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浙江大学学报（医学版）
浙江大学学报（医学版）  2021, Vol. 50 Issue (5): 553-560    DOI: 10.3724/zdxbyxb-2021-0056
专题报道     
地黄对阿尔茨海默病小鼠行为学的影响及其对血脑屏障的保护作用
赵大鹏,陆韵薇,于顾然()
南京中医药大学附属医院神经内科，江苏 南京 210029
Effects of Radix Rehmanniae on behavior and blood-brain barrier in Alzheimer’s disease mice
ZHAO Dapeng,LU Yunwei,YU Guran()
Department of Neurology, Affiliated Hospital of Nanjing University of Chinese Medicine, Nanjing 210029, China
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摘要：

目的：探讨中药地黄对阿尔茨海默病模型小鼠日常活动能力和恐惧记忆能力的行为学影响及其对血脑屏障的保护作用。方法：38只4月龄APP/PS1双转基因小鼠随机分为模型对照组（给予等渗氯化钠溶液）、地黄小剂量组（给予地黄5?g·kg–1·d–1）和地黄大剂量组（给予地黄12?g·kg–1·d–1），连续灌胃给药3个月，每日给药1次。分别采用筑巢实验和条件恐惧实验检测小鼠日常活动能力和恐惧记忆能力；硫黄素T染色检测小鼠皮层和海马CA1区β淀粉样蛋白（Aβ）沉积；免疫荧光双重标记染色检测小鼠皮层和海马CA1区血管内皮完整性及血脑屏障渗出情况。结果：与模型对照组比较，地黄小、大剂量组日常活动能力均明显改善（均P<0.01）；地黄大剂量组恐惧记忆能力明显提高（P<0.01）；地黄大剂量组皮层和海马CA1区Aβ沉积量明显减少，面积占比明显减小，地黄小剂量组海马CA1区Aβ沉积面积占比也减小（均P<0.05）；地黄小、大剂量组大脑皮层CD34阳性面积占比显著增加，纤维蛋白原阳性面积占比显著减小（均P<0.05）；地黄大剂量组海马CA1区CD34阳性面积占比显著增加，纤维蛋白原阳性面积占比显著减小（均P<0.05）。结论：中药地黄可以提高模型小鼠的日常活动能力及恐惧记忆功能，减少脑内Aβ沉积，且大剂量时效果更为显著；其机制可能与降低血脑屏障通透性、保护血脑屏障完整性有关。
关键词： 阿尔茨海默病;  地黄;  日常活动能力;  条件性恐惧记忆;  β淀粉样蛋白;  血脑屏障;  转基因小鼠    
Abstract:

Objective: To investigate the effects of Radix Rehmanniae on behavior and blood brain barrier (BBB) in Alzheimer’s disease mice. Methods: Thirty-eight 4-month-old APP/PS1 double transgenic mice were randomly divided into three groups: model group, Radix Rehmanniae low-dose group and Radix Rehmanniae high-dose group. Saline,Radix Rehmanniae 5?g·kg–1·d–1 andRadix Rehmanniae 12 g·kg–1·d–1 were given to each group by continuous gavage once a day for three months, respectively. The changes in activities of daily live and fear conditioning memory behavior of mice were examined by nesting behavior test and fear conditioning test, respectively. The β-amyloid protein (Aβ) depositions in cortex and hippocampal CA1 area of mice were detected by thioflavin T staining. The CD34 and activities fibrinogen (Fib) immunofluorescence double staining were used to determine the vascular endothelial integrity and BBB exudation. Results: Compared with model mice, activities of daily live were significantly improved in Radix Rehmanniae low-dose and high-dose groups (both P<0.01), the fear memory ability was significantly increased inRadix Rehmanniae high-dose group (P<0.01). The amount of Aβ deposition in cortex and hippocampal CA1 decreased significantly inRadix Rehmanniae high-dose group, the area ratio decreased significantly; the area ratio of Aβ deposition in hippocampal CA1 region inRadix Rehmanniae low-dose group also decreased (all P<0.05). The proportions of CD34 positive area of cortex in low and high doseRadix Rehmanniae groups increased, the percentage of fibrinogen positive area decreased (all P<0.05). The proportion of CD34 positive area in hippocampal CA1 region inRadix Rehmanniae high-dose group was significantly increased, the percentage of fibrinogen positive area decreased significantly (both P<0.05).Conclusions: Radix Rehmanniae, especially high-dose can improve the activities of daily live and fear conditioning memory function of APP/PS1 mice, reduce the deposition of Aβ in brain. The mechanism may be related to the reduction of BBB permeability and the protection of the integrity of BBB.
Key words: Alzheimer’s disease    Radix Rehmanniae    Activity of daily live    Fear conditioning memory    Amyloid β-protein    Blood-brain barrier    Transgenic mice
收稿日期: 2021-03-03 出版日期: 2021-12-29
:  R285  
基金资助: 国家自然科学基金（81573771）
通讯作者: 于顾然     E-mail: an
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引用本文:

