miR-124通过调控PI3K/AKT信号通路改善睡眠剥夺大鼠认知功能

doi:10.12122/j.issn.1673-4254.2025.02.15

摘要/Abstract

摘要：

目的探讨miR-124通过调控PI3K/AKT信号通路影响睡眠剥夺（SD）后大鼠认知功能的分子作用机制。方法分别转染miR-124 agomir/agomir NC， miR-124 antagomir/antagomir NC，建立睡眠剥夺模型。设置分组：正常对照组（CC组）、睡眠剥夺组（SD组）、SD+miR-124 agomir组、SD+agomir NC组、SD+miR-124 antagomir组、SD+antagomir NC组，9只/组。观察大鼠一般情况和体质量变化。Morris水迷宫（MWM）试验观察大鼠认知情况。HE染色法观察各组大鼠海马病理变化。qRT-PCR检测大鼠海马组织miR-124水平。Western blotting检测凋亡相关蛋白及信号通路蛋白的表达情况。结果造模后各造模组大鼠体质量均低于CC组（P<0.05）。MWM试验结果显示与CC组相比，SD组逃避潜伏期延长、穿越原平台次数以及在原平台所在象限游泳的时间和距离百分比减少（P<0.05）；与SD组相比，SD+miR-124 agomir组逃避潜伏期缩短、穿越原平台次数以及在第4象限游泳的时间和距离百分比增加，SD+miR-124 antagomir组逃避潜伏期延长、穿越原平台次数以及在第4象限游泳的时间和距离百分比减少（P<0.05）。HE染色结果显示与SD组比较，SD+miR-124 agomir组大鼠海马组织病理改变明显减轻， SD+miR-124 antagomir组明显加重（P<0.05）。qRT-PCR结果显示与CC组比较，SD组海马组织中miR-124的表达下降（P<0.05）；与SD组比较，SD+miR-124 agomir组中miR-124的表达明显升高（P<0.05）， SD+miR-124 antagomir组明显下降（P<0.05）。Western blotting结果显示与CC组比较，SD组PI3K、p-AKT/AKT、BCL-2蛋白表达明显降低（P<0.05），BAX、caspase-3蛋白表达明显升高（P<0.05）；与SD组比较，SD+miR-124 agomir组PI3K、p-AKT/AKT、BCL-2蛋白表达明显升高（P<0.05），BAX、caspase-3蛋白表达明显降低（P<0.05），SD+miR-124 antagomir组PI3K、p-AKT/AKT、BCL-2蛋白表达明显降低（P<0.05），BAX、caspase-3蛋白表达明显升高（P<0.05）。结论 miR-124通过激活PI3K/AKT通路缓解SD诱导的认知功能下降及神经元凋亡。

关键词: 睡眠剥夺, miR-124, PI3K/AKT信号通路, 认知功能, 大鼠

Abstract:

Objective To explore the molecular mechanism by which miR-124 affects cognitive function of sleep-deprived rats. Methods Fifty-four adult male SD rats were randomized into 6 groups (n=9), including a normal control group, a sleep deprivation (SD) model group, and 4 intracerebral microinjection groups in which the rats were subjected to stereotactic injection of miR-124 agomir, miR-124 agomir NC, miR-124 antagomir, or miR-124 antagomir into the lateral ventricle 7 days before SD modeling. The cognitive functions of the rats were evaluated with Morris water maze test, and pathological changes in the hippocampus were observed using HE staining. The expression level of miR-124 in hippocampal tissues of the rats was detected with qRT-PCR, and the expression level of apoptosis-related proteins and signaling pathway proteins were determined using Western blotting. Results In Morris water maze test, the SD rat models treated with miR-124 agomir showed a significantly shorter escape latency and fewer platform crossings with increased percentage of time and swimming distance in the fourth quadrant as compared with those in SD model group, while the rats treated with miR-124 antagomir exhibited worsened performance in the test. In the SD rat models, treatment with miR-124 agomir obviously lessened pathological changes in the hippocampus, while treatment with miR-124 antagomir significantly worsened the pathological changes. Compared with those in SD model group, the miR-124 agomir-treated rats showed an increased hippocampal expression of miR-124 with upregulated protein expressions of PI3K, p-AKT/AKT, and Bcl-2 and downregulated expressions of Bax and caspase-3 proteins, while rats treated with miR-124 antagomir showed significantly decreased hippocampal expression of miR-124 with lowered expressions of PI3K, p-AKT/AKT and Bcl-2 proteins and increased Bax and caspase-3 protein expressions. Conclusion High expression of miR-124 alleviates SD-induced cognitive decline and neuronal apoptosis in rats by activating the PI3K/AKT signaling pathway.

