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

doi:10.12122/j.issn.1673-4254.2024.10.20

Abstract

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

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.

Figures/Tables 10

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^{#^^^}

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

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.

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.

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.

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.

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.

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.

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.

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.

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