Article(id=1203002060628254882, tenantId=1146029695717560320, journalId=1189873630562394117, issueId=1203002056400396334, articleNumber=null, orderNo=null, doi=10.11855/j.issn.0577-7402.2023.06.0670, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1634659200000, receivedDateStr=2021-10-20, revisedDate=null, revisedDateStr=null, acceptedDate=1656950400000, acceptedDateStr=2022-07-05, onlineDate=1764747641951, onlineDateStr=2025-12-03, pubDate=1687881600000, pubDateStr=2023-06-28, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1764747641951, onlineIssueDateStr=2025-12-03, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1764747641951, creator=13701087609, updateTime=1764747641951, updator=13701087609, issue=Issue{id=1203002056400396334, tenantId=1146029695717560320, journalId=1189873630562394117, year='2023', volume='48', issue='6', pageStart='627', pageEnd='748', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=0, createTime=1764747640943, creator=13701087609, updateTime=1764747714497, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1203002364979540735, tenantId=1146029695717560320, journalId=1189873630562394117, issueId=1203002056400396334, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1203002364979540736, tenantId=1146029695717560320, journalId=1189873630562394117, issueId=1203002056400396334, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=670, endPage=675, ext={EN=ArticleExt(id=1203002060938633401, articleId=1203002060628254882, tenantId=1146029695717560320, journalId=1189873630562394117, language=EN, title=Effect of high mobility group protein B1 in exosomes from monocytes stimulated by heat stress, columnId=1190310110212751762, journalTitle=Medical Journal of Chinese People’s Liberation Army, columnName=Basic Research, runingTitle=null, highlight=null, articleAbstract=

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-1β expresion 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-1β in endothelial cells of heatstroke group were increased, which could be alleviated by the inhibitor of HMGB1, and the difference was statistically significant (P<0.05). Conclusion Heat stress increased the HMGB1 level in the monocyte-derived exosomes and might mediate endothelial cell inflammation via HMGB1.

, correspAuthors=Hua-Sheng Tong, authorNote=null, correspAuthorsNote=
* E-mail:
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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。Western blotting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NLRP3IL-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NLRP3IL-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。

, correspAuthors=童华生, authorNote=null, correspAuthorsNote=
童华生,E-mail:
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张明,博士研究生,副主任医师,主要从事中暑和脓毒症方面的临床与基础研究

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张明,博士研究生,副主任医师,主要从事中暑和脓毒症方面的临床与基础研究

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NTA. 纳米粒子跟踪分析仪;A. NTA测定外泌体直径和浓度;B. 根据外泌体来源单核细胞数量,校正并比较对照组和中暑组外泌体个数;C. Western blotting检测外泌体裂解物的表面抗原;D. 扫描电镜验证外泌体典型的双层膜结构;与对照组比较,(1)P<0.05

, figureFileSmall=ro4j67WyLjAdqUaF0/Mg5g==, figureFileBig=d/wQ9wPyPB1gu4foCZxSBQ==, tableContent=null), ArticleFig(id=1203008553310187708, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1203002060628254882, language=EN, label=Fig.2, caption=

Signal pathway of exosomal protein enrichment from monocytes under heat shock of rats

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HMGB1. 高迁移率族蛋白B1;A. Western blotting检测大鼠单核细胞来源外泌体内HMGB1水平(CD63为外泌体特异性蛋白);B. ELISA检测健康对照者及中暑患者单核细胞来源外泌体内HMGB1水平;与对照组比较,(1)P<0.05

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HMGB1. 高迁移率族蛋白B1;NLRP3. NOD样受体热蛋白结构域相关蛋白3;IL-1β. 白细胞介素-1β;EP. 丙酮酸乙酯;A. RT-qPCR检测内皮细胞NLRP3IL-1β的mRNA表达;B. Western blotting检测内皮细胞NLRP3、IL-1β蛋白表达;C. ELISA检测内皮细胞上清中活化的IL-1β水平;与对照组比较,(1)P<0.05;与中暑+EP组比较,(2)P<0.05

, figureFileSmall=F3oDkSiwYHBN0e7Kc+GgpA==, figureFileBig=wxfWuPq6wP0Cl6ER54FGRQ==, tableContent=null), ArticleFig(id=1203008553788338386, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1203002060628254882, language=EN, label=Tab.1, caption=

