1α,25-二羟基维生素D;通过TLR4信号通路抑制川崎病血清诱导的THP-1细胞炎症因子表达  

1α,25-Dihydroxyvitamin D;inhibits Kawasaki disease serum-induced expression of inflammatory factors in THP-1 cells via TLR4 signaling pathway

作　　者：周忠 田正 王锋 李洁滢 胡琳 杨艳娟 焦蓉 ZHOU Zhong;TIAN Zheng;WANG Feng;LI Jie-ying;HU Lin;YANG Yan-juan;JIAO Rong(Xiangyang No.1 People's Hospital,Hubei University of Medicine,Xiangyang 441000,China)

机构地区：[1]湖北医药学院附属襄阳市第一人民医院,湖北襄阳441000

出　　处：《中国病理生理杂志》2022年第1期130-138,共9页Chinese Journal of Pathophysiology

基　　金：湖北省卫生健康委科研项目(No.WJ2021F064);襄阳市科学技术局项目(No.2020ZD34);襄阳市第一人民医院科技创新项目(No.XYY2021M06);湖北医药学院研究生科技创新项目(No.YC2021031)。

摘　　要：目的:研究1α,25-二羟基维生素D_(3)[1,25-(OH)_(2)D_(3)]是否通过调控巨噬细胞Toll样受体4(TLR4)信号通路的活化影响川崎病炎症反应的发生。方法:收集襄阳市第一人民医院收治的73例川崎病患儿血清,另收集急性上呼吸道感染患儿(感染组)和儿保科体检儿童(对照组)的血清各30例,利用试剂盒检测各组25-羟基维生素D_(3)[25-(OH)D_(3)]水平的变化,并绘制受试者工作特征(ROC)曲线,确定诊断截断点。体外细胞实验,利用采集的血清诱导THP-1巨噬细胞构建川崎病固有免疫模型,实验分组为:15%正常血清组、15%川崎病血清组及15%川崎病血清+1,25-(OH)_(2)D_(3)(1 nmol/L、10 nmol/L和100 nmol/L)组[1,25-(OH)2D3均于血清刺激前加入]。加入血清刺激24h后,利用RT-qPCR检测TLR4、白细胞介素1β(IL-1β)和肿瘤坏死因子α(TNF-α)的mRNA表达水平,ELISA检测细胞培养上清液中TNF-α的含量;Western blot检测TLR4和p-P65的蛋白水平;免疫荧光技术检测TLR4蛋白的表达。结果:川崎病患儿体内白细胞数、中性粒细胞数、血小板数、丙氨酸转氨酶及C-反应蛋白均较感染组显著升高(P<0.05)。与对照组及感染组相比,川崎病患儿血清25-(OH)D_(3)水平显著降低(P<0.01),诊断截断点为27.55μg/L,ROC曲线下面积为0.922,预测的灵敏度为93.3%,特异度为75.0%。体外细胞实验显示,与正常血清组相比,川崎病血清可显著促进巨噬细胞TLR4、IL-1β和TNF-α的mRNA表达(P<0.05),增加巨噬细胞分泌TNF-α的含量(P<0.05);1,25-(OH)2D3预保护抑制血清诱导的炎症因子产生。川崎病血清显著提高巨噬细胞TLR4和p-P65蛋白水平(P<0.05),而1,25-(OH)_(2)D_(3)可显著抑制这一作用(P<0.05)。结论:川崎病患儿体内25-(OH)D_(3)水平显著降低,而补充1,25-(OH)_(2)D_(3)可通过调节TLR4信号通路来抑制川崎病血清诱导的THP-1细胞炎症反应。AIM:To investigate whether 1α,25-dihydroxyvitamin D_(3)[1,25-(OH)_(2)D_(3)]influences the inflammatory response of Kawasaki disease by regulating the activation of Toll-like receptor 4(TLR4)signaling pathway in macrophages.METHODS:Serum samples were collected from 73 children with Kawasaki disease admitted to Xiangyang No.1 People’s Hospital,30 children with acute upper respiratory tract infection(AURI)and 30 children examined in the department of child care.Serum 25-hydroxyvitamin D;[25-(OH)D;]levels were detected by the kit.Receiver operating characteristic(ROC)curve analysis was used to determine the diagnostic cut-off values.For in vitro experiments,the collected serum was used to induce THP-1 macrophages to construct an innate immune model of Kawasaki disease.The cells were divided into 15%normal serum group,15%Kawasaki disease serum group,and 15%Kawasaki disease serum+1,25-(OH)_(2)D_(3)(1,10 and 100 nmol/L)groups.1,25-(OH)_(2)D_(3)was added for pre-protection prior to serum stimulation.After24 h of stimulation with serum,the m RNA expression levels of TLR4,interleukin-1β(IL-1β)and tumor necrosis factor-α(TNF-α)were detected by RT-q PCR,and the content of TNF-αin cell culture supernatant was detected by ELISA.The protein levels of TLR4 and p-P65 were detected by Western blot,and the protein expression of TLR4 was detected by immunofluorescence.RESULTS:Leukocyte count,neutrophil count,platelet count,alanine aminotransferase and C-reactive protein were significantly higher in Kawasaki disease children than those in AURI group(P<0.05).Compared with control group and AURI group,the serum level of 25-(OH)D_(3)in children with Kawasaki disease was significantly decreased(P<0.01).The diagnostic cut-off point was 27.55μg/L,the area under ROC curve was 0.922,the predictive sensitivity was 93.3%,and the specificity was 75.0%.In vitro cell experiments showed that Kawasaki disease serum significantly promoted the m RNA expression of inflammatory factors TLR4,IL-1βand TNF-αin macrophages,and increased the cont

关 键 词：川崎病 1α 25-二羟基维生素D TLR4信号通路 巨噬细胞 炎症 

分 类 号：R725.5[医药卫生—儿科] R363.2[医药卫生—临床医学]