D-半乳糖诱导衰老相关认知功能障碍小鼠模型制备方法的优化

doi:10.13481/j.1671-587X.20250603

摘要/Abstract

摘要：

目的筛选适宜的D-半乳糖（D-gal）浓度和造模周期，建立其诱导的小鼠衰老相关认知功能障碍模型，并进行综合评价。方法 50只C57BL/6J小鼠随机分为对照组和100、200、400及800 mg·kg^-1 D-gal组，每组10只。各浓度D-gal组小鼠每日皮下注射相应浓度D-gal，每日1次，对照组小鼠注射等体积生理盐水。监测各组小鼠体质量及饮水量，采用前肢抓力值测试和爬杆运动能力实验中小鼠爬杆时间评估各组小鼠运动协调能力，通过新物体识别实验、Y迷宫实验及Morris水迷宫实验评价各组小鼠认知功能，HE染色和尼氏染色观察2组小鼠脑组织病理形态表现，免疫组织化学法检测各组小鼠脑组织中β-半乳糖苷酶（β-gal）蛋白表达情况，实时荧光定量PCR（RT-qPCR）法检测2组小鼠海马组织中白细胞介素（IL）-1β、肿瘤坏死因子α（TNF-α）、IL-18和IL-4 mRNA表达水平，Western blotting法检测2组小鼠海马组织中β-gal、p53和p16蛋白表达水平。结果对照组和各浓度D-gal组小鼠的体质量增长趋势一致，差异无统计学意义（P>0.05），饮水量比较差异无统计学意义（P>0.05）。皮下注射D-gal第8周后，与对照组比较，200和400 mg·kg^-1 D-gal组小鼠前肢抓力值均明显降低（P<0.05或P<0.01），200 mg·kg^-1 D-gal组小鼠爬杆时间明显延长（P<0.05），200和400 mg·kg^-1 D-gal组小鼠识别指数明显降低（P<0.01），100、200、400和800 mg·kg^-1 D-gal组小鼠自发交替率明显降低（P<0.05或P<0.01），逃避潜伏期明显增加（P<0.05）。空间探索实验，与对照组比较，200 mg·kg^-1 D-gal组小鼠逃避潜伏期明显增加（P<0.05）。HE染色和尼氏染色观察，与对照组比较，200 mg·kg^-1 D-gal组小鼠海马组织神经元排列紊乱、核浓染和核固缩明显、形态结构异常，尼氏染色阳性细胞数量明显减少。免疫组织化学法检测，与对照组比较，200 mg·kg^-1 D-gal组小鼠海马组织CA1区、CA3区和皮层（Cortex）区β-gal蛋白呈强阳性表达。RT-qPCR法检测，与对照组比较，200 mg·kg^-1 D-gal组小鼠海马组织中IL-1β、IL-18和TNF-α mRNA表达水平明显升高（P<0.05或P<0.01），IL-4 mRNA表达水平明显降低（P<0.01）。Western blotting法检测，与对照组比较，200 mg·kg^-1 D-gal组小鼠海马组织中β-gal、p53和p16蛋白表达水平均明显升高（P<0.05或P<0.01）。结论每日给小鼠皮下注射200 mg·kg^-1 D-gal、持续8周可制备衰老相关认知功能障碍小鼠模型。

关键词: 衰老, D-半乳糖, 认知功能障碍, 疾病模型, 小鼠, β-半乳糖苷酶

Abstract:

Objective To discuss the suitable concentration of D-galactose （D-gal） and modeling period， and establish its induced aging-related cognitive dysfunction model in the mice， and perform a comprehensive evaluation. Methods Fifty C57BL/6J mice were randomly divided into control group and 100， 200， 400， and 800 mg·kg^-1 D-gal groups， with 10 mice in each group. The mice in various D-gal groups were subcutaneously injected with the corresponding concentration of D-gal once daily； the mice in control group were injected with an equal volume of normal saline. The body mass and water consumption of the mice in various groups were monitored； forelimb grip strength test and experiment on the ability of pole climbing sports were used to evaluate the motor coordination ability of the mice in various groups； novel object recognition test， Y maze test， and Morris water maze test were used to evaluate the cognitive function of the mice in various groups； HE staining and Nissl staining were used to observe the pathomorphology of brain tissue of the mice in various groups； immunohistochemistry method was used to detect the expression of β-galactosidase （β-gal） protein in brain tissue of the mice in various groups； real-time fluorescence quantitative PCR （RT-qPCR） method was used to detect the mRNA expression levels of interleukin （IL）-1β， tumor necrosis factor-α （TNF-α）， IL-18， and IL-4 in hippocampus tissue of the mice in various groups； Western blotting method was used to detect the expression levels of β-gal， p53， and p16 proteins in hippocampus tissue of the mice in various groups. Results The body mass growth trends of the mice in control group and various D-gal groups were consistent and there was no statistically significant difference （P>0.05）， and there was no statistically significant difference in water consumption （P>0.05）. After 8 weeks of subcutaneous injection of D-gal， compared with control group， the forelimb grip strength values of the mice in 200 and 400 mg·kg?1 D-gal groups were significantly decreased （P<0.05 or P<0.01）； the pole-climbing time of the mice in 200 mg·kg?1 D-gal group was significantly prolonged （P<0.05）； the recognition indexes of the mice in 200 and 400 mg·kg?1 D-gal groups were significantly decreased （P<0.01）； the spontaneous alternation rate of the mice in 100， 200， 400， and 800 mg·kg?1 D-gal group was significantly decreased （P<0.05 or P<0.01）， the escape latency was significantly increased （P<0.05）.Spatial probe test showed that compared with control group， the escape latency of the mice in 200 mg·kg?1 D-gal group was significantly increased （P<0.05）. The HE staining and Nissl staining results showed that compared with control group， the hippocampus neurons of the mice in 200 mg·kg^-1 D-gal group were arranged disorderly， with obvious nuclear pyknosis， nuclear condensation， and abnormal morphology and structure， and the number of Nissl staining positive cells was significantly decreased. The immunohistochemistry results showed that compared with control group， the β-gal expressions in CA1 region， CA3 region， and cortex region of hippocampus tissue of the mice in 200 mg·kg?1 D-gal group were strongly positive. The RT-qPCR results showed that compared with control group， the expression levels of IL-1β，IL-18， and TNF-α mRNA in hippocampus tissue of the mice in 200 mg·kg?1 D-gal group were significantly increased （P<0.05 or P<0.01）， and the expression level of IL-4 mRNA was significantly decreased （P<0.01）. The Western blotting results showed that compared with control group， the expression levels of β-gal， p53， and p16 proteins in hippocampus tissue of the mice in 200 mg·kg?1 D-gal group were significantly increased （P<0.05 or P<0.01）. Conclusion The aging-related cognitive dysfunction model in the mice can be established by subcutaneous injection of 200 mg·kg?1 D-gal daily for 8 weeks.