赵大鹏,陆韵薇,于顾然. 地黄对阿尔茨海默病小鼠行为学的影响及其对血脑屏障的保护作用[J]. 浙江大学学报（医学版）, 2021, 50(5): 553-560.

ZHAO Dapeng,LU Yunwei,YU Guran. Effects of Radix Rehmanniae on behavior and blood-brain barrier in Alzheimer’s disease mice. J Zhejiang Univ (Med Sci), 2021, 50(5): 553-560.

链接本文:

https://www.zjujournals.com/med/CN/10.3724/zdxbyxb-2021-0056    或    https://www.zjujournals.com/med/CN/Y2021/V50/I5/553

组别

n

2?h

24?h

48?h

模型对照组

9

0.000（0.000，0.500）

0.500（0.250，1.000）

1.333±1.031

地黄小剂量组

12

0.500（0.500，0.500）*

1.750（1.000，0.250）**

2.458±0.620**

地黄大剂量组

13

0.500（0.000，0.500）

1.500（1.000，0.250）**

2.538±0.628**
表 1  筑巢实验中各组小鼠不同时间段的评分比较
图 1  各组大脑皮层和海马CA1区β淀粉样蛋白硫黄素T染色结果绿色荧光为β淀粉样蛋白阳性染色. 模型对照组皮层和海马CA1区β淀粉样蛋白沉积量较多，地黄小剂量组和地黄大剂量组皮层及海马CA1区β淀粉样蛋白沉积量较模型对照组减少，尤以地黄大剂量组减少更为明显. 标尺=100 μm.

组别

n

皮层

海马CA1区

沉积量

面积占比

沉积量

面积占比

模型对照组

6

6.00（4.00，10.00）

0.64（0.32，1.20）

3.00（2.00，7.00）

0.46±0.28

地黄小剂量组

6

4.00（3.25，10.00）

0.27（0.14，0.46）

1.00（0.75，2.50）

0.12±0.13*

地黄大剂量组

6

1.50（1.00，2.00）**##

0.13（0.00，0.28）*

1.00（1.00，1.25）*

0.12±0.08*
表 2  各组大脑皮层和海马CA1区β淀粉样蛋白沉积量和面积占比
图 2  各组大脑皮层和海马CA1区CD34、纤维蛋白原免疫荧光双重染色结果绿色荧光为CD34阳性表达，红色荧光为纤维蛋白原阳性表达. A：与模型对照组比较，地黄小剂量组和地黄大剂量组皮层CD34阳性面积明显增加，纤维蛋白原阳性面积明显减少；与地黄小剂量组比较，地黄大剂量组皮层CD34面积明显增加.B：与模型对照组比较，地黄小剂量组和地黄大剂量组海马CA1区CD34阳性面积增加，纤维蛋白原阳性面积减少，以地黄大剂量组更为显著. DAPI：4′，6-二脒基-2-苯基吲哚. 标尺=100 μm.

