and Aβ_1-42 levels in the brain tissue and serum, increased BBB permeability, upregulated RAGE expression, lowered expressions of VE-cadherin, LRP1, ZO-1, occludin, and AQP4 proteins, and reduced AQP4-expressing GFAP-positive cells. YGS treatment significantly improved the performance of the transgenic mice in Morris water maze test, reduced hippocampal and cortical pathologies and Aβ‑positive plaques, and ameliorated the abnormal changes in Aβ_1-40 and Aβ_1-42 levels, BBB permeability, protein expressions of RAGE, VE-cadherin, LRP1, ZO-1, occludin and AQP4, and the number of AQP4-expressing GFAP-positive cells. Conclusion YGS improves learning and memory changes in APP/PS1 mice by ameliorating neuronal damage and Aβ pathology in the brain and regulating brain fluid metabolism.

Key words: Yigong San, dementia, brain fluid metabolism system, APP/PS1 mice, aquaporin-4

曾静, 花雷, 阳勇, 张小梅, 魏江平, 李利生. 异功散通过调控脑水液代谢改善APP/PS1转基因小鼠的学习记忆能力[J]. 南方医科大学学报, 2024, 44(10): 2015-2023.

Jing ZENG, Lei HUA, Yong YANG, Xiaomei ZHANG, Jiangping WEI, Lisheng LI. Yigong San improves learning and memory functions of APP/PS1 transgenic mice by regulating brain fluid metabolism[J]. Journal of Southern Medical University, 2024, 44(10): 2015-2023.

图/表 10

表1 异功散对APP/PS1小鼠学习记忆能力的影响

Tab.1 Effect of Yigong San （YGS） on learning and memory ability of APP/PS1 mice (Mean±SD, n=8)

Group	Dosage (g/kg)	The escape latency (s)
Group	Dosage (g/kg)	First day	Second day	Third day	Fourth day	Fifth day
Control	-	118.95±2.36	80.73±28.54^{^}	47.48±16.79^{^^^}	29.50±10.43^{^^^}	14.98±5.30^{#^^^}
APP/PS1	-	112.11±23.66	99.90±35.32	83.94±29.68	66.07±23.36^{^^}	34.66±9.01*^{^^^}
APP/PS1+Donepezil	1.5×10^-3	120.00±0	92.39±32.66	55.98±19.79^{^^^}	38.79±13.71^{^^^}	16.10±5.69^{#^^^}
APP/PS1+YGS	7.5	120.00±0	102.07±35.20	78.92±27.90^{^}	42.27±14.94^{^^^}	18.23±6.44^{#^^^}

表2 异功散对APP/PS1小鼠空间探索能力的影响

Tab.2 Effect of YGS on spatial exploration ability of APP/PS1 mice (Mean±SD, n=8)

Group	Dosage (g/kg)	Platform quadrant residence time (s)	Platfrom crossing times (times)	Average swimming speed (cm/s)
Control	-	34.37±5.07^#	5.17±1.77	19.25±1.21
APP/PS1	-	25.69±5.01*	3.33±1.60	20.03±0.98
APP/PS1+Donepezil	1.5×10^-3	35.23±5.99^#	4.83±1.07	20.34±1.66
APP/PS1+YGS	7.5	34.17±4.08^#	4.83±0.67	20.58±1.32

图1 异功散对APP/PS1小鼠海马病理形态学的影响

Fig.1 Effect of YGS on hippocampal pathology of APP/PS1 mice (HE staining, original magnification: ×40). Red arrows indicate degenerative or necrotic neurons, and blue arrows indicate cell loss or enlarged intercellular space.

图2 异功散对APP/PS1小鼠海马和皮层受损神经细胞评分的影响

Fig.2 Effect of YGS on pathological score of damaged neurons in the hippocampus and cortex of APP/PS1 mice. **P<0.01, ***P<0.001 vs control group; #P<0.05, ##P<0.01, ###P<0.001 vs APP/PS1 group.

图3 异功散对APP/PS1小鼠海马皮层Aβ斑块数量的影响

Fig.3 Effect of YGS on the number of Aβ plaques in the hippocampus and cortex of APP/PS1 mice (immunohistochemistry, ×40). A: Immunohistochemical staining ofthe the hippocampal cortex in different groups (red arrows indicate amyloid plaques). B: Number of Aβ plaques in the hippocampus and cortex of the mice.*P<0.05, **P<0.01 vs control group; #P<0.05, ##P<0.01 vs APP/PS1 group.

