GSS抑制线粒体自噬减轻慢性脑缺血大鼠神经元损伤的研究  

GSS attenuating neuronal damage in rats with chronic cerebral ischaemia by inhibiting mitochondrial autophagy

作　　者：范梦瑶 邢亚康 袁志栋 罗维 陈琦晖 莫亦 黄志华 肖海 黎晓 FAN Meng-yao;XING Ya-kang;YUAN Zhi-dong;LUO Wei;CHEN Qi-hui;MO Yi;HUANG Zhi-hua;XIAO Hai;LI Xiao(School of Basic Medicine,Gannan Medical University;Biotechnology Department,School of Basic Medicine,Gannan Medical University;The First Clinical Medical School of Gannan Medical University;Physiology,School of Basic Medicine,Gannan Medical University;Department of Pathology,The First Affiliated Hospital of Gannan Medical University,Ganzhou,Jiangxi 341000)

机构地区：[1]赣南医科大学基础医学院,江西赣州341000 [2]赣南医科大学基础医学院生物技术教研室,江西赣州341000 [3]赣南医科大学第一临床医学院,江西赣州341000 [4]赣南医科大学基础医学院生理学教研室,江西赣州341000 [5]赣南医科大学第一附属医院病理科,江西赣州341000

出　　处：《赣南医科大学学报》2024年第11期1093-1099,共7页JOURNAL OF GANNAN MEDICAL UNIVERSITY

基　　金：国家自然科学基金项目(31760290,82160688);江西省自然科学基金项目(20202BABL206058);赣州市指导性科技计划项目(2022B-SF9554);赣南医学院本科生科技创新项目(NO.10);赣南医学院神经生物研究创新团队项目(TD2021JC06)。

摘　　要：目的:探讨染料木素磺酸钠(Genisteinsodium sulfonate,GSS)对神经元的保护作用及可能机制,为慢性脑缺血的治疗提供潜在靶点。方法:采用改良型双侧颈总动脉结扎法(Modified bilateral common carotid artery occlusion,2-VO)制备慢性脑缺血大鼠模型,大鼠分为Sham组、2-VO组、2-VO+GSS组(1 mg·kg^(-1)GSS),持续8周后取大鼠脑组织,运用iTRAQ方法鉴定各组大鼠脑组织中差异表达的蛋白质,并对这些差异表达蛋白进行GO注释富集分析和MCODE分析;同时以“脑损伤+线粒体自噬”为关键词检索GEO数据库获取数据集进行生物信息富集通路分析;随后运用qPCR和Western blot(WB)等方法检测大鼠脑组织中HIF-1α、CypD和LC3Ⅱ/Ⅰ的mRNA及(或)蛋白表达水平。结果:蛋白质组学技术测定与分析结果提示,GSS可以改善2-VO大鼠脑组织中的线粒体功能,包括改善线粒体ATP的合成、电子传递链、氧化磷酸化和细胞色素复合体、呼吸链复合体和线粒体内膜细胞器生成等;生物信息富集通路分析结果提示,线粒体自噬参与了缺血、衰老和神经元的损伤过程,其中以蛋白质和酶的结合过程最显著。qPCR和WB检测结果显示,GSS显著下调脑组织中线粒体CypD、LC3Ⅱ/Ⅰ的表达水平及HIF-1α的mRNA与蛋白表达水平。结论:GSS对慢性脑缺血后神经元的保护作用可能通过抑制线粒体过度自噬,进而减轻氧化应激损伤实现。Objective:To investigate the protective effects of genisteinsodium sulfonate(GSS)on neurons and the possible mechanisms,and to provide a potential target for the treatment of chronic cerebral ischemia.Methods:A rat model of chronic cerebral ischemia was set up by modified bilateral common carotid artery occlusion(2-VO).The rats were divided into Sham group,2-VO+Vehicle group,and 2-VO+GSS group(1 mg·kg^(-1)GSS).After eight week,the brain tissues were taken from the rats.And the differentially expressed proteins in the brain tissues of rats in each group were identified by iTRAQ,and the differentially expressed proteins were subjected to GO annotation enrichment analysis and MCODE analysis.Meanwhile,the GEO database was searched with the keywords"brain injury"+"mitochondrial autophagy"to obtain the dataset for the bioconcentration pathway analysis.Subsequently,the mRNA and/or protein expression levels of HIF-1α,CypD and LC3Ⅱ/Ⅰin the brain tissues of rats were detected by qPCR and Western blot(WB).Results:The results of proteomic determination and analysis suggested that GSS improved mitochondrial functions in brain tissue of 2-VO rats,including improving mitochondrial ATP synthesis,electron transport chain,oxidative phosphorylation procedure,cytochrome complex,respiratory chain complex,inner mitochondrial membrane organelle generation,etc.The result of the bioconcentration pathway analysis showed that mitochondrial autophagy was involved in the process of ischemia,aging and neuronal damage,among which the binding process of proteins or enzymes was the most significant.The result of qPCR and WB showed that GSS could markedly down-regulate the expression levels of mitochondrial CypD and LC3Ⅱ/Ⅰ,and the mRNA and protein expression levels of HIF-1αin brain tissues.Conclusion:The protective effect of GSS on neurons after chronic cerebral ischemia may be achieved by inhibiting mitochondrial excessive autophagy and thereby attenuating oxidative stress damage.

关 键 词：慢性脑缺血 染料木素磺酸钠 线粒体自噬 缺氧诱导因子-1Α 

分 类 号：R338[医药卫生—人体生理学]