Characterization of exosomes derived from monocytes of rats

, figureFileSmall=null, figureFileBig=null, tableContent=
基因名称蛋白质名称上调/下调倍数P
Cxcl10C-X-C基序趋化因子1022.510.0001
Hmgb1高迁移率族蛋白B118.630.0001
Csfgm粒细胞集落刺激因子15.230.0001
Cldn1Claudin-112.780.0001
Cxcl6C-X-C基序趋化因子610.560.0001
Il18bp白细胞介素18结合蛋白9.240.0001
Tnfsf2肿瘤坏死因子8.250.0001
Ptx3Pentraxin-related protein 37.050.0001
Cxcl2C-X-C基序趋化因子27.640.0001
Psma7蛋白酶体亚基76.570.0001
Igll1免疫球蛋白λ样多肽10.210.0001
C3补体30.280.0160
Plg血纤维蛋白溶酶原0.290.0280
Saa3血清淀粉样蛋白A30.320.0350
IL-4白细胞介素40.430.0410
), ArticleFig(id=1203008553884807383, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1203002060628254882, language=CN, label=表1, caption=

大鼠单核细胞源性外泌体内含蛋白鉴定

, figureFileSmall=null, figureFileBig=null, tableContent=
基因名称蛋白质名称上调/下调倍数P
Cxcl10C-X-C基序趋化因子1022.510.0001
Hmgb1高迁移率族蛋白B118.630.0001
Csfgm粒细胞集落刺激因子15.230.0001
Cldn1Claudin-112.780.0001
Cxcl6C-X-C基序趋化因子610.560.0001
Il18bp白细胞介素18结合蛋白9.240.0001
Tnfsf2肿瘤坏死因子8.250.0001
Ptx3Pentraxin-related protein 37.050.0001
Cxcl2C-X-C基序趋化因子27.640.0001
Psma7蛋白酶体亚基76.570.0001
Igll1免疫球蛋白λ样多肽10.210.0001
C3补体30.280.0160
Plg血纤维蛋白溶酶原0.290.0280
Saa3血清淀粉样蛋白A30.320.0350
IL-4白细胞介素40.430.0410
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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
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张明 1 , 杨经文 1 , 李悦 2 , 施学智 2 , 李华楚 1 , 童华生 2, *
解放军医学杂志 | 基础研究 2023,48(6): 670-675
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解放军医学杂志 | 基础研究 2023, 48(6): 670-675
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
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张明1, 杨经文1, 李悦2, 施学智2, 李华楚1, 童华生2, *
作者信息
  • 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
  • 张明,博士研究生,副主任医师,主要从事中暑和脓毒症方面的临床与基础研究

通讯作者:

童华生,E-mail:
Effect of high mobility group protein B1 in exosomes from monocytes stimulated by heat stress
Ming Zhang1, Jing-Wen Yang1, Yue Li2, Xue-Zhi Shi2, Hua-Chu Li1, Hua-Sheng Tong2, *
Affiliations
  • 1Department of Critical Care Medicine, the Sixth Affiliated Hospital of Guangzhou Medical University, Qingyuan People's Hospital, Qingyuan, Guangdong 511500, China
  • 2Department 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
出版时间: 2023-06-28 doi: 10.11855/j.issn.0577-7402.2023.06.0670
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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。Western blotting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NLRP3IL-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NLRP3IL-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。

中暑  /  外泌体  /  单核细胞  /  高迁移率族蛋白B1

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-1β expresion 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-1β in endothelial cells of heatstroke group were increased, which could be alleviated by the inhibitor of HMGB1, and the difference was statistically significant (P<0.05). Conclusion Heat stress increased the HMGB1 level in the monocyte-derived exosomes and might mediate endothelial cell inflammation via HMGB1.

