NOX2作为miR-532-3p的靶点介导其对脑缺血再灌注诱导SH-SY5Y细胞铁死亡的抑制作用

Mediation by NOX2 of the suppressive effect of miR-532-3p on ferroptosis induced by cerebral ischemia-reperfusion in SH-SY5Y cells

摘要

目的 缺血性脑卒中后,神经细胞铁死亡是导致神经功能缺损的关键病理环节。早期干预这一可逆性死亡过程,对于减轻急性期脑损伤、改善长期预后具有重要意义。微小RNA(MicroRNA,miRNA)在脑缺血后表达谱发生显著变化,然而,miR-532-3p在脑缺血再灌注后表达变化的生物学意义尚不明确,其能否通过靶向氧化应激关键酶NADPH氧化酶2(NADPH oxidase 2, NOX2)进而影响铁死亡通路未见报道。本研究基于前期课题组测序数据,从MCAO/R大鼠皮层组织中筛选出变化显著的miR-532-3p及其潜在靶基因NOX2,旨在阐明二者的靶向调控关系,并进一步明确miR-532-3p-NOX2调控轴在缺血性卒中神经细胞铁死亡中的作用。方法 线栓法构建大鼠中动脉闭塞再灌注模型(middle cerebral artery occlusion and reperfusion, MCAO/R),通过行为学评分及全脑2,3,5-氯化三苯基四氮唑(2,3,5-triphenyltetrazolium chloride, TTC)染色指标评定缺血再灌注模型,应用逆转录实时荧光定量聚合酶链式反应(reverse transcription quantitative polymerase chain reaction, RT-qPCR)及蛋白质印迹(Western blot)验证miR-532-3p与NOX2的表达相关性。预测miR-532-3p与NOX2的结合位点并比对相关物种保守性,双荧光素酶报告实验验证二者结合位点特异性。构建SH-SY5Y细胞系的氧糖剥夺/再灌注(oxygen glucose deprivation/reperfusion, OGD/R)模型并加入miR-532-3p模拟物(mimic)及抑制剂(inhibitor)和NOX2小干扰RNA(small interfering RNA, siRNA)验证miR-532-3p对NOX2的负向表达调控及对体外缺血再灌注模型中氧化应激水平、铁死亡标志物、线粒体状态及细胞活力的影响。结果 研究结果显示,在缺血脑组织中,miR-532-3p的表达水平显著降低(P<0.05)。该分子能够特异性识别并结合NOX2 mRNA的编码序列(coding sequence, CDS)区,进而负调控NOX2 mRNA翻译效率。在体外模拟缺血再灌注的OGD/R模型中,过表达miR-532-3p可有效抑制NOX2的表达(P<0.05)。这一调控作用引发了一系列保护性效应:细胞的氧化还原状态得到显著改善(P<0.05),表现为NADPH/NADP⁺比值(P<0.05)与GSH/GSSG比值(P<0.05)升高;同时,铁死亡进程受到抑制,具体为GPX4表达上调(P<0.05),而细胞内Fe²⁺水平及脂质过氧化产物水平下降(P<0.05)。此外,线粒体损伤得到缓解(P<0.05),神经细胞的存活能力也显著提升(P<0.05)。结论 本研究表明,miR-532-3p可能通过直接结合并负向调控NOX2的表达,从而减弱NOX2驱动的氧化应激反应,这一过程或许进一步限制了铁死亡通路的激活。上述发现提示,该调控轴有望成为缺血性脑卒中干预研究的候选方向。