图4 异功散对APP/PS1小鼠海马和皮层Aβ蛋白表达的影响

Fig.4 Effects of YGS on Aβ protein expression in the hippocampus and cortex of APP/PS1 mice (immunofluorescence staining, ×20 or ×100). A: Thioflavin-S staining. B: Aβ plaque area in the hippocampus and cortex of the mice. *P<0.05 vs control group; #P<0.05 vs APP/PS1 group.

图5 异功散对APP/PS1小鼠血清和脑组织Aβ1-40和Aβ1-42浓度的影响

Fig.5 Effect of YGS on Aβ1-40 and Aβ1-42 levels in the serum and brain tissue of APP/PS1 mice. A: Serum Aβ1-40 levels in the mice. B: Serum Aβ1-42 levels in the mice. C: Brain tissue Aβ1-40 level in the mice. D: Brain tissue Aβ1-42 level in the mice. *P<0.05, **P<0.01 vs control group; #P<0.05, ##P<0.01 vs APP/PS1 group.

图6 异功散对APP/PS1小鼠海马BBB通透性(n=3)和连接蛋白表达(n=5)的影响

Fig.6 Effects of YGS on BBB permeability and connexin expression in the hippocampus of APP/PS1 mice (n=5). A: Blood-brain barrier permeability of the mice (n=3). B-E: Western blotting for detecting expressions of ZO-1, VE-cadherin and occludin and their relative expression levels. *P<0.05, **P<0.01 vs control group; #P<0.05, ##P<0.01 vs APP/PS1 group.

图8 异功散对APP/PS1小鼠脑组织中AQP4极化的影响

Fig.8 Effect of YGS on AQP4 polarization in the brain tissue of APP/PS1 mice. A: Expression of AQP4 in astrocytes (Red is GFAP; green is AQP4; and blue is DAPI. The yellow arrows indicate AQP4-expressing GFAP-positive cells). B: Numbers of AQP4+GFAP in the brain tissue of the mice (n=5). *P<0.05, **P<0.01 vs control group; #P<0.05, ##P<0.01 vs APP/PS1 group.

图9 异功散对APP/PS1小鼠脑组织APP、PS1、BACE1和IDE蛋白表达的影响

Fig.9 Effect of YGS on protein expressions of APP, PS1, BACE1 and IDE in the brain tissue of APP/PS1 mice (n=5). A-E: Western blotting for detecting expressions of APP, PS1, BACE1 and IDE and their relative expression levels. *P<0.05, **P<0.01 vs control group; #P<0.05, ##P<0.01 vs APP/PS1 group.

