Drp1在星形胶质细胞A1型活化中的作用  

Role of Drp1 in A1 activation of astrocytes

作　　者：周龙云 陈旭青[2] 方露 姚敏[3] 刘书芬[3] ZHOU Longyun;CHEN Xuqing;FANG Lu;YAO Min;LIU Shufen(The First Affiliated Hospital of Nanjing Medical University,Nanjing 210029,China;Affiliated Hospital of Nanjing University of Chinese Medicine,Nanjing 210029,China;Longhua Hospital Affiliated to Shanghai University of Traditional Chinese Medicine,Shanghai 200032,China)

机构地区：[1]南京医科大学第一附属医院,江苏南京210029 [2]南京中医药大学附属医院,江苏南京210029 [3]上海中医药大学附属龙华医院,上海200032

出　　处：《中国病理生理杂志》2025年第1期64-71,共8页Chinese Journal of Pathophysiology

基　　金：国家自然科学青年科学基金项目(No.82205148);国家自然科学基金面上项目(No.82374476;No.82374527)。

摘　　要：目的:探讨发动蛋白相关蛋白1(dynamin-related protein 1,Drp1)在星形胶质细胞A1型活化中的作用,揭示星形胶质细胞异常活化的内在机制。方法:将大鼠星形胶质细胞CTX-TNA2分为对照组、星形胶质细胞条件培养基(astrocyte-conditioned medium,ACM)组及5、10和25μmol/L线粒体分裂抑制剂1(mitochondrial division inhibitor-1,Mdivi-1;选择性抑制Drp1)+ACM组。其中,ACM组以含白细胞介素1α(interleukin-1α,IL-1α)、肿瘤坏死因子α(tumor necrisos factor-α,TNF-α)及补体1q(complement 1q,C1q)的条件培养基刺激24 h,诱导A1型活化;Mdivi-1+ACM组于对应浓度Mdivi-1预处理2 h,而后以条件培养基刺激24 h。以RT-qPCR检测各组干预后细胞A1型活化相关指标IL-1β、TNF-α和IL-10的mRNA表达;以细胞免疫荧光检测各组细胞A1型活化标志性分子补体C3、诱导型一氧化氮合成酶(inducible nitric oxide synthase,iNOS)、S100钙结合蛋白A10(S100 calcium binding protein A10,S100A10)表达;采用MitoSOX Red荧光探针和流式细胞术检测各组干预后细胞线粒体活性氧(reactive oxygen species,ROS)水平;以MICA全场景显微成像分析平台观察各组细胞线粒体形态;结合免疫印迹分析,测定各组细胞线粒体分裂蛋白1(mitochondrial fission protein 1,FIS1)表达及Drp1活化水平。结果:RT-qPCR及免疫荧光结果示,与对照组比较,ACM组细胞IL-1β、TNF-αmRNA水平及C3、iNOS蛋白表达显著升高,而IL-10 mRNA水平及S100A10蛋白表达则有所下降,差异具有统计学意义(P<0.05);10和25μmol/L Mdivi-1干预则能有效抑制ACM诱导的IL-1β、TNF-αmRNA水平及C3、iNOS蛋白表达的升高,且能一定程度上回升S100A10蛋白的表达。MitoSOX Red染色流式定量分析显示,ACM刺激下,星形胶质细胞线粒体ROS水平显著提升;而Mdivi-1干预则能有效逆转这一病理性改变。MICA全场景显微成像分析平台显示,ACM刺激可诱导星形胶质细胞胞内大量圆球状线粒体生成,而10和25μmol/L Mdivi-1干AIM:This study aims to investigate the role of dynamin-related protein 1(Drp1)in the A1 type activation of astrocytes and elucidate the underlying mechanism of abnormal astrocyte activation.METHODS:Rat astrocyte line CTX-TNA2 was divided into control group,astrocyte-conditioned medium(ACM)group,and 5,10 and 25μmol/L mitochondrial division inhibitor-1(Mdivi-1;a selective inhibitor of Drp1)+ACM group.The cells in ACM group were exposed to ACM containing interleukin-1α(IL-1α),tumor necrosis factor-α(TNF-α)and complement 1q(C1q)for 24 h to induce type A1 activation,while those in Mdivi-1+ACM group were pre-treated with various concentrations of Mdivi-1 for 2 h before stimulation with ACM for 24 h.RT-qPCR was used to detect the expression levels of A1 type activationrelated indicators IL-1β,TNF-αand IL-10 mRNA in each group.Immunofluorescence was utilized to assess the expression levels of A1 type activation marker molecules C3,inducible nitric oxide synthase(iNOS)and S100 calcium binding protein A10(S100A10).The level of mitochondrial reactive oxygen species(ROS)was measured using MitoSOX Red staining and flow cytometry analysis.The mitochondrial morphology was observed using the MICA full-field imaging analysis platform.Lastly,the expression level of mitochondrial fission protein 1(FIS1)and the activation level of Drp1 in each group were evaluated through immunoblotting analysis.RESULTS:The RT-qPCR and immunofluorescence results indicated that the ACM group exhibited significantly elevated levels of IL-1βand TNF-αmRNA,and C3 protein expression compared with control group,along with increased iNOS protein expression and reduced IL-10 mRNA and S100A10 protein expression(P<0.05).Interventions with 10 and 25μmol/L Mdivi-1 effectively inhibited the rise in IL-1βand TNF-αmRNA,and C3 and iNOS protein expression induced by ACM,while promoting S100A10 expression.MitoSOX Red staining revealed a significant increase in mitochondrial ROS levels in astrocytes stimulated by ACM,which was effectively reversed by Mdivi-

关 键 词：发动蛋白相关蛋白1 线粒体分裂 星形胶质细胞 A1型活化 

分 类 号：R741.02[医药卫生—神经病学与精神病学] Q421[医药卫生—临床医学] R363[生物学—神经生物学]