交泰丸通过激活PI3K/AKT信号通路改善阿尔茨海默病模型小鼠大脑的葡萄糖代谢

doi:10.12122/j.issn.1673-4254.2024.05.11

摘要/Abstract

摘要：

目的探讨交泰丸对阿尔茨海默病模型小鼠大脑胰岛素PI3K/AKT通路的影响。方法 50只3月龄雄性APP/PS1双转基因小鼠随机分为5组：模型组、交泰丸低剂量组（2.1 g/kg）、交泰丸中剂量组（4.2 g/kg）、交泰丸高剂量组（8.4 g/kg）、多奈哌齐组（3 mg/kg），C57BL/6J雄性小鼠为正常组，10只/组。给药4周后，采用水迷宫和旷场实验评估各组小鼠学习记忆能力，采用HE染色、尼氏染色观察小鼠脑组织神经元病理改变，免疫组化法观察小鼠脑组织Aβ淀粉样斑块沉积情况，ELISA试剂盒测定小鼠空腹血清胰岛素水平，采用Western blot和RT-qPCR检测小鼠脑组织中Aβ₄₂、胰岛素PI3K/AKT通路以及下游GLUTs等相关指标蛋白和mRNA表达水平。结果与正常组小鼠比较，模型组小鼠学习记忆能力受损，小鼠海马神经元细胞数量减少，排列稀疏，小鼠脑组织Aβ淀粉样斑块沉积显著增多（P<0.001），Aβ₄₂蛋白表达水平升高（P<0.05），胰岛素抵抗指数升高（P<0.001），小鼠海马PI3K蛋白以及mRNA表达均降低（P<0.01），海马与皮质AKT蛋白表达（P<0.05）、AKT mRNA表达（P<0.001）、InR蛋白表达（P<0.05），GLUT1、GLUT3、GLUT4蛋白表达（P<0.05）及mRNA表达（P<0.001）均降低，GSK3β蛋白表达（P<0.01）及GSK3β mRNA表达（P<0.001）显著升高。与模型组比较，交泰丸各组和多奈哌齐组小鼠记忆能力明显改善，尤其是交泰丸中剂量组，小鼠海马神经元细胞数量增多，小鼠海马、皮质Aβ淀粉样斑块沉积减少（P<0.01），Aβ₄₂蛋白表达下调（P<0.001），胰岛素抵抗指数降低（P<0.001），小鼠海马、皮质PI3K、AKT、InR、GLUT1、GLUT3、GLUT4蛋白（P<0.05）及mRNA（P<0.01）表达不同程度升高，GSK3β蛋白及mRNA表达降低。结论交泰丸可通过激活APP/PS1双转基因小鼠大脑胰岛素PI3K/AKT通路，调控其下游GLUTs表达水平，提高大脑葡萄糖代谢能力，改善小鼠学习记忆能力，延缓阿尔茨海默病发展进程。

关键词: 阿尔茨海默病, 交泰丸, 葡萄糖代谢, 胰岛素PI3K/AKT通路, 葡萄糖转运蛋白

Abstract:

Objective To investigate the effect of Jiaotaiwan on brain insulin-PI3K/AKT pathway in a mouse model of Alzheimer's disease (AD). Methods Fifty 3-month-old male APP/PS1 double transgenic mice were randomized into AD model group, low-, medium- and high-dose Jiaotaiwan treatment groups, and donepezil treatment group. Cognitive functions of the mice were assessed using water maze and open field tests, and neuronal pathologies were observed with HE staining and Nissl staining; immunohistochemistry was used to detect amyloid Aβ deposition in the brain. Fasting serum insulin levels of the mice were measured, and the expressions of Aβ₄₂, insulin-PI3K/AKT pathway components and downstream glucose transporters in the brain tissue were detected with RT-qPCR and Western blotting. Results The AD mouse models exhibited obvious impairment of learning and memory abilities, significantly reduced hippocampal neurons, and obvious Aβ amyloid plaques in the brain tissue with increased Aβ₄₂ protein expression (P<0.05) and insulin resistance index, decreased hippocampal PI3K expressions, lowered expressions of AKT and InR, reduced expressions of GLUT1, GLUT3, and GLUT4, and increased expression of GSK3β in both the hippocampus and cortex. Treatment with Jiaotaiwan and donepezil both effectively improved memory ability of the mouse models, increased the number of hippocampal neurons, reduced Aβ amyloid plaques and increased the expressions of PI3K, AKT, InR, GLUT1, GLUT3 and GLUT4 in the hippocampus and cortex. Conclusion Jiaotaiwan improves learning and memory abilities of APP/PS1 double transgenic mice and delay the development of AD by activating the PI3K/AKT pathway and regulating the expression levels of its downstream GLUTs in the brain.