Abstract

Objective After ischemic stroke, ferroptosis of neural cells is a key pathological process leading to neurological impairment. Early intervention in this reversible death process is of great significance for reducing acute-phose brain injury and improving long-term prognosis. The expression profile of microRNAs (miRNAs) changes significantly after cerebral ischemia, but the biological significance of the expression changes of miR-532-3p after cerebral ischemia-reperfusion remains unclear. Whether miR-532-3p can affect the ferroptosis pathway by targeting the key enzyme of oxidative stress, NADPH oxidase 2 (NOX2), has not been reported. Based on the research groups previous sequencing data, this study identified the significantly changed miR-532-3p and its potential target gene NOX2 from the cortical tissues of MCAO/R rats, aimming to clarify the targeted regulatory relationship between them and further determine the role of the miR-532-3p-NOX2 regulatory axis in iron death of neural cells in ischemic stroke. Methods A rat model of middle cerebral artery occlusion and Reperfusion (MCAO/R) was established by the suture method. The ischemia-reperfusion model was evaluated by behavioral scoring and whole brain 2,3,5-triphenyltetrazolium chloride (TTC) staining indicators. The correlation between miR-532-3p and NOX2 expression was verified by Reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western Blot. The binding sites of miR-532-3p and NOX2 were predicted and the conservation of related species was compared. The specificity of the binding sites was verified by dual-luciferase reporter experiments. The Oxygen Glucose Deprivation/Reperfusion (OGD/R) model of SH-SY5Y cell line was constructed and miR-532-3p mimic and inhibitor and NOX2 small interfering RNA (siRNA) were added to verify the negative expression regulation of miR-532-3p on NOX2 and its effects on oxidative stress levels, ferroptosis markers, mitochondrial status and cell viability in the in vitro ischemia-reperfusion model. Results The results showed that the expression level of miR-532-3p was significantly decreased in ischemic brain tissue (P<0.05). This molecule could specifically recognize and bind to the coding sequence (CDS) region of NOX2 mRNA, thereby inhibiting its expression. In the OGD/R model simulating ischemia-reperfusion in vitro, overexpression of miR-532-3p effectively inhibited the expression of NOX2 (P<0.05). This regulatory effect triggered a series of protective effects: the redox state of cells was significantly improved (P<0.05), manifested as an increase in the NADPH/NADP⁺ ratio (P<0.05) and the GSH/GSSG ratio (P<0.05); at the same time, the ferroptosis process was inhibited, specifically, the expression of GPX4 was upregulated (P<0.05), while the intracellular Fe²⁺ level and lipid peroxidation product levels decreased (P<0.05). In addition, mitochondrial damage was alleviated (P<0.05), and the survival ability of neural cells was significantly enhanced (P<0.05). Conclusion This study indicates that miR-532-3p may directly bind to and negatively regulate the expression of NOX2, thereby weakening the oxidative stress response driven by NOX2. This process may further limit the activation of the ferroptosis pathway. These findings suggest that this regulatory axis is expected to be a candidate direction for intervention studies in ischemic stroke.

