大电导钙依赖性钾通道参与脓毒症炎症应答的机制研究  

Mechanism of large-conductance calcium-activated potassium channel involved in inflammatory response in sepsis

作　　者：武坤[1] 赵凌峰[2] 王雨平[3] 刘盼 程沈菊 杨潇[3] 王英[3] 祝艳翠[3] Wu Kun;Zhao Lingfeng;Wang Yuping;Liu Pan;Cheng Shenju;Yang Xiao;Wang Ying;Zhu Yancui(Department of Medical Laboratory,First Affiliated Hospital of Kunming Medical University,Kunming 650032,Yunnan,China;Department of Vascular Surgery,First Affiliated Hospital of Kunming Medical University,Kunming 650032,Yunnan,China;Department of Critical Medicine,First Affiliated Hospital of Kunming Medical University,Kunming 650032,Yunnan,China;Department of Medical Laboratory,the Third People's Hospital of Honghe Hani and Yi Autonomous Prefecture,Gejiu 661000,Yunnan,China)

机构地区：[1]昆明医科大学第一附属医院医学检验科,云南昆明650032 [2]昆明医科大学第一附属医院血管外科,云南昆明650032 [3]昆明医科大学第一附属医院重症医学科,云南昆明650032 [4]云南红河哈尼族彝族自治州第三人民医院医学检验科,云南个旧661000

出　　处：《中华危重病急救医学》2023年第5期469-475,共7页Chinese Critical Care Medicine

基　　金：云南省科技厅科技计划项目(202101AY070001-110);云南省教育厅科学研究基金项目(2019J1230)。

摘　　要：目的探讨大电导钙依赖性钾通道(BKCa)参与脓毒症发病的机制。方法收集脓毒症患者(28例)、普通感染者(25例)和健康体检者(25例)血清,采用酶联免疫吸附试验(ELISA)测定BKCa水平;并分析脓毒症患者血清BKCa水平与急性生理学与慢性健康状况评分Ⅱ(APACHEⅡ)的相关性。培养RAW 264.7细胞,用脂多糖(LPS)刺激,部分实验以尼日尼亚菌素(Nigericin)作为第二刺激信号构建脓毒症细胞模型;采用实时荧光定量聚合酶链反应(RT-qPCR)、蛋白质免疫印迹试验(Western blotting)测定不同浓度LPS(0、50、100、1000μg/L)刺激后细胞BKCa的mRNA和蛋白表达。RAW 264.7细胞转染BKCa的小干扰RNA(siRNA-BKCa),用Western blotting测定细胞天冬氨酸特异性半胱氨酸蛋白酶1前体(pro-caspase-1)、白细胞介素-1β前体(pro-IL-1β),细胞培养液中caspase-1 p20、IL-1βp17,以及NOD样受体蛋白3(NLRP3)、核转录因子-κB(NF-κB)水平。碘化丙啶(PI)染色后荧光显微镜下观察凋亡情况,测定乳酸脱氢酶(LDH)释放率,Western blotting测定细胞凋亡蛋白Gasdermin D(GSDMD)表达以评估沉默BKCa对细胞焦亡的影响。结果脓毒症患者血清BKCa明显高于普通感染者和健康体检者(ng/L:165.2±25.9比102.5±25.9、98.8±20.0,均P<0.05),且脓毒症患者血清BKCa水平与APACHEⅡ评分呈显著正相关(r=0.453,P=0.013)。用LPS构建脓毒症细胞模型,显示LPS呈浓度依赖地促进BKCα的mRNA和蛋白表达,1000μg/L刺激后细胞BKCa的mRNA和蛋白表达水平均较空白组(0μg/L)明显增加〔BKCa mRNA(2^(-ΔΔCt)):3.00±0.36比1.00±0.16,BKCa/β-actin:1.30±0.16比0.37±0.09,均P<0.05〕。与对照组比较,模型组细胞caspase-1 p20/pro-caspase-1比值、IL-1βp17/pro-IL-1β比值均明显升高(caspase-1 p20/pro-caspase-1比值:0.83±0.12比0.27±0.05,IL-1βp17/pro-IL-1β比值:0.77±0.12比0.23±0.12,均P<0.05);但转染siRNA-BKCa后二者均较模型组明显下降(caspase-1 p20/pro-caspase-1比值:0.23±0.12比0.83±0.12,IL-1βpObjective To explore the mechanisms of large-conductance calcium-activated potassium channel(BKCa)involved in inflammatory response in sepsis.Methods The serum levels of BKCa were measured by enzyme-linked immunosorbent assay(ELISA)in patients with sepsis(28 cases),patients with common infection(25 cases)and healthy people(25 cases).The relationship between levels of BKCa and acute physiology and chronic health evaluationⅡ(APACHEⅡ)were analyzed.Cultured RAW 264.7 cells were stimulated by lipopolysaccharide(LPS).In some experiments,a cell model of sepsis was constructed using Nigericin as the second stimulus signal.The mRNA and protein expressions of BKCa in RAW 264.7 cells stimulated with LPS(0,50,100,1000μg/L)were measured by real-time fluorescence quantitative polymerase chain reaction(RT-qPCR)and Western blotting.RAW 264.7 cells were transfected with small interfering RNA of BKCa(siRNA-BKCa),and the levels of caspase-1 precursor(pro-caspase-1),interleukin-1βprecursor(pro-IL-1β)in cell,and the levels of caspase-1 p20,IL-1βp17 of cell culture medium,and NOD-like receptor protein 3(NLRP3),nuclear factor-κB(NF-κB)were measured by Western blotting.The apoptosis were detected by staining with propidium iodide(PI),the release rate of lactate dehydrogenase(LDH)were measured,and the expression of apoptotic protein Gasdermin D(GSDMD)was measured by Western blotting to evaluate the effect of silencing BKCa on cell pyrosis.Results The level of serum BKCa in patients with sepsis was significantly higher than that in patients with common infection and health peoples(ng/L:165.2±25.9 vs.102.5±25.9,98.8±20.0,both P<0.05).In addition,the level of serum BKCa in patients with sepsis was significantly positively correlated with APACHEⅡscore(r=0.453,P=0.013).LPS could construct a sepsis cell model by which LPS could promote BKCa expression in mRNA and protein with a concentration-dependent manner.The mRNA and protein expressions of BKCa in the cells stimulated by 1000μg/L LPS were significantly higher than that in t

关 键 词：大电导钙依赖性钾通道 脓毒症 核转录因子-ΚB NOD样受体蛋白3 天冬氨酸特异性半胱氨酸蛋白酶1 

分 类 号：R459.7[医药卫生—急诊医学]