黄精丸的配伍提取液对学习记忆障碍大鼠海马齿状回区干细胞影响的研究

doi:10.12114/j.issn.1007-9572.2024.0007

摘要/Abstract

摘要： 背景阿尔茨海默病（AD）是一类以学习记忆障碍为主的神经退行性疾病，给家庭和社会造成了沉重负担。中医药在防治AD方面具有重要潜力，当前应积极从中医药宝库中挖掘有效治疗方药。目的探讨古代名方黄精丸的配伍提取液（CHP）对D-半乳糖联合东莨菪碱所致学习记忆障碍大鼠海马齿状回（DG）区干细胞增殖的影响。方法 2023年1—12月选取90只雄性SD大鼠，随机分成正常对照组、AD模型组、多奈哌齐组、黄精丸配伍低、中、高剂量组，每组15只。制备CHP，通过皮下注射1% D-半乳糖液+东莨菪碱制备AD大鼠模型，多奈哌齐组给予0.5 mg·kg-1·d-1多奈哌齐灌胃，黄精丸配伍低、中、高剂量组分别给予1、3、9 g·kg-1·d-1剂量的黄精丸配伍提取液灌胃，共干预8周。通过跳台实验检测学习、记忆能力。取材后采用实时荧光定量聚合酶链式反应（RT-qPCR）检测大鼠海马细胞周期蛋白D1（CyclinD1）、巢蛋白（Nestin）mRNA表达水平，采用免疫印迹法（Western blot）检测大鼠CyclinD1、Nestin蛋白表达水平。通过5-溴-2-脱氧尿嘧啶核苷（BrdU）免疫组化、免疫荧光检测免疫阳性细胞数量。结果跳台实验结果显示AD模型组较正常对照组大鼠反应时间延长，跳台学习成绩与跳台记忆成绩的错误次数增多，潜伏时间缩短；多奈哌齐组及CHP各剂量组较AD模型组大鼠跳台学习成绩的反应时间及错误次数、跳台记忆成绩的错误次数均减少，潜伏时间增加（P<0.05）。RT-qPCR结果显示AD模型组较正常对照组的大鼠海马CyclinD1、Nestin mRNA表达水平降低，多奈哌齐组及CHP各剂量组较AD模型组大鼠海马CyclinD1、Nestin mRNA表达水平均升高，CHP高剂量组CyclinD1、Nestin mRNA表达水平高于正常对照组（P<0.05）。Western blot结果显示AD模型组较正常对照组的大鼠海马CyclinD1、Nestin蛋白表达水平降低，多奈哌齐组及CHP各剂量组较AD模型组的大鼠海马CyclinD1、Nestin蛋白表达水平均升高，CHP高剂量组CyclinD1、Nestin蛋白表达水平高于正常对照组（P<0.05）。BrdU免疫组化、免疫荧光结果显示AD模型组较正常对照组的大鼠海马DG区阳性细胞数量均明显减少；多奈哌齐组及CHP各剂量组较AD模型组的大鼠海马DG区阳性细胞数量均明显增多，CHP中、高剂量组阳性细胞数量高于正常对照组（P<0.05）。结论 CHP可促进学习记忆障碍大鼠海马神经干细胞增殖，促进海马神经功能恢复进而改善AD认知障碍，具有临床推广价值。

关键词: 阿尔茨海默病, 记忆障碍, 黄精丸, 齿状回, 神经干细胞

Abstract:

Background

Alzheimer's disease (AD) is a type of neurodegenerative disorder primarily characterized by learning and memory impairments, posing a significant burden to families and society. Traditional Chinese Medicine (TCM) holds considerable potential in preventing and treating AD, and it is crucial to actively explore effective therapeutic methods from the treasury of TCM.

Objective

This study aimed to investigate the effects of the compatibility extract of Huangjing pill (CHP) on the proliferation of stem cells in the dentate gyrus (DG) region of the hippocampus in rats with learning and memory impairments induced by D-galactose combined with scopolamine.

Methods

From January to December 2023, 90 male SD rats were randomly divided into a normal control group, an AD model group, a donepezil group, and CHP low, medium, and high dose groups, with 15 rats in each group. The donepezil group was given 0.5 mg·kg^-1·d^-1 donepezil by gavage. The CHP low, medium, and high dose groups were given 1, 3 and 9 g·kg^-1·d^-1 doses of the combined extract of CHP by gavage respectively. The intervention lasted for 8 weeks. The AD rat model was prepared by subcutaneous injection of 1% D-galactose solution combined with scopolamine. The learning and memory capabilities were assessed through the step-down test. After sampling, the expression levels of hippocampal cell cycle protein D1 (CyclinD1) and nestin mRNA were measured by real-time fluorescent quantitative polymerase chain reaction (RT-qPCR), and the expression levels of CyclinD1 and nestin proteins were detected by Western blot. The number of immunopositive cells was determined by 5-bromo-2-deoxyuridine (BrdU) immunohistochemistry and immunofluorescence.

