大鼠miR-664-1-5p靶向PTEN/Akt/Nrf2通路减轻缺氧/复氧PC12细胞氧化损伤的机制研究

doi:10.3969/j.issn.1673-5765.2026.05.012

中国卒中杂志 ›› 2026, Vol. 21 ›› Issue (5): 612-623.DOI: 10.3969/j.issn.1673-5765.2026.05.012

大鼠miR-664-1-5p靶向PTEN/Akt/Nrf2通路减轻缺氧/复氧PC12细胞氧化损伤的机制研究

彭艳¹，冯文战²，刘沁²，唐红梅³，刘祎²，高立²，汪思嘉²，白雪³

1泸州 646102 泸县中医医院科教科
2泸州 646000 西南医科大学附属中医医院全科医学科
3泸州 646000 西南医科大学附属中医医院神经内科

收稿日期:2025-03-20 修回日期:2025-10-29 接受日期:2025-12-18 出版日期:2026-05-20 发布日期:2026-05-20
通信作者: 白雪 bx7246@163.com
基金资助:
四川省中医药管理局科学技术研究专项课题（2024MS422）
泸州市科技计划项目（2022-SYF-35；2025JYJ057）

Mechanistic Study on Rat miR-664-1-5p Targeting the PTEN/Akt/Nrf2 Pathway to Attenuate Oxidative Damage in PC12 Cells Induced by Hypoxia/Reoxygenation

PENG Yan¹, FENG Wenzhan², LIU Qin², TANG Hongmei³, LIU Yi², GAO Li², WANG Sijia², BAI Xue³

1Department of Medical Education & Research, Luxian Hospital of TCM, Luzhou 646102, China
2Department of General Practice, The Affiliated Traditional Chinese Medicine Hospital of Southwest Medical University, Luzhou 646000, China
3Department of Neurology, The Affiliated Traditional Chinese Medicine Hospital of Southwest Medical University, Luzhou 646000, China

Received:2025-03-20 Revised:2025-10-29 Accepted:2025-12-18 Online:2026-05-20 Published:2026-05-20
Contact: BAI Xue, E-mail: bx7246@163.com

摘要/Abstract

摘要： 目的探讨大鼠小分子核糖核酸（micro ribonucleic acid，miRNA）-664-1-5p（以下简称miR-664-1-5p）对缺氧/复氧后肾上腺嗜铬细胞瘤（adrenal pheochromocytoma，PC12）细胞损伤的影响及作用机制。
方法体外培养PC12细胞，分为空白组、缺氧/复氧组、缺氧/复氧+阴性对照（negative control，NC）模拟物（mimic）组、缺氧/复氧+miR-664-1-5p mimic组。采用实时荧光定量PCR检测miR-664-1-5p的表达水平；使用细胞计数试剂盒-8检测细胞活性；采用流式细胞术检测细胞凋亡率；采用酶联免疫吸附试验检测超氧化物歧化酶（superoxide dismutase，SOD）含量和丙二醛（malondialdehyde，MDA）含量；应用免疫印迹法检测磷酸酶及张力蛋白同源物（phosphatase and tensin homolog，PTEN）、蛋白激酶B（protein kinase B，Akt）、磷酸化Akt（phosphorylated Akt，p-Akt）、核转录因子红系2相关因子2（nuclear factor-erythroid 2-related factor 2，Nrf2）的蛋白相对表达量；采用双萤光素酶报告基因检测验证大鼠miR-664-1-5p与PTEN的靶向结合关系；通过PTEN过表达验证大鼠miR-664-1-5p与PTEN的调控关系。
结果与空白组相比，缺氧/复氧组PC12细胞中大鼠miR-664-1-5p表达（P<0.01）、细胞存活率（P<0.05）、SOD含量（P<0.05）、Nrf2（P<0.05）和p-Akt（P<0.01）蛋白相对表达量降低，MDA含量（P<0.05）、细胞凋亡率（P<0.05）及PTEN（P<0.01）蛋白相对表达量升高。大鼠miR-664-1-5p过表达可逆转上述指标的变化。双萤光素酶报告基因实验表明，大鼠miR-664-1-5p与PTEN存在靶向结合关系，且可抑制萤光素酶活性。进一步转染质粒-巨细胞病毒DNA-PTEN过表达质粒后，PC12细胞SOD含量、Nrf2和p-Akt蛋白相对表达量降低，MDA含量及细胞凋亡率升高（均P<0.01）。
结论大鼠miR-664-1-5p可通过靶向抑制PTEN表达，激活Akt/Nrf2信号通路，从而减轻缺氧/复氧诱导的PC12细胞氧化应激损伤及细胞凋亡，为脑缺血再灌注损伤的防治提供新的潜在靶点。