组 别

n

皮层

海马CA1区

CD34

纤维蛋白原

CD34

纤维蛋白原

模型对照组

6

0.124（0.083，0.200）

0.313（0.167，0.444）

0.136（0.087，0.217）

0.209±0.025

地黄小剂量组

6

0.295（0.197，0.358）*

0.111（0.067，0.146）**

0.202（0.148，0.415）

0.117±0.073

地黄大剂量组

6

0.431（0.324，0.551）**##

0.092（0.080，0.104）**

0.442（0.313，0.665）*

0.110±0.043*
表 3  各组大脑皮层和海马CA1区CD34和纤维蛋白原阳性面积占比比较
1 FAROUK F M, OOI L, LAW C S W, et al. Dual‐target‐directed ligand displaying selective butyrylcholinesterase inhibitory and neurite promoting activities as a potential therapeutic for Alzheimer’s disease[J]. Chemistry Select, 2020, 5(36): 11229-11236
2 周仲英. 中医内科学[M]. 北京: 中国中医药出版社, 2003: 180
ZHOU Zhongying. Internal medicine of traditional Chinese medicine[M]. Beijing: China Press of Traditional Chinese Medicine, 2003: 180. (in Chinese)
3 芦锰, 周雨慧, 李晓宁, 等. 基于数据挖掘中医药治疗阿尔茨海默病用药规律研究[J]. 中国中药杂志,2021, 46(6): 1558-1563
LU Meng, ZHOU Yuhui, LI Xiaoning, et al. Research on regularity of traditional Chinese medicine in treatment of Alzheimer’s disease based on data mining[J]. China Journal of Chinese Materia Medica, 2021, 46(6): 1558-1563. (in Chinese)
4 TROMPETEROA, GORDILLOA, DEL PILARM C, et al.Alzheimer’s disease and Parkinson’s disease: a review of current treatment adopting a nanotechnology approach[J]Curr Pharm Des, 2018, 24( 1): 22-45.
doi: 10.2174/1381612823666170828133059
5 王丹丹. “补肾益精方”含药血清对血脑屏障的影响及该方治疗轻度认知功能障碍的疗效评价[D]. 南京: 南京中医药大学, 2016
WANG Dandan. Evaluate the effect of serum containing Bushenyijingfang on the blood-brain barrier and the clinical study of Bushenyijingfang on mild cognitive impairment[D]. Nanjing: Nanjing University of Chinese Medicine, 2016. (in Chinese)
6 刘晨阳. 梓醇对纤维状Aβ1-42介导的体外血脑屏障的损伤具有保护作用[D]. 南京: 南京中医药大学, 2018
LIU Chenyang. Catalpol provides a protective effect on fibrillary Aβ1-42-induced barrier disruption in an in vitro model of the blood-brain barrier[D]. Nanjing: Nanjing University of Chinese Medicine, 2018. (in Chinese)
7 陆韵薇, 于顾然, 符布清. 生地黄水煎剂对H2O2诱导的PC12细胞凋亡的保护作用研究[J]. 江苏中医药, 2016, 48(10): 103-105
LU Yunwei, YU Guran, FU Buqing. Protective effect of Radix Rehmanniae Recen water decoction on H2O2-induced apoptosis of PC12 cells[J]. Jiangsu Journal of Traditional Chinese Medicine, 2016, 48(10): 103-105. (in Chinese)
8 National Research Council (US), Committee for the Update of the Guide for the Care and Use of Laboratory Animals, Institute for Laboratory Animal Research (US),et al. Guide for the care and use of laboratory animals[M]. 8th ed. Washington (DC): National Academies Press, 2011
9 陈奇. 中药药理研究方法学[M]. 第3版. 北京: 人民卫生出版社, 2011: 1261-1263
CHEN Qi. Research Methods in Pharmacology of Chinese Materia Medica[M]. 3rd ed. Beijing: China Press of Traditional Chinese Medicine, 2011: 1261-1263. (in Chinese)
10 WESSOND W, WILSOND A. Age and gene overexpression interact to abolish nesting behavior in Tg2576 amyloid precursor protein (APP) mice[J]Behav Brain Res, 2011, 216( 1): 408-413.
doi: 10.1016/j.bbr.2010.08.033
11 LEWARCHC L, HOEKSTRAH E. The evolution of nesting behaviour in Peromyscus mice[J]Anim Behaviour, 2018, 103-115.
doi: 10.1016/j.anbehav.2018.03.008
12 崔瑛. 怀庆熟地黄益智作用研究[D]. 北京: 北京中医药大学, 2002
