作者：袁莉，向军军，李丽琴，陈炜，胡跃强，姚春

单位：

1.广西中医药大学2.广西中医药大学第一附属医院脑病一区

单位（英文）：

1.Guangxi University of Traditional Chinese Medicine, Nanning 530012, China 2.Encephalopathy Department, the First Affiliated Hospital of Guangxi University of Traditional Chinese Medicine, Nanning 530020, China

关键词：

再灌注损伤; 缺血/再灌注损伤; 大脑; miR-335; F-box和富含亮氨酸的重复蛋白5; 核受体辅激活蛋白4;

关键词（英文）：

Reperfusion injury; Ischemia/reperfusion injury; Cerebrum; miR-335; F-box and leucine-rich repeat protein 5; Nuclear receptor coactivator 4

中图分类号：

DOI：

10.12114/j.issn.1008-5971.2023.00.097

基金项目：

国家自然科学基金资助项目（81973768）； 广西自然科学基金重点项目（2019GXNSFDA245006）； 广西中医脑病临床研究中心项目（桂科AD20238028）； 广西研究生教育创新计划项目（YCSZ2022015）；

摘要：

目的 探究miR-335通过调控铁死亡对大鼠脑缺血/再灌注损伤的保护作用及机制。方法 本实验时间为2022年3—9月。将180只成年雄性Sprague Dawley大鼠随机分为假手术组、模型组、miR-335模拟物组、miR-335模拟物对照组、miR-335抑制物组、miR-335抑制物对照组，每组30只。假手术组大鼠仅暴露右侧大脑中动脉并缝合。模型组大鼠制备大脑中动脉闭塞/再灌注（MCAO/R）损伤模型。miR-335模拟物组大鼠在大脑中动脉脑立体定位注射miR-335模拟物腺相关病毒2μl,7 d后制备MCAO/R损伤模型。miR-335模拟物对照组大鼠在大脑中动脉脑立体定位注射miR-335模拟物空载体，7 d后制备MCAO/R损伤模型。miR-335抑制物组大鼠在大脑中动脉脑立体定位注射miR-335抑制物腺相关病毒2μl,7 d后制备MCAO/R损伤模型。miR-335抑制物对照组大鼠在大脑中动脉脑立体定位注射miR-335抑制物空载体，7 d后制备MCAO/R损伤模型。模型制备后12、24、72 h，分别在各组随机选取10只大鼠并将其处死，取缺血半暗带区脑组织以进行后续实验。采用Zea Longa评分评估各组大鼠神经功能缺损程度，采用qRT-PCR检测各组大鼠脑组织miR-335、F-box和富含亮氨酸的重复蛋白5(FBXL5)mRNA、核受体共激活因子4(NCOA4)mRNA相对表达量，采用Western blot法检测各组大鼠脑组织FBXL5、NCOA4蛋白相对表达量。结果 本研究大鼠均建模成功。假手术组大鼠Zea Longa评分为0分。模型制备后12、24、72 h,miR-335模拟物组大鼠Zea Longa评分低于miR-335模拟物对照组，miR-335抑制物组大鼠Zea Longa评分高于miR-335抑制物对照组（P<0.05）。模型制备后12、24、72 h，模型组大鼠脑组织miR-335相对表达量低于假手术组，miR-335模拟物组大鼠脑组织miR-335相对表达量高于miR-335模拟物对照组，miR-335抑制物组大鼠脑组织miR-335相对表达量低于miR-335抑制物对照组（P<0.05）。模型制备后12、24、72 h，模型组大鼠脑组织FBXL5 mRNA、蛋白相对表达量低于假手术组，miR-335模拟物组大鼠脑组织FBXL5 mRNA、蛋白相对表达量高于miR-335模拟物对照组，miR-335抑制物组大鼠脑组织FBXL5 mRNA、蛋白相对表达量低于miR-335抑制物对照组（P<0.05）；模型制备后12、24、72 h，模型组大鼠脑组织NCOA4 mRNA、蛋白相对表达量高于假手术组，miR-335模拟物组大鼠脑组织NCOA4 mRNA、蛋白相对表达量低于miR-335模拟物对照组，miR-335抑制物组大鼠脑组织NCOA4 mRNA、蛋白相对表达量高于miR-335抑制物对照组（P<0.05）。结论上调miR-335表达可有效减轻MCAO/R损伤模型大鼠神经功能缺损程度，其机制可能与miR-335促进FBXL5表达、抑制NCOA4表达，调控铁自噬与铁代谢，进而影响铁死亡有关。