Key words: Alzheimer's disease, Jiaotaiwan, glucose metabolism, insulin PI3K/AKT pathway, glucose transporter proteins

王妍, 阮毓卿, 崔璨, 王秀. 交泰丸通过激活PI3K/AKT信号通路改善阿尔茨海默病模型小鼠大脑的葡萄糖代谢[J]. 南方医科大学学报, 2024, 44(5): 894-903.

Yan WANG, Yuqing RUAN, Can CUI, Xiu WANG. Jiaotaiwan improves brain glucose metabolism in a mouse model of Alzheimer's disease by activating the PI3K/AKT signaling pathway[J]. Journal of Southern Medical University, 2024, 44(5): 894-903.

图/表 7

图1 各组小鼠Morris水迷宫实验以及旷场实验结果比较

Fig.1 Results of Morris water maze test and open field test. A: Comparison of escape latency of the mice among the groups (Mean±SD, n=8). B: Swimming trajectories during escape latency in each group. C: Open field behavior trajectories of the mice in each group. ***P<0.001 vs Control group; ##P<0.01, ###P<0.001 vs model group.

表1 各组小鼠水迷宫空间探索实验以及旷场实验比较

Tab.1 Results of water maze spatial exploration test and open field test in each group of mice (Mean±SD, n=8)

Group	Times of platform crossing	Time of first platform crossing (s)	Time in platform quadrant (s)	Number of central area entry	Time in the central zone (s)
Control	4.25±0.83	24.17±2.72	31.68±2.56	14.88±1.45	32.91±1.78
Model	1.63±0.70***	37.69±2.65***	20.11±1.99***	6.25±1.85***	16.21±1.28***
JTWL	3.13±1.05^#	32.99±2.25^##	23.21±2.81	9.00±1.41^#	18.10±2.56
JTWM	4.00±0.71^###	28.12±1.05^###	26.89±3.00^###	12.25±1.85^###	20.91±1.99^###
JTWH	3.25±0.83^#	31.27±2.02^###	25.52±2.25^##	10.63±1.58^###	19.77±2.02^#
DON	3.38±0.99^##	33.71±1.41^#	21.01±2.16	9.75±1.98^##	19.9±1.66^#

图2 交泰丸对各组小鼠海马神经元形态的影响

Fig.2 Effect of Jiaotaiwan on hippocampal neuron morphology in each group. A: Representative HE staining results of the hippocampus in each group. B: Nissl staining of the hippocampus in each group (scale bar=100 μm).

图3 交泰丸对各组小鼠海马、皮质Aβ斑块聚集和Aβ42含量的影响

Fig.3 Impact of Jiaotaiwan on aggregation of Aβ plaques and Aβ42 level in the hippocampus and cortex of the mice (Mean±SD, n=4). A: Immunohistochemistry showing Aβ plaques in the hippocampus and cortex (scale bar=100 μm. B: Quantification of Aβ42 protein expressions in the hippocampus and cortex using Western blotting. *P<0.05,***P<0.001 vs Control group; ##P<0.01,###P<0.001 vs Model group.

表2 交泰丸对各组小鼠胰岛素抵抗的影响比较

Tab.2 Effects of Jiaotaiwan on insulin resistance of the mice in each group (Mean±SD)

Group	Fasting glucose (mmol/L)	Fasting insulin (mIU/L)	HOMA-IR
Control	5.76±0.57	5.54±0.90	1.40±0.17
Model	11.68±1.34***	16.41±1.43***	8.52±1.24***
JTWL	8.98±2.44	13.98±1.16	5.57±1.53^###
JTWM	7.51±1.41^##	12.00±1.61^###	3.97±0.72^###
JTWH	9.60±3.71	13.43±3.07^#	5.29±1.12^###
DON	7.40±1.34^##	12.85±1.25^###	4.19±0.69^###

图4 交泰丸对各组小鼠海马、皮质胰岛素PI3K/AKT通路相关指标的影响

Fig.4 Effects of Jiaotaiwan on insulin-PI3K/AKT pathway in the hippocampus and cortex of the mice in each group. A: Western blots of the proteins in the insulin-PI3K/AKT pathway in the hippocampus and cortex in each group. B: Comparison of PI3K, AKT, GSK3β, and InR protein and mRNA expressions in the hippocampus and cortex among the groups (Mean±SD).*P<0.05,**P<0.01,***P<0.001 vs Control group; #P<0.05,##P<0.01,###P<0.001 vs Model group.

图5 交泰丸对各组小鼠海马、皮质葡萄糖转运相关指标的影响

Fig.5 Effects of Jiaotaiwan on glucose transport in the hippocampus and cortex of the mice in each group. A: Western blots of the proteins involved in glucose transport in the hippocampus and cortex in each group. B: Expression levels of GLUT1, GLUT3, and GLUT4 proteins and mRNAs in the hippocampus and cortex in each group (Mean±SD).*P<0.05, **P<0.01, ***P<0.001 vs Control group; #P<0.05, ##P<0.01, ###P<0.001 vs Model group.

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