Key words: Aging, D-galactose, Cognitive dysfunction, Disease models, Mouse, β-galactosidase

中图分类号:

R965.1

孙菡,孙伟伦,王荟沣,马文丽,徐华丽,付雯雯. D-半乳糖诱导衰老相关认知功能障碍小鼠模型制备方法的优化[J]. 吉林大学学报(医学版), 2025, 51(6): 1464-1474.

Han SUN,Weilun SUN,Huifeng WANG,Wenli MA,Huali XU,Wenwen FU. Optimization of preparation method for D-galactose-induced mouse model of aging-related cognitive dysfunction[J]. Journal of Jilin University(Medicine Edition), 2025, 51(6): 1464-1474.

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Primer	Sequence（5'-3'）
IL-1β	F: TGCCACCTTTTGACAGTGAT R: TGTGCTGCTGCGAGATTTGA
IL-18	F: CAACGATGATGCACTTGCAGA R: TGACTCCAGCTTATCTCTTGGT
TNF-α	F: AGGCACTCCCCCAAAAGATG R: CCACTTGGTGGTTTGTGAGTG
IL-4	F: GGGTTGCCAAGCCTTATCGG R: AGACACCTTGGTCTTGGAGCTTATT
GAPDH	F: GGAGAGTGTTTCCTCGTCCC R: ATGAAGGGGTCGTTGATGGC

Group	Grip strength (F/N)			Pole-climbing time (t/s)
Group	（week） 4	6	8	（week） 4	6	8
Control	87.53±6.75	87.42±4.31	86.76±5.14	5.93±1.60	8.36±3.86	7.11±2.73
D-gal
100 mg·kg^-1	83.54±4.10	85.65±4.62	80.04±5.45	6.12±2.33	8.42±3.74	11.37±2.42
200 mg·kg^-1	85.57±4.53	82.24±3.96	77.84±5.36^**	6.96±2.28	10.82±7.76	14.05±4.41^*
400 mg·kg^-1	83.62±5.61	89.50±4.97	78.05±6.71^*	5.32±1.27	7.30±4.73	10.56±6.15
800 mg·kg^-1	86.33±3.43	85.56±2.98	81.57±5.26	5.41±2.42	8.08±3.22	9.78±8.34

Group	Total distance (l/cm)			Recognition index (η/%)
Group	（week） 4	6	8	（week） 4	6	8
Control	77.42±14.93	71.25±18.91	76.68±35.37	59.65±23.02	48.19±13.11	65.54±9.30
D-gal
100	83.57±19.89	73.03±20.90	75.96±27.37	54.39±14.74	46.01±13.77	54.06±9.49
200 mg·kg^-1	85.95±23.83	67.40±15.37	75.81±31.01	59.21±16.96	46.64±15.27	43.46±11.37^*
400 mg·kg^-1	84.51±16.15	76.07±17.35	75.35±40.42	58.67±16.45	46.76±18.49	44.19±10.36^*
800 mg·kg^-1	86.86±12.73	71.79±17.11	77.22±30.89	60.46±7.93	48.90±16.71	57.58±15.47

Group	Spontaneous alternation rate
Group	（week） 4	6	8
Control	63.84±9.81	64.76±4.90	68.78±3.60
D-gal
100 mg·kg^-1	61.51±7.80	60.35±4.11	60.23±7.25^*
200 mg·kg^-1	62.94±4.89	60.63±8.37	55.78±5.14^**
400 mg·kg^-1	64.98±9.70	64.74±6.74	60.89±5.25^*
800 mg·kg^-1	69.19±7.21	60.12±10.12	58.73±6.06^**

Group		IL-1β	IL-18	TNF-α	IL-4
Control		1.00±0.00	1.00±0.00	1.00±0.00	1.00±0.00
200 mg·kg^-1 D-gal		3.20±0.44^**	2.53±0.46^*	2.16±0.57^**	0.60±0.05^**