heatstroke  /  exosome  /  monocyte  /  high mobility group protein B1
张明, 杨经文, 李悦, 施学智, 李华楚, 童华生. 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. 解放军医学杂志, 2023 , 48 (6) : 670 -675 . DOI: 10.11855/j.issn.0577-7402.2023.06.0670
Ming Zhang, Jing-Wen Yang, Yue Li, Xue-Zhi Shi, Hua-Chu Li, Hua-Sheng Tong. Effect of high mobility group protein B1 in exosomes from monocytes stimulated by heat stress[J]. Medical Journal of Chinese People’s Liberation Army, 2023 , 48 (6) : 670 -675 . DOI: 10.11855/j.issn.0577-7402.2023.06.0670
重症中暑是一种高温高湿环境诱发的疾病,可引起包括中枢神经系统在内的多器官组织损伤[1]。对中暑诱导的器官损伤缺乏治疗手段,患者死亡风险明显增高,是中暑治疗的难题[2-3]。作为一种以炎症损伤为本质的疾病,中暑导致器官损伤的原因难以被简单地解释为热的直接作用,而更可能是炎症传导对靶器官的间接伤害[4]。清楚地认识炎症的传导方式及具体机制,有助于探索控制中暑病情进展的新途径。外泌体是广泛存在的细胞外微囊泡,能够在细胞间传递活性分子,在多种疾病的发生发展中起重要作用[5-6],这些活性分子主要为包括炎性因子在内的功能调节蛋白。越来越多的证据表明,外泌体内含蛋白发生改变可参与炎症传导及靶器官损伤的过程,被认为是潜在的疾病监测及干预靶点[7]。但目前尚不明确外泌体及其内含物是否参与了中暑导致器官损伤的炎症传导过程。本研究分析热打击后单核细胞来源外泌体关键蛋白的变化规律,旨在探讨热打击对炎症的潜在影响。
健康雄性SPF级SD大鼠,体重220~240 g,购自广东医学实验动物中心[粤(2019)05073]。Ficoll密度梯度离心法试剂盒购自Sigma-Aldrich公司,ExoQuick提取试剂盒购自美国SBI公司,NanoDrop 2000超微量分光光度计购自美国Thermo公司,反转录及qPCR试剂盒购自TaKaRa公司,兔来源CD63、β-actin、NLRP3、IL-1β一抗购自美国Abcam公司。
将SD大鼠分为对照组与中暑组,每组6只。中暑大鼠的制模方法参照既往研究,将大鼠逐只转移至温度为(39.5±0.5)℃、湿度为(60±3)%的高温培养箱内,以中心体温超过42 ℃、精神变差且血压从最高点开始下降作为制模成功的标志[8]。对照组大鼠逐只移动到常温常湿的培养箱内,1 h后转移回原笼内。操作流程符合实验动物伦理规范。
造模1 h后,两组大鼠接受麻醉并自腹主动脉取血,置于肝素抗凝采血管中,按照试剂盒操作流程分离单核细胞。吸取单核细胞层,转移至含10%去外泌体血清的培养皿中培养,细胞接种密度约为1×107个/皿。沉淀12 h去除悬浮细胞并更换培养基,获得以贴壁单核细胞为主的细胞群,继续培养12 h并收集上清。
采用ExoQuick提取试剂盒提取外泌体。将大鼠血液中分离的单核细胞培养液通过离心保留上清,按照操作说明低温离心沉淀并吸取外泌体层,重悬至200 μl PBS溶液中。采用纳米粒子跟踪分析仪(nanoparticle tracking analysis,NTA)读取外泌体的直径及浓度,扫描电镜观察外泌体形态及结构。
将两组大鼠来源的外泌体蛋白样本加入裂解液,使用超滤管酶解蛋白,离心后收集滤液,并用超微量分光光度计进行肽段定量。各组样品取100 μg肽段,按照试剂盒操作流程进行稳定同位素标记蛋白质组学技术(isobaric tags for relative and absolute quantitation,iTRAQ)标记,使用Q-Exactive Plus质谱仪进行质谱分析。使用Mascot 2.5及Proteome Discoverer 2.1软件对蛋白进行鉴定及定量。
采用基因本体联合会数据库(Gene Ontology,GO)分析确定特定基因的作用,京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)分析蛋白通路注解。应用Fisher精确检验比较GO分类及通路分布的差异。
以2020年5月1日-11月1日入住南部战区总医院的中暑患者作为研究对象。入组前已向患者及志愿者充分告知研究的内容及意义,研究过程符合医学伦理学规范。以6名健康志愿者作为对照组,中暑患者入组例数达到6例后停止入组。纳入标准:(1)自愿加入研究的中暑患者(在高湿高温环境中活动后,中心体温超过40 ℃且器官功能异常);(2)发病后24 h内可获得全血样本。排除标准:合并慢性炎症、感染、心血管疾病等基础疾病的患者。采血及提取单核细胞外泌体的方法同上,采用酶联免疫吸附法(enzyme linked immunosorbent assay,ELISA)测定人源标本的外泌体HMGB1水平。
