Nek6通过mTOR信号通路调控自噬,减轻脑缺血再灌注损伤。

IF 3.3 3区 医学 Q2 NEUROSCIENCES Molecular Brain Pub Date : 2024-12-19 DOI:10.1186/s13041-024-01166-7
Qingzhi Wang, Xinjing Liu, Jing Yuan, Ting Yang, Lan Ding, Bo Song, Yuming Xu
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引用次数: 0

摘要

目的:脑缺血再灌注损伤(CIRI)是缺血性脑卒中临床治疗后神经功能恢复的主要障碍。本研究旨在探讨Nek6通过脑缺血后自噬减轻CIRI的分子机制。材料与方法:采用大脑中动脉闭塞法(MCAO)建立小鼠CIRI模型。TUNEL染色观察神经元细胞凋亡情况。采用缺氧加再氧建立氧葡萄糖剥夺/再氧(OGD/R)模型。检测细胞凋亡及活性。Western blot检测自噬相关蛋白、蛋白激酶B (Akt)/哺乳动物雷帕霉素靶蛋白(mTOR)和腺苷5′-单磷酸活化蛋白激酶(AMPK)/mTOR信号通路相关蛋白的表达。用荧光法观察细胞自噬通量。放线菌素D处理检测Nek6 mRNA的稳定性。甲基化RNA免疫沉淀技术检测Nek6甲基化水平。结果:Nek6在MCAO和OGD/R模型中表达均升高。OGD/R过表达Nek6抑制细胞凋亡,降低LC3II和Beclin-1表达,增加p62表达,发生溶酶体功能障碍。干扰Nek6则会产生相反的结果。Nek6过表达促进p-Akt和p-mTOR蛋白表达,抑制p-AMPK和p- unc -51样激酶1蛋白表达和细胞凋亡,而LY294002、雷帕霉素或RSVA405处理逆转了这一作用。甲基转移酶样蛋白3 (METTL3)在CIRI中的异常表达增强了m6A的修饰,促进了Nek6的表达水平。结论:本研究证实Nek6通过mTOR信号通路调控自噬,缓解CIRI,为未来缺血性脑卒中患者提供新的治疗策略。
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Nek6 regulates autophagy through the mTOR signaling pathway to alleviate cerebral ischemia-reperfusion injury.

Objective: Cerebral ischemia-reperfusion injury (CIRI) is a major obstacle to neurological recovery after clinical treatment of ischemic stroke. The aim of this study was to investigate the molecular mechanism of Nek6 alleviating CIRI through autophagy after cerebral ischemia.

Materials and methods: A mouse model of CIRI was constructed by middle cerebral artery occlusion (MCAO). TUNEL staining was used to observe the apoptosis of neuronal cells. The oxygen glucose deprivation/reoxygenation (OGD/R) model was established by hypoxia and reoxygenation. The cell apoptosis and activity was detected. Western blot was performed to detect the expression of autophagy-related proteins, protein kinase B (Akt)/mammalian target of rapamycin (mTOR) and adenosine 5'-monophosphate-activated protein kinase (AMPK)/mTOR signaling pathway-related proteins. Cellular autophagy flux was observed by fluorometric method. NIMA-related kinase 6 (Nek6) mRNA stability was detected by actinomycin D treatment. Methylation RNA immunoprecipitation technique was used to detect Nek6 methylation level.

Results: Nek6 expression was increased in both MCAO and OGD/R models. Overexpression of Nek6 in OGD/R inhibited apoptosis, decreased LC3II and Beclin-1 expression, increased p62 expression, and occurred lysosome dysfunction. Interference with Nek6 has opposite results. Nek6 overexpression promoted p-Akt and p-mTOR protein expressions, inhibited p-AMPK and p-UNC-51-like kinase 1 protein expressions and cell apoptosis, while LY294002, Rapamycin or RSVA405 treatment reversed this effect. Abnormal methyltransferase·like protein 3 (METTL3) expression in CIRI enhanced m6A modification and promoted Nek6 expression level.

Conclusion: This study confirmed that Nek6 regulates autophagy and alleviates CIRI through the mTOR signaling pathway, which provides a novel therapeutic strategy for patients with ischemic stroke in the future.

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来源期刊
Molecular Brain
Molecular Brain NEUROSCIENCES-
CiteScore
7.30
自引率
0.00%
发文量
97
审稿时长
>12 weeks
期刊介绍: Molecular Brain is an open access, peer-reviewed journal that considers manuscripts on all aspects of studies on the nervous system at the molecular, cellular, and systems level providing a forum for scientists to communicate their findings. Molecular brain research is a rapidly expanding research field in which integrative approaches at the genetic, molecular, cellular and synaptic levels yield key information about the physiological and pathological brain. These studies involve the use of a wide range of modern techniques in molecular biology, genomics, proteomics, imaging and electrophysiology.
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