Tu Limin, Cheng Wei, Wang Xudong, Li Zhixin, Li Xing
School of Medicine, Wuhan University of Science and Technology, Wuhan, 430081, Hubei, China.
Department of Neurology, Wuhan Puren Hospital affiliated to Wuhan University of Science and Technology, Wuhan, 430081, Hubei, China.
Transpl Immunol. 2024 Jun;84:102042. doi: 10.1016/j.trim.2024.102042. Epub 2024 Mar 26.
Exogenous neural cell transplantation may be therapeutic for stroke, cerebral ischemic injury. Among other mechanisms, increasing findings indicated circular RNAs (circRNAs) regulate the pathogenesis progression of cerebral ischemia. Mmu_circ_0015034 (circEfnb2) was upregulated in focal cortical infarction established by middle cerebral artery occlusion (MCAO) in mice. Our study was designed to probe the molecular mechanism of circEfnb2 in the oxygen-glucose deprivation/reperfusion (OGD/R)-induced neuronal damage in cerebral ischemia.
We established an in vitro OGD/R cell model. CircEfnb2 and microRNA-202-5p (miR-202-5p) levels were detected using real-time quantitative polymerase chain reaction (RT-qPCR). Lactate dehydrogenase (LDH), malondialdehyde (MDA), and reactive oxygen species (ROS) levels were assessed using specific kits. Tumor necrosis factor-α (TNF-α) and Interleukin-1β (IL-1β) levels were examined using an Enzyme-linked immunosorbent assay (ELISA). Flow cytometry analysis evaluated cell apoptosis. Protein levels of B-cell lymphoma-2 (Bcl-2), Bcl-2 related X protein (Bax), cleaved caspase 3, and Tumor necrosis factor receptor-associated factor 3 (TRAF3) were determined using Western blot assay.
Overall, circEfnb2 was highly expressed whereas miR-202-5p was decreased in OGD/R-treated mouse hippocampal neuronal HT22 cells compared to normal controls (both p > 0.05). From an in vitro functional perspective, circEfnb2 knockdown attenuated an OGD/R-triggered neuronal injury compared to controls (p > 0.05). Mechanically, circEfnb2 acted as a sponge of miR-202-5p; downregulation of miR-202-5p annulled the inhibitory roles of circEfnb2 silencing in an OGD/R-caused neuronal injury model. Our analysis showed that miR-202-5p directly targeted TRAF3 as enhanced TRAF3 abolished the effects of miR-202-5p in the OGD/R-induced neuronal injury. In vivo, lentivirus with a short hairpin (sh)-circEfnb2 inhibited cerebral injury, when injected into cerebral cortex in MCAO mice (p > 0.05).
Our results suggest that circEfnb2 deficiency may decrease OGD/R-induced HT22 cell damage by modulating the miR-202-5p/TRAF3 axis. This explanation may provide a new direction for cerebral infarction potential therapeutic targets.
外源性神经细胞移植可能对中风、脑缺血性损伤具有治疗作用。在诸多机制中，越来越多的研究结果表明环状RNA（circRNAs）可调节脑缺血的发病机制进展。在小鼠大脑中动脉闭塞（MCAO）建立的局灶性皮质梗死模型中，Mmu_circ_0015034（circEfnb2）表达上调。我们的研究旨在探究circEfnb2在氧糖剥夺/再灌注（OGD/R）诱导的脑缺血神经元损伤中的分子机制。
我们建立了体外OGD/R细胞模型。使用实时定量聚合酶链反应（RT-qPCR）检测circEfnb2和微小RNA-202-5p（miR-202-5p）的水平。使用特定试剂盒评估乳酸脱氢酶（LDH）、丙二醛（MDA）和活性氧（ROS）水平。使用酶联免疫吸附测定（ELISA）检测肿瘤坏死因子-α（TNF-α）和白细胞介素-1β（IL-1β）水平。通过流式细胞术分析评估细胞凋亡。使用蛋白质免疫印迹法测定B细胞淋巴瘤-2（Bcl-2）、Bcl-2相关X蛋白（Bax）、裂解的半胱天冬酶3和肿瘤坏死因子受体相关因子3（TRAF3）的蛋白质水平。
总体而言，与正常对照相比，在OGD/R处理的小鼠海马神经元HT22细胞中，circEfnb2高表达而miR-202-5p降低（均p>0.05）。从体外功能角度来看，与对照相比，circEfnb2敲低减轻了OGD/R引发的神经元损伤（p>0.05）。机制上，circEfnb2作为miR-202-5p的海绵；miR-202-5p的下调消除了circEfnb2沉默在OGD/R诱导的神经元损伤模型中的抑制作用。我们的分析表明，miR-202-5p直接靶向TRAF3，因为TRAF3的增强消除了miR-202-5p在OGD/R诱导的神经元损伤中的作用。在体内，将短发夹（sh）-circEfnb2慢病毒注射到MCAO小鼠的大脑皮质中可抑制脑损伤（p>0.05）。
我们的结果表明，circEfnb2缺陷可能通过调节miR-202-5p/TRAF3轴来减少OGD/R诱导的HT22细胞损伤。这一解释可能为脑梗死潜在治疗靶点提供新的方向。