关键词

miR-532-3p / NOX2 / MCAO/R / OGD/R / 铁死亡

Key words

miR-532-3p / NOX2 / MCAO/R / OGD/R / ferroptosis

[1] Feigin V L, Brainin M, Norrving B, et al. World stroke organization: Global stroke fact sheet 2025[J]. Int J Stroke, 2025, 20(2): 132-144.
[2] Fei Y, Leng Q. Research progress of ferroptosis in cerebral infarction[J]. Brain Behav, 2026, 16(1): e71192.
[3] Chalet L, Boutelier T, Christen T, et al. Clinical imaging of the penumbra in ischemic stroke: From the concept to the era of mechanical thrombectomy[J]. Front Cardiovasc Med, 2022(9): 861913.
[4] Wassélius J, Arnberg F, Von Euler M, et al. Endovascular thrombectomy for acute ischemic stroke[J]. J Intern Med, 2022, 291(3): 303-316.
[5] Hou L, Li X, Su C, et al. Current status and prospects of research on ischemia-reperfusion injury and ferroptosis[J]. Front Oncol, 2022(12): 920707.
[6] Hu X, Bao Y, Li M, et al. The role of ferroptosis and its mechanism in ischemic stroke[J]. Exp Neurol, 2024(372): 114630.
[7] Kamal F Z, Lefter r, Jaber H, et al. The role of potential oxidative biomarkers in the prognosis of acute ischemic stroke and the exploration of antioxidants as possible preventive and treatment options[J]. Int J Mol Sci, 2023, 24(7):6389.
[8] Delgado-martín S, Martínez-ruiz A. The role of ferroptosis as a regulator of oxidative stress in the pathogenesis of ischemic stroke[J]. FEBS Lett, 2024, 598(17): 2160-2173.
[9] Ye Y, Xie X, Bi Y, et al. Nrf2 alleviates acute ischemic stroke induced ferroptosis via regulating xCT/GPX4 pathway[J]. Free Radic Biol Med, 2025(231): 153-162.
[10] Zhou F, Wang Y K, Zhang C G, et al. miR-19a/b-3p promotes inflammation during cerebral ischemia/reperfusion injury via SIRT1/FoxO3/SPHK1 pathway[J]. J Neuroinflammation, 2021, 18(1): 122.
[11] Su W, Lyu M, Wang D, et al. Tanshinone IIA alleviates traumatic brain injury by reducing ischemia-reperfusion via the miR-124-5p/FoxO1 axis[J]. Mediators Inflamm, 2024(24): 7459054.
[12] Wang X, Xu Q, Wang S. Overexpression of miR-149-5p attenuates cerebral Ischemia/Reperfusion (I/R) injury by targeting notch2[J]. Neuromolecular Med, 2022, 24(3): 279-289.
[13] Wang C, Yang Y, Xu M, et al. Deep sequencing of the rat MCAO cortexes reveals crucial circRNAs involved in early stroke events and their regulatory networks[J]. Neural Plast, 2021(20): 9942537.
[14] Sun Y, Wu D, Hu Q. NADP(+)/NADPH in metabolism and its relation to cardiovascular pathologies[J]. Curr Med Chem, 2025, 32(23): 4671-4687.
[15] Qin C, Yang S, Chu Y H, et al. Signaling pathways involved in ischemic stroke: Molecular mechanisms and therapeutic interventions[J]. Signal Transduct Target Ther, 2022, 7(1): 215-223.
[16] Sun Y Y, Zhu H J, Zhao R Y, et al. Remote ischemic conditioning attenuates oxidative stress and inflammation via the Nrf2/HO-1 pathway in MCAO mice[J]. Redox Biol, 2023(66): 102852.
[17] Ni X C, Wang H F, Cai Y Y, et al. Ginsenoside Rb1 inhibits astrocyte activation and promotes transfer of astrocytic mitochondria to neurons against ischemic stroke[J]. Redox Biol, 2022(54): 102363.
[18] Guo J, Tuo Q Z, Lei P. Iron, ferroptosis, and ischemic stroke[J]. J Neurochem, 2023, 165(4): 487-520.
[19] Hilkens N A, Casolla B, Leung T W, et al. Stroke[J]. Lancet, 2024, 403(10446): 2820-2836.
[20] Yuan Y, Zhai Y, Chen J, et al. Kaempferol ameliorates oxygen-glucose deprivation/reoxygenation-induced neuronal ferroptosis by activating Nrf2/SLC7A11/GPX4 axis[J]. Biomolecules, 2021, 11(7):923.
[21] Wang Y, Liu Z, Li L, et al. Anti-ferroptosis exosomes engineered for targeting M2 microglia to improve neurological function in ischemic stroke[J]. J Nanobiotechnology, 2024, 22(1): 291-298.
[22] Wu W, Luo Z, Shen D, et al. IL-10 protects against OPC ferroptosis by regulating lipid reactive oxygen species levels post stroke[J]. Redox Biol, 2024(69): 102982.
[23] Dustin C M, Shiva S S, Vazquez A, et al. NOX2 in Alzheimer's and Parkinson's disease[J]. Redox Biol, 2024(78): 103433.
[24] Wang Q, Liu J, Zhang Y, et al. Microglial CR3 promotes neuron ferroptosis via NOX2-mediated iron deposition in rotenone-induced experimental models of Parkinson's disease[J]. Redox Biol, 2024(77): 103369.
[25] Feng Y, Wang X, Li P, et al. Exogenous hydrogen sulfide and NOX2 inhibition mitigate ferroptosis in pressure-induced retinal ganglion cell damage[J]. Biochim Biophys Acta Mol Basis Dis, 2025, 1871(3): 167705.
[26] Wang Z, Yuan Y, Zhang Z, et al. Inhibition of miRNA-27b enhances neurogenesis via AMPK activation in a mouse ischemic stroke model[J]. FEBS Open Bio, 2019, 9(5): 859-869.
[27] Xue L X, Shu L Y, Wang H M, et al. miR-181b promotes angiogenesis and neurological function recovery after ischemic stroke[J]. Neural Regen Res, 2023, 18(9): 1983-1989.
[28] Zhang Y, Liu Z, Chopp M, et al. Small extracellular vesicles derived from cerebral endothelial cells with elevated microRNA 27a promote ischemic stroke recovery[J]. Neural Regen Res, 2025, 20(1): 224-233.
[29] Zhang K, Zhang X, Cai Z, et al. A novel class of microRNA-recognition elements that function only within open reading frames[J]. Nat Struct Mol Biol, 2018, 25(11): 1019-1027.

基金资助

国家自然科学基金项目(32260142);新疆生产建设兵团区域创新引导计划项目(2021BB002)