热打击单核细胞外泌体高迁移率族蛋白B1对内皮细胞炎症的作用  

Effect of high mobility group protein B1 in exosomes from monocytes stimulated by heat stress

作　　者：张明[1] 杨经文[1] 李悦 施学智 李华楚 童华生 Zhang Ming;Yang Jing-Wen;Li Yue;Shi Xue-Zhi;Li Hua-Chu;Tong Hua-Sheng(Department of Critical Care Medicine,the Sixth Affiliated Hospital of Guangzhou Medical University,Qingyuan People's Hospital,Qingyuan,Guangdong 511500,China;Department of Intensive Care Unit,General Hospital of Southern Theatre Command of PLA/Key Laboratory of Trauma and Tissue Repair of Tropical Area of PLA,Guangzhou,Guangdong 510010,China)

机构地区：[1]广州医科大学附属第六医院/清远市人民医院重症医学科一区,广东清远511500 [2]南部战区总医院重症医学科/解放军热区损伤与组织修复重点实验室,广东广州510010

出　　处：《解放军医学杂志》2023年第6期670-675,共6页Medical Journal of Chinese People's Liberation Army

基　　金：广东省医学科研基金(A2021404);广东省中医药局科研项目(20212292);广东省清远市科技计划项目(200715164560747)。

摘　　要：目的观察热打击单核细胞外泌体高迁移率族蛋白B1(HMGB1)对内皮细胞炎症的作用。方法制备中暑大鼠模型,将大鼠分为对照组(n=6)与中暑组(n=6),分离单核细胞提取外泌体并进行鉴定;对外泌体蛋白进行质谱分析并采用注释数据库进行功能描述;采用Western blotting及酶联免疫吸附法(ELISA)分别验证中暑大鼠及患者单核细胞来源外泌体HMGB1的表达变化;将脐静脉内皮细胞(HUVECs)分为对照组、中暑组以及中暑+丙酮酸乙酯(ethyl pyruvate,EP)组,分别添加来自对照组、中暑组大鼠外周血单核细胞上清中提取的外泌体及EP,采用RT-qPCR及Western blotting检测内皮细胞NOD样受体热蛋白结构域相关蛋白3(NLRP3)及白细胞介素-1β(IL-1β)的表达水平;采用ELISA检测健康志愿者及中暑患者外泌体NLRP3及IL-1β的表达水平。结果与对照组比较,中暑组大鼠单核细胞来源外泌体数量(×104/单核细胞)明显增多(11.24±2.66 vs.1.52±0.21,P<0.001)。蛋白质组学分析表明中暑组外泌体HMGB1表达较对照组上调18.63倍(P<0.001),且在NOD样受体等信号通路富集。Westernblotting检测结果显示,中暑组大鼠外泌体内含HMGB1表达量较对照组明显升高(4.13±0.22 vs.1.00±0.15,P<0.001)。ELISA检测结果显示,中暑组患者外泌体内含HMGB1水平(ng/ml)较对照组明显升高(0.29±0.11 vs.0.12±0.04,P=0.006)。RT-qPCR及Western blotting检测结果显示,与对照组比较,中暑组内皮细胞中NLRP3及IL-1β的mRNA和蛋白表达水平均明显升高,与中暑组比较,中暑+EP组NLRP3及IL-1β的mRNA和蛋白表达水平则明显降低,差异有统计学意义(P<0.05)。结论热打击可提高单核细胞来源外泌体HMGB1水平,并可能通过HMGB1介导内皮细胞的炎症反应。Objective To profile the high mobility group protein B1(HMGB1)in exosomes derived from heat-stressed monocytes and its effect on endothelial cell inflammation.Methods The heatstroke model was established.The rats were divided into control group(n=6)and heatstroke group(n=6).Monocytes from blood were isolated and exosomes were extracted.Isobaric tags for relative and absolute quantification technology were used to identify the exosomal protein components,such as HMGB1,before functional description with annotation databases.Then,Western blotting and enzyme-linked immunosorbent assay(ELISA)were used to verify the changes of monocyte-derived exosomes HMGB1 in rats and patients.Umbilical vein endothelial cells(HUVECs)were treated with exosomes from rats in control group,heatstroke group and heatstorke+ethyl pyruvate group.The expression levels of NOD-like receptor thermal protein domain associated protein 3(NLRP3)and interleukin-1β(IL-1β)were detected with RT-qPCR and Western blotting.HMGB1,NLRP3 and IL-1βexpresion levels in exosomes of patient and healthy donors,were detected with ELISA.Results In heatstroke group,the number of exosomes(×104/monocyte)derived from monocytes significantly increased than that in control group(11.24±2.66 vs.1.52±0.21,P<0.001).Proteomic analysis showed that the expression of exosome HMGB1 in heatstroke group was up regulated than control group(18.63 times,P<0.001),and these proteins were enriched in NOD-like receptors and other signaling pathways.Western blotting showed that the relative expression of HMGB1 in exosomes of rats in heatstroke group was significantly higher than that in control group(4.13±0.22 vs.1.00±0.15,P<0.001).ELISA also demonstrated that the level of HMGB1 in exosomes of patients in heatstroke group increased than that in control group[(0.29±0.11)ng/ml vs.(0.12±0.04)ng/ml,P=0.006].qPCR and Western blotting showed that compared with control group,the mRNA and protein levels of NLRP3 and IL-1βin endothelial cells of heatstroke group were increased,which could

关 键 词：中暑 外泌体 单核细胞 高迁移率族蛋白B1 

分 类 号：R594.13[医药卫生—内科学]