脓毒症老年小鼠树突状细胞高尔基体应激和自噬水平变化及其对免疫功能的影响  

Alterations in Golgi stress response and autophagy of dendritic cells in elderly mice and its impact on sepsis-induced immunosuppression

作　　者：樊琪 董宁[2] 吴瑶 童森 贺鹏翼 祝筱梅[2] 姚咏明[1,2] Fan Qi;Dong Ning;Wu Yao;Tong Sen;He Pengyi;Zhu Xiaomei;Yao Yongming(Emergency Medicine Center,the First Affiliated Hospital of Wenzhou Medical University,Wenzhou 325015,China)

机构地区：[1]温州医科大学附属第一医院急诊医学中心,浙江温州325015 [2]解放军总医院医学创新研究部转化医学研究中心第四医学中心,北京100853

出　　处：《中国急救医学》2023年第11期868-874,共7页Chinese Journal of Critical Care Medicine

基　　金：国家自然科学基金项目(82272200,82241062);国家老年疾病临床医学研究中心开放课题(NCRCG-PLAH-2022005)。

摘　　要：目的 探究脓毒症状态下老年小鼠脾脏树突状细胞(dendritic cell,DC)功能状态的异常改变及其与高尔基体应激反应和自噬反应的内在联系。方法 不同年龄小鼠(青年鼠8周龄,老年鼠18月龄)分别随机分为假手术组与脓毒症模型组(CLP组)(n=4)。CLP组小鼠于术后24 h处死并取脾脏组织。采用免疫磁珠法分离纯化脾脏DC,流式细胞术检测各组DC表面标志物及共刺激分子(CD80、CD86、MHC-Ⅱ)表达水平。分离纯化不同年龄小鼠脾脏DC,各自分为空白对照组、LPS刺激组(1μg/mL)(n=4)。免疫印迹法检测各组细胞高尔基体应激反应相关蛋白——高尔基体磷蛋白3(Golgi phosphoprotein 3,GOLPH3)、高尔基体重组堆积蛋白2(Golgi reassembly stacking protein of 55 kDa,GRASP55)以及高尔基体自噬相关蛋白高尔基体蛋白亚家族A成员2(Golgi matrix protein,GM130)、微管相关蛋白1A和1B(microtubule-associated proteins 1A and 1B,MAP1LC3B)表达水平。激光共聚焦显微镜检测高尔基体红色荧光探针(Golgi-tracker Red)在各空白对照组中的显示情况。进一步应用抑制高尔基体蛋白转运的莫能菌素(monensin,MON)(1μg/mL)预处理,检测扰乱高尔基体稳态对小鼠DC功能分化的影响。结果 与青年小鼠比较,老年脓毒症模型小鼠脾脏DC活化障碍显著。CLP术后24 h老年小鼠脾脏DC表面标志分子CD80、CD86、MHC-Ⅱ表达升高水平明显低于青年小鼠(P<0.05)。对比健康小鼠脾脏DC胞内高尔基体的结构功能状态发现,与青年小鼠比较,老年DC胞内高尔基体结构蛋白GRASP55、GM130呈高表达(P<0.001),提示高尔基体结构肿胀;高尔基体外膜蛋白GOLPH3表达减弱(P<0.001),提示蛋白修饰及转运功能低下。分离提取不同年龄小鼠脾脏DC进行体外培养并给予LPS刺激,发现脓毒症老年小鼠DC胞内高尔基体应激反应增强、自噬明显,与DC功能障碍密切相关。给予MON抑制高尔基体蛋白转运,观察到青年、老年小鼠DCObjective To investigate the abnormal alterations of the functional status of splenic dendritic cells(DCs)in elderly mice in sepsis and its intrinsic connection with Golgi stress response and autophagy.Methods Mice of different ages(8 weeks old for young mice and 18 months old for elderly mice)were randomly divided into sham group and sepsis model group(CLP group)(n=4).Mice in the CLP group were executed 24 h after the operation and the splenic tissues were collected.Splenic DCs were isolated and the purified by immunomagnetic bead method,and the expressions of DC surface markers as well as co-stimulatory molecules including CD80,CD86,and MHC-II were determined by flow cytometry in each group.Splenic DCs from mice of different ages were isolated and purified,and each group was further divided into a blank control group and a lipopolysaccharide(LPS)-stimulated group(1μg/mL)(n=4).Western blotting was used to measure expression levels of Golgi stress-related protein—Golgi phosphoprotein 3(GOLPH3),Golgi reassembly stacking protein of 55 kDa(GRASP55)as well as Golgi autophagy-associated proteins Golgi matrix protein 130(GM130)as the member of Golgi protein subfamily A2,and microtubule-associated proteins 1A and 1B light chain 3B(MAP1LC3B).Laser confocal microscopy was used to detect the display of Golgi red fluorescent probe in each blank control group.Moreover,pretreatment with monensin(MON)(1μg/mL),which inhibited Golgi protein transport,was applied to assess the effect of disturbing Golgi homeostasis on the functional differentiation of murine DCs.Results The activation of splenic DCs was significantly impaired in the elderly septic mice compared with the young mice.24 h after CLP surgery,increased levels of splenic DC surface marker molecules(CD80,CD86,and MHC-II)were significantly lower in the elderly group compared with the young group(P<0.05).The structural-functional status of the intracellular Golgi in splenic DCs of healthy mice revealed that intracellular Golgi structural proteins GRASP55 and GM130 were

关 键 词：老年 脓毒症 免疫抑制 树突状细胞(DC) 高尔基体自噬 高尔基体应激 内毒素 莫能菌素 

分 类 号：R459.7[医药卫生—急诊医学]