采用含10%胎牛血清的DMEM高糖培养基体外培养脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)。将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10 μg外泌体,在37 ℃、5% CO2培养箱中孵育细胞9 h;再分别使用裂解液及RNAiso plus提取试剂处理细胞,提取蛋白及RNA。中暑+EP组在添加相应外泌体之前添加HMGB1抑制剂EP预刺激1 h。
使用RNAiso plus提取试剂提取总RNA,使用反转录及qPCR试剂盒检测NOD样受体热蛋白结构域相关蛋白3(NOD-like receptor thermal protein domain associated protein 3,NLRP3)、白细胞介素-1β(interleukin-1β,IL-1β)的mRNA表达水平,采用2–ΔΔCt方法进行结果相对定量分析。
将收取的大鼠来源外泌体裂解液分别加入上样缓冲液煮沸变性,常规凝胶电泳分离,转移至PVDF膜上。用BSA封闭后在4 ℃条件下孵育一抗,在摇床上使用TBST洗脱未结合的抗体,并用二抗孵育,其中抗体使用兔来源β-actin、NLRP3、IL-1β一抗及山羊来源抗兔IgG二抗。最后使用ECL化学发光试剂显影。
收集外泌体并稀释至相同终末浓度,检测HMGB1、IL-1β蛋白表达水平,操作流程按照说明书进行。
采用SPSS 20.0软件分析数据。使用Kolmogorov-Smirnov检验检测正态性,符合正态分布的计量资料以$\bar{x}±s$表示。三组数据间比较采用单因素方差分析,进一步两两比较采用Bonferroni方法。P<0.05为差异有统计学意义。
从单核细胞上清中收集外泌体,NTA测量结果显示,大部分颗粒直径为70~150 nm,符合外泌体外观特点(图1A);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<0.05,图1B)。采用Western blotting方法验证三组外泌体典型抗原CD63,均显示CD63表达阳性,β-actin表达阴性(图1C);扫描电镜观察显示其具有外泌体典型的双层膜结构(图1D)。
蛋白质质谱分析共鉴定出782种蛋白质,其中中暑组来源蛋白中有包括HMGB1在内的73种较对照组表达上调,45种表达下调(P<0.05)。上调倍数超过2.0或下调倍数超过0.5且表达差异明显的部分蛋白见表1
基于GO数据库的分析显示,外泌体含有的差异蛋白最常见的生物学过程是对刺激的反应、生物学调节过程、多细胞组织过程等。分子功能分析显示,蛋白参与了信号转导、分子功能及分子转导活动等。通过KEGG数据库对蛋白进行功能注释,结果显示,在热打击下单核细胞外泌体内富集的信号通路包括趋化因子信号通路、细胞因子与细胞因子受体的相互作用、NOD样受体信号通路、Toll样受体信号通路、TNF信号通路,富集信号通路主要与细胞介导的炎症反应有关(图2)。
Western blotting检测结果显示,中暑组大鼠单核细胞来源外泌体中HMGB1表达相对值明显高于对照组(4.13±0.22 vs. 1.00±0.15),差异有统计学意义(t=–20.26,P<0.001)。ELISA结果表明,中暑患者全血分离单核细胞上清中收集的外泌体内HMGB1水平(ng/ml)明显高于对照组(0.29±0.11 vs. 0.12±0.04),差异有统计学意义(t=3.472,P=0.006;图3)。
RT-qPCR及Western blotting检测显示,与对照组比较,中暑组内皮细胞NLRP3IL-1β的mRNA及蛋白表达水平均明显升高(图4A、B)。ELISA结果显示,中暑组内皮细胞上清IL-1β水平亦明显增高。额外添加HMGB1抑制剂EP到中暑组内皮细胞,可降低内皮细胞NLRP3IL-1β的mRNA及蛋白表达水平,且细胞上清IL-1β水平亦有所下降,差异均有统计学意义(P<0.05,图4)。
目前认为,重症中暑引发的器官损伤很难完全用热的直接破坏来解释,其更主要的致病机制是炎症反应被激活后持续放大,造成靶器官炎症性损伤。在既往研究中,学者们已发现多种炎症相关的关键蛋白在重症中暑个体中呈高表达,主要包括白细胞介素家族[9]、干扰素[10]、肿瘤坏死因子[11]、HMGB1[12]等,这揭示了炎性因子及炎症反应信号通路对中暑疾病发生发展的重要作用。然而,上述蛋白如何作用于靶细胞微环境,以及蛋白改变的全局状态如何仍需要进一步明确。