Key words: sleep deprivation, miR-124, PI3K/AKT signaling pathway, cognitive function, rats

裴月娇, 刘慧敏, 昕宇, 刘波. miR-124通过调控PI3K/AKT信号通路改善睡眠剥夺大鼠认知功能[J]. 南方医科大学学报, 2025, 45(2): 340-346.

Yuejiao PEI, Huimin LIU, Yu XIN, Bo LIU. High expression of miR-124 improves cognitive function of sleep-deprived rats by modulating the PI3K/AKT signaling pathway[J]. Journal of Southern Medical University, 2025, 45(2): 340-346.

图/表 7

表1 miR-124引物序列

Tab.1 Primers sequences for qRT-PCR in this study

Name

Primer sequence (5'-3')

rno-miR-124-3p-RT

CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGGGCATTCA

rno-miR-124-3p-S

ACACTCCAGCTGGGTAAGGCACGCGGTG

U6-A

U6-S

AACGCTTCACGAATTTGCGT

CTCGCTTCGGCAGCACA

表2 各组大鼠训练5 d后体质量比较

Tab.2 Comparison of body weight （BW）among the 6 groups of rats at the end of sleep deprivation (SD) for 5 days (Mean±SD, n=6)

Group	Initial BW(g)	Final BW(g)
CC	267.2±24.4	296.0±22.7
SD	269.7±24.9^a	245.2±20.0^b
SD+miR-124 agomir	268.2±28.0^a	241.3±26.7^b
SD+agomir NC	268.8±32.9^a	242.8±28.6^b
SD+miR-124 antagomir	284.7±26.1^a	259.2±26.5^b
SD+antagomir NC	277.5±20.8^a	255.0±20.4^b
F	0.345	3.558
P	0.881	0.012

表3 miR-124对小鼠MWM空间参考记忆的影响

Tab.3 Effect of miR-124 on spatial reference memory of the rats in Morris mater maze test (Mean±SD, n=6)

Groups	Escape latency (s)	Number of crossings through the original platform	Percentage of target quadrant dwell time (%)	Target quadrant distance percentage (%)
CC	4.23±0.88	8.17±1.67	0.37±0.02	0.35±0.02
SD	14.91±4.09^a	4.50±0.96^a	0.24±0.03^a	0.23±0.03^a
SD+miR-124 agomir	6.39±1.42^b	6.83±1.57^b	0.33±0.01^b	0.31±0.01^b
SD+agomir NC	14.32±5.85^ac	4.83±1.07^ac	0.25±0.02^ac	0.23±0.02^ac
SD+miR-124 antagomir	32.46±8.86^ab	2.00±0.58^ab	0.16±0.02^ab	0.16±0.02^ab
SD+antagomir NC	13.04±4.54^ad	4.17±1.07^ad	0.24±0.02^ad	0.24±0.02^ad
F	19.469	15.890	58.233	52.981
P	<0.001	<0.001	<0.001	<0.001

图1 各组大鼠海马组织HE染色图

Fig.1 HE staining of the hippocampus of the rats in each group. A: CC group. B: SD group. C: SD+miR-124 agomir group. D: SD+agomir NC group.E: SD+miR-124 antagomir group. F: SD+antagomir NC group.

图2 各组大鼠海马miR-124-3p表达比较分析

Fig.2 Comparison of hippocampal miR-124-3p expression levels in the rats among the 6 groups (Mean±SD). ***P<0.001.

图3 各组大鼠BAX、BCL-2和Caspase-3水平比较

图4 各组大鼠海马PI3K和p-AKT/AKT水平比较

Fig.4 Comparison of hippocampal expression levels of PI3K and p-AKT/AKT among the 6 groups. A: Western blotting for detecting protein expressions of PI3K and p-AKT/AKT proteins. B, C: Relative expressions of PI3K and p-AKT/AKT proteins in each group (Mean±SD, n=3). **P<0.01, ***P<0.001.

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