英文摘要：

Objective To explore the protective effect and mechanism of miR-335 on cerebral ischemia/reperfusion injury in rats by regulating ferroptosis. Methods The experiment was conducted from March to September 2022. One hundred and eighty adult male Sprague Dawley rats were randomly divided into sham operation group, model group, miR-335 mimic group,miR-335 mimic control group, miR-335 inhibitor group and miR-335 inhibitor control group, with 30 rats in each group. In the sham operation group, only the right middle cerebral artery was exposed and sutured. The middle cerebral artery occlusion/ reperfusion (MCAO/R) injury model was prepared in the model group. Rats in the miR-335 mimic group were stereotaxically injected with 2 μl of miR-335 mimic adeno-associated virus in the middle cerebral artery, and MCAO/R injury model was prepared after 7 days. Rats in the miR-335 mimic control group were stereotaxically injected with miR-335 mimic empty vector in the middle cerebral artery, and MCAO/R injury model was prepared after 7 days. Rats in the miR-335 inhibitor group were stereotaxically injected with 2 μl of miR-335 inhibitor adeno-associated virus in the middle cerebral artery, and MCAO/R injury model was prepared after 7 days. Rats in the miR-335 inhibitor control group were stereotaxically injected with miR-335 inhibitor empty vector in the middle cerebral artery, and MCAO/R injury model was prepared after 7 days. At 12, 24 and 72 h after model preparation, 10 rats in each group were randomly selected and sacrificed, and the ischemic penumbra brain tissue was taken for subsequent experiments. Zea Longa score was used to evaluate the degree of neurological deficit in each group. The relative expression of miR-335, F-box and leucine-rich repeat protein 5 (FBXL5) and nuclear receptor coactivator 4 (NCOA4) mRNA in brain tissue of each group was detected by qRT-PCR. The relative expression of FBXL5 and NCOA4 protein in brain tissue of each group was detected by Western blot. Results The rats in this study were successfully modeled. The Zea Longa score of the sham operation group was 0. At 12, 24 and 72 h after model preparation, the Zea Longa score of miR-335 mimic group was lower than that of miR-335 mimic control group, and the Zea Longa score of miR-335 inhibitor group was higher than that of miR-335 inhibitor control group (P < 0.05) . At 12, 24 and 72 h after model preparation, the relative expression of miR-335 in brain tissue of model group was lower than that of sham operation group, the relative expression of miR-335 in miR-335 mimic group was higher than that in miR-335 mimic control group, and the relative expression of miR-335 in miR-335 inhibitor group was lower than that in miR-335 inhibitor control group (P < 0.05) . At 12, 24, and 72 h after model preparation, the relative expression of FBXL5 mRNA and protein in brain tissue of model group was lower than that of sham operation group, the relative expression of FBXL5 mRNA and protein in brain tissue of miR-335 mimic group was higher than that of miR-335 mimic control group, and the relative expression of FBXL5 mRNA and protein in brain tissue of miR-335 inhibitor group was lower than that of miR-335 inhibitor control group (P < 0.05) . At 12, 24, and 72 h after model preparation, the relative expression of NCOA4 mRNA and protein in brain tissue of model group was higher than that of sham operation group, the relative expression of NCOA4 mRNA and protein in brain tissue of miR-335 mimic group was lower than that of miR-335 mimic control group, and the relative expression of NCOA4 mRNA and protein in brain tissue of miR-335 inhibitor group was higher than that of miR-335 inhibitor control group (P < 0.05) . Conclusion Up-regulation of miR-335 expression can effectively reduce the degree of neurological deficit in MCAO/R injury model rats. The mechanism may be related to miR-335 promoting FBXL5 expression, inhibiting NCOA4 expression, regulating iron autophagy and iron metabolism, and then affecting ferroptosis.

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