ATP6V1A缺失导致脓毒症心脏驻留巨噬细胞清除能力损伤  

ATP6V1A deletion leads to impaired clearance of septic cardiac resident macrophages

作　　者：杨顺心 周远群 向鑫明 刘良明 李涛 胡弋 YANG Shunxin;ZHOU Yuanqun;XIANG Xinming;LIU Liangming;LI Tao;HU Yi(Department of Anesthesiology,Army Medical Center of PLA,Chongqing,400042;State Key Laboratory of Trama and Chemical Poisoning,Department of War Wound Shock and Transfusion,Institute of Surgery Research,Chongqing,400042)

机构地区：[1]陆军特色医学中心麻醉科,重庆400042 [2]野战外科研究部战伤休克与输血研究室创伤与化学中毒全国重点实验室,重庆400042

出　　处：《陆军军医大学学报》2024年第23期2599-2607,共9页Journal of Army Medical University

基　　金：国家自然科学基金面上项目(82270523)。

摘　　要：目的 探讨脓毒症后心脏驻留巨噬细胞的清除能力及其机制。方法 利用盲肠穿孔结扎术建立脓毒症小鼠模型,将30只雄性C57BL/6小鼠(8周龄,体质量20~25 g)按随机数字抽样法分为假手术组(Sham组,n=15)和脓毒症模型组(Sepsis组,n=15)。使用免疫荧光技术标记心肌细胞与巨噬细胞观察小鼠心肌细胞凋亡及心脏驻留巨噬细胞对心肌细胞的吞噬情况。提取心脏驻留巨噬细胞进行转录组学测序,观察脓毒症后心脏驻留巨噬细胞的功能变化。在细胞水平建立心脏驻留巨噬细胞系,即正常组(RAC组);用LPS处理RAC组细胞后,即脓毒症组(RAC+LPS组)。观察2组间对凋亡心肌细胞清除能力的差异;使用DQ-BSA-RED溶酶体活性检测探针、Lyso-Sensor yellow/bule染料、ELISA以及蛋白质印迹实验法检测心脏驻留巨噬细胞溶酶体功能、溶酶体重要水解酶活性及蛋白表达、囊泡型腺苷三磷酸酶(vacuolar-type adenosine triphosphatases,V-ATPase)含量及其相关亚基的改变。结果 与Sham组相比,Sepsis组小鼠心肌细胞凋亡数量增加(P<0.05),心脏巨噬细胞对心肌细胞的吞噬增加(P<0.05),转录组学结果分析发现脓毒症后心脏驻留巨噬细胞的溶酶体相关功能显著失调。体外实验结果显示,与RAC组相比,RAC+LPS组对凋亡心肌细胞的碎片化能力降低,溶酶体的黄色荧光强度明显减弱(P<0.05),溶酶体水解酶活性降低(P<0.05)。此外,LPS处理显著降低了心脏驻留巨噬细胞V-ATPase及其主要亚基ATP6V1A的活性和表达(P<0.05)。结论 脓毒症后心脏驻留巨噬细胞对凋亡心肌细胞的清除能力降低,其机制可能与其溶酶体V-ATPase重要亚基ATP6V1A和溶酶体水解酶活性降低有关。Objective To investigate the clearance capacity of cardiac resident macrophages in post-sepsis and its underlying mechanism.Methods A mouse model of sepsis was established using cecum perforation ligation.Thirty male C57BL/6 mice(8 weeks old,weighing 20~25 g) were randomly and equally divided into a sham operation group(sham group) and a model group(sepsis group).Immunofluorescence assay was employed to label the cardiomyocytes and macrophages to observe the apoptosis of cardiomyocytes and the phagocytosis by cardiac resident macrophages.Cardiac resident macrophages were extracted for transcriptomic sequencing to determine the functional changes of the cells after sepsis.Cardiac resident macrophage cell lines were established at the cellular level and served as the normal group(RAC group),and the RAC cells treated with LPS were subjected as the sepsis group(RAC+LPS group).Then the differences in the ability to clear apoptotic cardiomyocytes between the 2 groups were observed.Then DQ-BSA-RED lysosomal activity detection probe,Lyso-Sensor yellow/bule dye,ELISA,and Western blotting were applied to detect the lysosomal function of cardiac resident macrophages,activity and expression of important lysosomal hydrolases,changes in contents and related subunits of vacuolar-type adenosine triphosphatases(V-ATPase).Results Compared with the sham group,the sepsis group had larger number of apoptotic cardiomyocytes(P<0.05) and increased phagocytosis of cardiomyocytes by cardiac macrophages(P<0.05).The results of transcriptomic sequencing revealed a significant dysfunction of lysosome-associated functions of cardiac-resident macrophages after sepsis.In in vitro experiments,the RAC+LPS group had a reduced fragmentation capacity of apoptotic cardiomyocytes,reduction in the intensity of yellow fluorescence of lysosomes(P<0.05),and decrease in lysosomal hydrolase activity(P<0.05) when compared with the RAC group.In addition,LPS treatment significantly decreased the activity and expression of V-ATPase and its major subunit ATP6V1A

关 键 词：脓毒症心脏功能障碍 心脏驻留巨噬细胞 溶酶体 囊泡型腺苷三磷酸酶 

分 类 号：R322.11[医药卫生—人体解剖和组织胚胎学] R345[医药卫生—基础医学] R631.1