外泌体传递物质具有稳定性高、不易降解、器官特异性高等特点,更符合生物学高效低耗的基本原理,是机体组织间信号转导的重要媒介。本课题组前期研究证实,肝细胞在热打击后,内含物中的外泌体发生了明显改变,可诱发炎症反应[13],这意味着外泌体可能在中暑的炎症传导过程中发挥作用。本研究首先通过检测外泌体数量,初步提示外泌体在热刺激环境中可能发生变化,然后尝试探索外泌体在炎性因子及信号转导中的作用,利用蛋白组学手段观察蛋白质表达谱的整体变化规律。鉴定结果表明,单核细胞外泌体中包含多种炎症相关蛋白。进一步的生物信息学分析结果显示,这些具有表达差异的蛋白倾向于参与炎症反应,其中包括NOD样受体等早已被发现参与中暑疾病的关键信号通路[14]。上述结果提示了外泌体及内含蛋白介导中暑相关炎症损伤的可能性。为验证外泌体的功能变化,本研究应用被证明在中暑疾病中发生损伤的内皮细胞作为靶细胞,验证了外泌体能够引起细胞内NOD样受体信号通路表达发生变化。
单核细胞来源外泌体内表达差异明显的蛋白中包括重要的损伤相关分子模式HMGB1蛋白。前人研究还指出,HMGB1在中暑疾病中能激活下游NOD样受体相关炎症通路,引起炎症反应[14]。在作用于内皮细胞的实验中,使用HMGB1抑制剂确实可在一定程度上缓解这些外泌体造成的损伤,表明HMGB1是发挥功能的主要成分之一。为验证外泌体内HMGB1水平的变化,本研究分别用Western blotting及ELISA检测大鼠及成人血液样本单核细胞来源外泌体HMGB1相对表达量的变化,结果显示,接受热打击后HMGB1蛋白的富集程度明显提高,该变化趋势与前人报道的HMGB1变化规律一致[12]
既往关于免疫细胞介导中暑组织损伤的研究多以组织内定植或浸润的免疫细胞为研究对象,如肝巨噬细胞、肺泡巨噬细胞等。本研究尝试探讨血液循环系统中的单核细胞作为致炎性免疫细胞的可能性,结果发现,循环血中的单核细胞能够导致炎症反应,并引起远处组织器官损伤,与向周围微环境释放炎性因子不同的是,这种介导炎症反应的过程可通过单核细胞释放的外泌体完成[15-16]。也有研究显示,单核细胞在热打击后能被激活,并对炎症反应具有重要作用[17]。当然,本研究并不能排除单核细胞受刺激后分化成巨噬细胞或其他种类单核细胞发挥作用的可能性,这可能涉及免疫细胞亚型转换等更加复杂的问题。
最后,为治疗单核细胞来源外泌体介导的炎症损伤,本研究尝试使用HMGB1抑制剂作为细胞实验的干预剂,评估此类药物对大鼠单核细胞来源外泌体HMGB1表达的影响,结果显示其能降低HMGB1表达水平,并伴随着炎症水平下降,提示HMGB1抑制剂可能通过改变外泌体功能发挥作用。因此,基于抑制HMGB1的新一代小分子药物可能存在进一步开发的价值。
综上所述,本研究初步表明,来源于热打击下单核细胞的外泌体内含蛋白HMGB1表达水平升高并起到促炎作用,使用HMGB1抑制剂能减轻与其相关的炎症反应。
  • 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)
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2023年第48卷第6期
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doi: 10.11855/j.issn.0577-7402.2023.06.0670
  • 接收时间:2021-10-20
  • 首发时间:2025-12-03
  • 出版时间:2023-06-28
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  • 收稿日期:2021-10-20
  • 录用日期:2022-07-05
基金
Medical Research Foundation of Guangdong Province(A2021404)
广东省医学科研基金(A2021404)
Traditional Chinese Medicine Scientific Research Project of the Guangdong Traditional Chinese Medicine Bureau(20212292)
广东省中医药局科研项目(20212292)
Science and Technology Planning Project of Qingyuan City(200715164560747)
广东省清远市科技计划项目(200715164560747)
作者信息
    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

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2种不同金属材料的力学参数

Family
属数
Number of
genus
种数
Number of
species
占总种数比例
Percentage of
total species (%)

Genus
种数
Number of
species
占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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