Yu Yuanfang, Li Pan, Chen Mengyuan, Zhan Wenfeng, Zhu Ting, Min Ling, Liu Hao, Lv Bo
Guangdong Cardiovascular Institute, Guangdong Provincial People's Hospital, Guangdong Academy of Medical Sciences, Guangzhou, China.
Department of General Practice, Guangdong Geriatrics Institute, Guangdong Provincial People's Hospital (Guangdong Academy of Medical Sciences), Southern Medical University, Guangzhou, China.
Front Neurol. 2024 May 10;15:1395833. doi: 10.3389/fneur.2024.1395833. eCollection 2024.
Ischemic stroke (IS) is a neurological disease with significant disability and mortality. MicroRNAs were proven to be associated with cerebral ischemia. Previous studies have demonstrated miR-122 downregulation in both animal models of IS and the blood of IS patients. Nonetheless, the role and mechanism of miR-122-5p in IS remain unclear.
We established primary human and mouse astrocytes, along with HT22 mouse hippocampal neuronal cells, through oxygen-glucose deprivation/reoxygenation (OGD/R) treatment. To assess the impact of miR-122, we employed CCK8 assays, flow cytometry, RT-qPCR, western blotting, and ELISA to evaluate cell viability, apoptosis, reactive oxygen species (ROS) generation, and cytokine expression. A dual-luciferase reporter gene assay was employed to investigate the interaction between miR-122 and sPLA2-IIA.
Overexpression of miR-122 resulted in decreased apoptosis, reduced cleaved caspase-3 expression, and increased cell viability in astrocytes and HT22 cells subjected to OGD/R. RT-qPCR and ELISA analyses demonstrated a decrease in mRNA and cytokine levels of interleukin (IL)-6 and tumor necrosis factor (TNF)-α in both astrocytes and HT22 cells following miR-122 overexpression. Moreover, miR-122 overexpression reversed OGD/R-induced ROS levels and 8-OHdG formation in astrocytes. Additionally, miR-122 overexpression decreased the mRNA and protein expression of inducible nitric oxide synthase (iNOS). Furthermore, we found that miR-122 attaches to the 3'-UTR of sPLA2-IIA, thereby downregulate its expression.
Our study demonstrates that miR-122-mediated inhibition of sPLA2-IIA attenuates OGD/R-induced neuronal injury by suppressing apoptosis, alleviating post-ischemic inflammation, and reducing ROS production. Thus, the miR-122/sPLA2-IIA axis may represent a promising target for IS treatment.
缺血性中风（IS）是一种具有显著致残率和死亡率的神经疾病。微小RNA已被证明与脑缺血有关。先前的研究表明，在IS动物模型和IS患者血液中均存在miR-122下调。然而，miR-122-5p在IS中的作用和机制仍不清楚。
我们通过氧糖剥夺/复氧（OGD/R）处理建立了原代人星形胶质细胞、小鼠星形胶质细胞以及HT22小鼠海马神经元细胞。为了评估miR-122的影响，我们采用CCK8检测、流式细胞术、RT-qPCR、蛋白质印迹法和酶联免疫吸附测定（ELISA）来评估细胞活力、凋亡、活性氧（ROS）生成和细胞因子表达。采用双荧光素酶报告基因检测来研究miR-122与分泌型磷脂酶A2-IIA（sPLA2-IIA）之间的相互作用。
在经历OGD/R的星形胶质细胞和HT22细胞中，miR-122过表达导致凋亡减少、裂解的半胱天冬酶-3表达降低以及细胞活力增加。RT-qPCR和ELISA分析表明，miR-122过表达后，星形胶质细胞和HT22细胞中白细胞介素（IL）-6和肿瘤坏死因子（TNF）-α的mRNA和细胞因子水平均降低。此外，miR-122过表达逆转了OGD/R诱导的星形胶质细胞中的ROS水平和8-羟基脱氧鸟苷（8-OHdG）形成。另外，miR-122过表达降低了诱导型一氧化氮合酶（iNOS）的mRNA和蛋白质表达。此外，我们发现miR-122附着于sPLA2-IIA的3'-非翻译区（3'-UTR），从而下调其表达。
我们的研究表明，miR-122介导的对sPLA2-IIA的抑制通过抑制凋亡、减轻缺血后炎症和减少ROS产生来减轻OGD/R诱导的神经元损伤。因此，miR-122/sPLA2-IIA轴可能是IS治疗的一个有前景的靶点。
