Zhu Xin, Yang Mei, Yang Liu
The First Affiliated Hospital of Dalian Medical University, China.
The First Affiliated Hospital of Dalian Medical University, China.
Neuroscience. 2023 May 1;517:26-36. doi: 10.1016/j.neuroscience.2023.01.018. Epub 2023 Jan 25.
The purpose of the study was to investigate the effect of isoflurane postconditioning on neuron injury in MCAO (middle cerebral artery occlusion) rats and its molecular mechanism of affecting autophagy through miR-384-5p/ATG5 (autophagy-related protein 5). HT22 cells (mouse hippocampal neuronal cell line) were exposed to 1.5% isoflurane for 30 min after OGD/R (oxygen-glucose deprivation/reoxygenation). Flow cytometry and CCK-8 kit were used to analyze changes in apoptosis and cell viability. The level of miR-384-5p was detected by qRT-PCR. Targetscan database prediction combined with dual luciferase reporter gene assay confirmed ATG5 as a target molecule downstream of miR-384-5p. In addition, western blot results confirmed that isoflurane postconditioning regulated miR-384-5p/ATG5 and significantly inhibited the expression of apoptosis-related proteins. Meanwhile, immunofluorescence staining for LC3II positivity combined with western blot results revealed that isoflurane postconditioning significantly inhibited autophagy. In vivo, MCAO induced neuronal injury for 90 min, followed by 24-h reperfusion. Isoflurane postconditioning (Iso) group underwent 1.5% isoflurane postconditioning for 60 min after reperfusion. Neurological scoring and TTC staining were used to evaluate the protective effect of isoflurane post-treatment on neurological injury, respectively. TUNEL staining and western blot results confirmed that isoflurane post-conditioning could regulate miR-384-5p and inhibit apoptosis. Immunofluorescence staining and western blot results confirmed that isoflurane post-conditioning inhibited autophagy in MCAO rats. Based on the above results, we speculated that the molecular mechanism of isoflurane post-conditioning to alleviate ischemic neuronal injury may be related to the regulation of miR-384-5p/ATG5-mediated autophagy.
本研究旨在探讨异氟烷后处理对大脑中动脉闭塞（MCAO）大鼠神经元损伤的影响及其通过miR-384-5p/自噬相关蛋白5（ATG5）影响自噬的分子机制。氧糖剥夺/复氧（OGD/R）后，将HT22细胞（小鼠海马神经元细胞系）暴露于1.5%异氟烷中30分钟。采用流式细胞术和CCK-8试剂盒分析细胞凋亡和细胞活力的变化。通过qRT-PCR检测miR-384-5p的水平。Targetscan数据库预测结合双荧光素酶报告基因检测证实ATG5是miR-384-5p下游的靶分子。此外，蛋白质免疫印迹结果证实异氟烷后处理调节miR-384-5p/ATG5并显著抑制凋亡相关蛋白的表达。同时，LC3II阳性的免疫荧光染色结合蛋白质免疫印迹结果显示异氟烷后处理显著抑制自噬。在体内，MCAO诱导神经元损伤90分钟，随后进行24小时再灌注。异氟烷后处理（Iso）组在再灌注后接受1.5%异氟烷后处理60分钟。分别采用神经功能评分和TTC染色评估异氟烷后处理对神经损伤的保护作用。TUNEL染色和蛋白质免疫印迹结果证实异氟烷后处理可调节miR-384-5p并抑制凋亡。免疫荧光染色和蛋白质免疫印迹结果证实异氟烷后处理抑制MCAO大鼠的自噬。基于上述结果，我们推测异氟烷后处理减轻缺血性神经元损伤的分子机制可能与miR-384-5p/ATG5介导的自噬调节有关。