关键词: 缺氧/复氧; 肾上腺嗜铬细胞瘤细胞; 大鼠miR-664-1-5p; 磷酸酶及张力蛋白同源物; 蛋白激酶B通路; 氧化应激

Abstract: Objective To investigate the effect and mechanism of rat micro ribonucleic acid (miRNA) -664-1-5p (hereinafter referred to as miR-664-1-5p) on injury induced by hypoxia/reoxygenation in adrenal pheochromocytoma (PC12) cells.
Methods PC12 cells were cultured in vitro and divided into the control group, the hypoxia/reoxygenation group, the hypoxia/reoxygenation+negative control (NC) mimic group, and the hypoxia/reoxygenation+miR-664-1-5p mimic group. The expression level of miR-664-1-5p was detected by real-time fluorogenic quantitative PCR. Cell viability was measured using cell counting kit-8. Apoptosis rate was determined by flow cytometry. The levels of superoxide dismutase (SOD) and malondialdehyde (MDA) were detected by enzyme linked immunosorbent assay. The protein relative expression levels of phosphatase and tensin homolog (PTEN), protein kinase B (Akt), phosphorylated Akt (p-Akt), and nuclear factor-erythroid 2-related factor 2 (Nrf2) were detected by Western blotting. The targeted binding between miR-664-1-5p and PTEN was verified by dual-luciferase reporter assay. The regulatory relationship between miR-664-1-5p and PTEN was confirmed by PTEN overexpression.
Results Compared with the control group, the expression of miR-664-1-5p (P<0.01), cell viability (P<0.05), SOD level (P<0.05), and protein expression of Nrf2 (P<0.05) and p-Akt (P<0.01) were significantly decreased in the hypoxia/reoxygenation group, while MDA level (P<0.05), apoptosis rate (P<0.05), and PTEN protein expression (P<0.01) were significantly increased. Overexpression of miR-664-1-5p reversed these changes. Dual-luciferase reporter assay confirmed that miR-664-1-5p directly targeted PTEN and inhibited luciferase activity. Furthermore, transfection with the plasmid cytomegalovirus DNA-PTEN overexpression plasmid reduced SOD level, Nrf2 and p-Akt protein expression, and elevated MDA level and apoptosis rate in PC12 cells (all P<0.01).
Conclusions Rat miR-664-1-5p alleviates oxidative stress injury and apoptosis induced by hypoxia/reoxygenation in PC12 cells by targeting PTEN and activating the Akt/Nrf2 signaling pathway, which provides a novel potential target for the prevention and treatment of cerebral ischemia reperfusion injury.

Key words: Hypoxia/reoxygenation; Adrenal pheochromocytoma cell; Rat miR-664-1-5p; PTEN; Protein kinase B pathway; Oxidative stress

中图分类号:

彭艳, 冯文战, 刘沁, 唐红梅, 刘祎, 高立, 汪思嘉, 白雪. 大鼠miR-664-1-5p靶向PTEN/Akt/Nrf2通路减轻缺氧/复氧PC12细胞氧化损伤的机制研究[J]. 中国卒中杂志, 2026, 21(5): 612-623.

PENG Yan, FENG Wenzhan, LIU Qin, TANG Hongmei, LIU Yi, GAO Li, WANG Sijia, BAI Xue. Mechanistic Study on Rat miR-664-1-5p Targeting the PTEN/Akt/Nrf2 Pathway to Attenuate Oxidative Damage in PC12 Cells Induced by Hypoxia/Reoxygenation[J]. Chinese Journal of Stroke, 2026, 21(5): 612-623.

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https://www.chinastroke.org.cn/CN/Y2026/V21/I5/612

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大鼠miR-664-1-5p靶向PTEN/Akt/Nrf2通路减轻缺氧/复氧PC12细胞氧化损伤的机制研究

Mechanistic Study on Rat miR-664-1-5p Targeting the PTEN/Akt/Nrf2 Pathway to Attenuate Oxidative Damage in PC12 Cells Induced by Hypoxia/Reoxygenation

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