CUI Ying. Experimental study on mechanism of promoting intelligence with Huaiqing Shudihuang[D]. Beijing: Beijing University of Chinese Medicine, 2002. (in Chinese)
13 KOOKS Y, SEOK HONGH, MOONM, et al.Disruption of blood-brain barrier in Alzheimer disease pathogenesis[J/OL]Tissue Barriers, 2013, 1( 2): e23993.
doi: 10.4161/tisb.23993
14 WILCOCKD M, COLTONC A. Anti-amyloid-β immunotherapy in alzheimer’s disease: relevance of transgenic mouse studies to clinical trials[J]J Alzheimer Dis, 2008, 15( 4): 555-569.
doi: 10.3233/jad-2008-15404
15 SZCZYPKAM S, KWOKK, BROTM D, et al.Dopamine production in the caudate putamen restores feeding in dopamine-deficient mice[J]Neuron, 2001, 30( 3): 819-828.
doi: 10.1016/S0896-6273(01)00319-1
16 DEACONR M, CROUCHERA, RAWLINSJ N. Hippocampal cytotoxic lesion effects on species-typical behaviours in mice[J]Behav Brain Res, 2002, 132( 2): 203-213.
doi: 10.1016/s0166-4328(01)00401-6
17 UYSJ D, STEIND J, DANIELSW M, et al.Animal models of anxiety disorders[J]Curr Psychiatry Rep, 2003, 5( 4): 274-281.
doi: 10.1007/s11920-003-0056-7
18 吴江. 神经病学[M]. 北京: 人民卫生出版社, 2010: 331
WU Jiang. Neurology[M]. Beijing: People’s Medical Publishing House, 2010: 331. (in Chinese)
19 SELKOE D J, HARDY J. The amyloid hypothesis of Alzheimer’s disease at 25 years[J]. EMBO Mol Med, 2016, 8(6): 595-608
20 HARDYJ A, HIGGINSG A. Alzheimer’s disease: the amyloid cascade hypothesis[J]Science, 1992, 256( 5054): 184-185.
doi: 10.1126/science.1566067
21 STROUD J C, LIU C, TENG P K, et al. Toxic fibrillar oligomers of amyloid-β have cross-β structure[J]. Proc Natl Acad Sci U S A, 2012, 109(20): 7717-7722
22 DANEMANR, PRATA. The blood-brain barrier[J]Cold Spring Harb Perspect Biol, 2015, 7( 1): a020412.
doi: 10.1101/cshperspect.a020412
23 常铮, 许继平. 脑血管内皮细胞与神经系统变性病关系的研究进展[J]. 临床医学工程, 2010, 3(17): 151-153
CHANG Zheng, XU Jiping. Progress in study on relationship between brain microvascular endothelial cells and nervous system degenerative diseases[J]. Clinical Medicine & Engineering, 2010, 3(17): 151-153. (in Chinese)
24 YAMAZAKIY, KANEKIYOT. Blood-brain barrier dysfunction and the pathogenesis of Alzheimer’s disease[J]Int J Mol Sci, 2017, 18( 9): 1965.
doi: 10.3390/ijms18091965
25 MONTAGNEA, BARNESS R, SWEENEYM D, et al.Blood-brain barrier breakdown in the aging human hippocampus[J]Neuron, 2015, 85( 2): 296-302.
doi: 10.1016/j.neuron.2014.12.032
26 SAGARE A P, BELL R D, ZLOKOVIC B V. Neurovascular dysfunction and faulty amyloid β-peptide clearance in Alzheimer disease[J]. Cold Spring Harb Perspect Med, 2012, 2(10): a011452
27 柏树令, 赵丹. CD34抗原的生物学特性及其临床应用[J]. 解剖科学进展, 2005, 11(1): 54-56, 60
BAI Shuling, ZHAO Dan. Biological features of CD34 antigen and its clinical application[J]. Progress of Anatomical Sciences, 2005, 11(1): 54-56, 60. (in Chinese)
28 RYUJ K, DAVALOSD, AKASSOGLOUK. Fibrinogen signal transduction in the nervous system[J]J Thromb Haemost, 2009, 151-154.
doi: 10.1111/j.1538-7836.2009.03438.x
29 田书娟, 吴卫平. 纤维蛋白沉积与多发性硬化和周围神经损伤关系研究现状[J]. 脑与神经疾病杂志, 2008, 16(2): 149-151
TIAN Shujuan, WU Weiping. The relationship between fibrin deposition and peripheral nerve injury in multiple sclerosis[J]. Journal of Brain and Nervous Diseases, 2008, 16(2): 149-151. (in Chinese)
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