Results

The step-down test showed that the reaction time was prolonged, the number of errors in step-down learning and memory scores increased, and the latency time significantly decreased in the AD model group compared to the normal control group. The donepezil group and each dosage group of CHP significantly reduced the reaction time and the number of errors in step-down learning and memory scores, and significantly increased the latency time compared to the AD model group (P<0.05). RT-qPCR results indicated that the expression levels of CyclinD1 and nestin mRNA in the hippocampus of the AD model group were significantly lower than those in the normal control group. The donepezil group and each dosage group of CHP showed increased expression levels of hippocampal CyclinD1 and nestin mRNA compared to the AD model group, with the high dosage group of CHP showing higher levels than the normal control group (P<0.05). Western blot results mirrored these findings, with significant increases in the expression levels of CyclinD1 and nestin proteins in the donepezil group and each dosage group of CHP compared to the AD model group, especially in the high dosage group of CHP, which were higher than those in the normal control group (P<0.05). BrdU immunohistochemistry and immunofluorescence showed that the number of positive cells in the hippocampal DG region significantly decreased in the AD model group compared to the normal control group; however, it significantly increased in the donepezil group and each dosage group of CHP, with the medium and high dosage groups of CHP having higher numbers than the normal control group (P<0.05) .

Conclusion

CHP can promote the proliferation of neural stem cells in the hippocampus of rats with learning and memory impairments, facilitating the recovery of hippocampal neural functions and thereby improving cognitive impairments in AD, which highlights its value for clinical application.

Key words: Alzheimer disease, Memory disorders, Huangjing pill, Dentate gyrus, Neural stem cells

廖可欣,肖移生. 黄精丸的配伍提取液对学习记忆障碍大鼠海马齿状回区干细胞影响的研究[J]. 中国全科医学, 2025, 28(24): 3019-3025. DOI: 10.12114/j.issn.1007-9572.2024.0007.
LIAO Kexin,XIAO Yisheng. Effects of the Compatibility Extract of Huangjing Pill on the Hippocampal DG Region Stem Cells in Rats with Learning and Memory Impairments[J]. Chinese General Practice, 2025, 28(24): 3019-3025. DOI: 10.12114/j.issn.1007-9572.2024.0007.

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组别	只数	跳台学习成绩		跳台记忆成绩
组别	只数	反应时间（s）	错误次数（次）	潜伏时间（s）	错误次数（次）
正常对照组	15	23.47±4.05	1.67±0.70	246.13±31.78	1.33±0.60
AD模型组	15	160.73±12.44^a	8.47±1.63^a	79.73±10.45^a	6.87±1.26^a
多奈哌齐组	15	133.93±9.23^b	6.53±0.96^b	108.67±12.25^b	5.33±0.87^b
CHP低剂量组	15	151.40±11.26^b	8.07±1.77^b	83.13±10.62^b	6.53±1.09^b
CHP中剂量组	15	132.47±8.75^b	6.60±1.20^b	98.60±10.17^b	5.07±0.93^b
CHP高剂量组	15	84.80±6.83^b	5.40±1.08^b	120.93±12.42^b	4.73±0.77^b
F值		454.24	51.53	199.05	61.31
P值		<0.05	<0.05	<0.05	<0.05

组别	只数	跳台学习成绩		跳台记忆成绩
组别	只数	反应时间（s）	错误次数（次）	潜伏时间（s）	错误次数（次）
正常对照组	15	23.47±4.05	1.67±0.70	246.13±31.78	1.33±0.60
AD模型组	15	160.73±12.44^a	8.47±1.63^a	79.73±10.45^a	6.87±1.26^a
多奈哌齐组	15	133.93±9.23^b	6.53±0.96^b	108.67±12.25^b	5.33±0.87^b
CHP低剂量组	15	151.40±11.26^b	8.07±1.77^b	83.13±10.62^b	6.53±1.09^b
CHP中剂量组	15	132.47±8.75^b	6.60±1.20^b	98.60±10.17^b	5.07±0.93^b
CHP高剂量组	15	84.80±6.83^b	5.40±1.08^b	120.93±12.42^b	4.73±0.77^b
F值		454.24	51.53	199.05	61.31
P值		<0.05	<0.05	<0.05	<0.05

组别	只数	CyclinD1	Nestin
正常对照组	7	1.00±0.00	1.00±0.00
AD模型组	7	0.23±0.04^a	0.17±0.03^a
多奈哌齐组	7	0.43±0.04^b	0.46±0.04^b
CHP低剂量组	7	0.53±0.05^b	0.55±0.02^b
CHP中剂量组	7	0.71±0.10^b	0.75±0.05^b
CHP高剂量组	7	1.25±0.10^ab	1.39±0.05^ab
F值		199.42	891.74
P值		<0.05	<0.05

组别	只数	CyclinD1	Nestin
正常对照组	7	1.00±0.00	1.00±0.00
AD模型组	7	0.23±0.04^a	0.17±0.03^a
多奈哌齐组	7	0.43±0.04^b	0.46±0.04^b
CHP低剂量组	7	0.53±0.05^b	0.55±0.02^b
CHP中剂量组	7	0.71±0.10^b	0.75±0.05^b
CHP高剂量组	7	1.25±0.10^ab	1.39±0.05^ab
F值		199.42	891.74
P值		<0.05	<0.05

组别	只数	CyclinD1	Nestin
正常对照组	7	0.49±0.03	0.52±0.04
AD模型组	7	0.11±0.02^a	0.15±0.02^a
多奈哌齐组	7	0.28±0.02^b	0.23±0.04^b
CHP低剂量组	7	0.32±0.03^b	0.35±0.03^b
CHP中剂量组	7	0.41±0.04^b	0.47±0.03^b
CHP高剂量组	7	0.57±0.04^ab	0.59±0.04^ab
F值		163.84	168.68
P值		<0.05	<0.05