参考文献 26

1	张景明, 陈震霖. 论津液与脏腑关系及其抗病御邪作用[J]. 中国中医基础医学杂志, 2007, 13(1): 13-4.
2	陈路军, 孙智玲, 陈文静, 等. 从 “津液代谢” 探讨变通小青龙汤在慢性阻塞性肺疾病急性期的应用[J]. 中医药临床杂志, 2022, 34(10): 1811-4.
3	徐世军, 赵宜军, 张文生. 从中医脑络功能演变谈轻度认知障碍的病机[J]. 中医杂志, 2011, 52(19): 1627-9. DOI: 10.1038/cdd.2010.68
4	田恺, 张向宇, 牛博真, 等. 基于 “毒损脑络” 理论中医脑病病因病机和辨证施治的研究进展[J]. 中西医结合心脑血管病杂志, 2021, 19(8): 1308-10.
5	魏江平, 赵子瑄, 曾静, 等. 异功散调控CXCL12/CXCR4信号减少谷氨酸释放改善衰老模型小鼠认知下降[J]. 中国中药杂志, 2023, 48(23): 6483-91.
6	Mestre H, Kostrikov S, Mehta RI, et al. Perivascular spaces, glymphatic dysfunction, and small vessel disease[J]. Clin Sci, 2017, 131(17): 2257-74.
7	Mamtilahun M, Wei ZY, Qin C, et al. DL-3n-butylphthalide improves blood-brain barrier integrity in rat after middle cerebral artery occlusion[J]. Front Cell Neurosci, 2020, 14: 610714.
8	Wei JP, Dai Y, Wen W, et al. Blood-brain barrier integrity is the primary target of alcohol abuse[J]. Chem Biol Interact, 2021, 337: 109400.
9	Shi ZS, Zhu LH, Li TT, et al. Neuroprotective mechanisms of Lycium barbarum polysaccharides against ischemic insults by regulating NR2B and NR2A containing NMDA receptor signaling pathways[J]. Front Cell Neurosci, 2017, 11: 288.
10	Gonneaud J, Arenaza-Urquijo EM, Mézenge F, et al. Increased florbetapir binding in the temporal neocortex from age 20 to 60 years[J]. Neurology, 2017, 89(24): 2438-46.
11	吴东南, 刘玲, 明淑萍, 等. 基于“Aβ异常沉积” 浅析中医“从痰论治” 阿尔茨海默病[J]. 中华中医药杂志, 2019, 34(10): 4699-702.
12	Yang AC, Vest RT, Kern F, et al. A human brain vascular atlas reveals diverse mediators of Alzheimer’s risk[J]. Nature, 2022, 603(7903): 885-92.
13	Montagne A, Barnes SR, Sweeney MD, et al. Blood-brain barrier breakdown in the aging human hippocampus[J]. Neuron, 2015, 85(2): 296-302.
14	Wei JP, Qin LX, Fu Y, et al. Long-term consumption of alcohol exacerbates neural lesions by destroying the functional integrity of the blood-brain barrier[J]. Drug Chem Toxicol, 2022, 45(1): 231-8.
15	Wang Q, Huang XM, Su YX, et al. Activation of Wnt/β-catenin pathway mitigates blood-brain barrier dysfunction in Alzheimer's disease[J]. Brain, 2022, 145(12): 4474-88.
16	Hu MY, Li TM, Ma XM, et al. Macrophage lineage cells-derived migrasomes activate complement-dependent blood-brain barrier damage in cerebral amyloid angiopathy mouse model[J]. Nat Commun, 2023, 14(1): 3945.
17	Xie P, Kancherla K, Chandramohan S, et al. Involvement of single nucleotide polymorphisms of junction adhesion molecule with small vessel vascular dementia[J]. Aging Med, 2023, 6(4): 347-52.
18	Yan SD, Chen X, Fu J, et al. RAGE and amyloid-beta peptide neurotoxicity in Alzheimer's disease[J]. Nature, 1996, 382(6593): 685-91.
19	Sagare A, Deane R, Bell RD, et al. Clearance of amyloid-beta by circulating lipoprotein receptors[J]. Nat Med, 2007, 13(9): 1029-31.
20	Lee J, Lee H, Lee H, et al. ANKS1A regulates LDL receptor-related protein 1 (LRP1)‑mediated cerebrovascular clearance in brain endothelial cells[J]. Nat Commun, 2023, 14(1): 8463.
21	Kanekiyo T, Cirrito JR, Liu CC, et al. Neuronal clearance of amyloid‑β by endocytic receptor LRP1[J]. J Neurosci, 2013, 33(49): 19276-83.
22	Burfeind KG, Murchison CF, Westaway SK, et al. The effects of noncoding aquaporin-4 single-nucleotide polymorphisms on cognition and functional progression of Alzheimer's disease[J]. Alzheimers Dement, 2017, 3(3): 348-59.
23	Zeppenfeld DM, Simon M, Haswell JD, et al. Association of perivascular localization of aquaporin-4 with cognition and alzheimer disease in aging brains[J]. JAMA Neurol, 2017, 74(1): 91-9.
24	Iliff JJ, Wang MH, Liao YH, et al. A paravascular pathway facilitates CSF flow through the brain parenchyma and the clearance of interstitial solutes, including amyloid Β[J]. Sci Transl Med, 2012, 4(147): 147ra111.
25	Galvão F Jr, Grokoski KC, da Silva BB, et al. The amyloid precursor protein (APP) processing as a biological link between Alzheimer's disease and cancer[J]. Ageing Res Rev, 2019, 49: 83-91.
26	Rammes G. Molecular mechanism of Alzheimer’s disease[J]. Int J Mol Sci, 2023, 24(23): 16837.

异功散通过调控脑水液代谢改善APP/PS1转基因小鼠的学习记忆能力

Yigong San improves learning and memory functions of APP/PS1 transgenic mice by regulating brain fluid metabolism

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