摘要
目的探讨白细胞介素-1受体相关激酶(interleukin-1 receptor associated kinase,IRAK)在细菌脂多糖(lipopolysaccharide,LPS)诱导的气道黏液高分泌中的作用及其机制。方法采用特异性小干扰RNA(small interfering RNA,siRNA)转染,下调Toll样受体(Toll-like receptor,TLR)-4、IRAK在人气道上皮细胞株BEAS-2B中的表达,采用逆转录–聚合酶链反应(reverse transcription polymerase chain reaction,RT-PCR)检测细胞内黏蛋白5AC(mucin 5AC,MUC5AC)mRNA转录水平,酶联免疫吸附试验(enzyme linked immunosorbent assay,ELISA)检测细胞上清液中MUC5AC分泌量。利用蛋白印迹法检测胞内信号蛋白IRAK磷酸化水平及核因子(nuclear factor,NF)-κB p65的核转位情况。结果经LPS刺激后BEAS-2B细胞内IRAK磷酸化水平显著增高,使用siRNA下调TLR-4表达,可部分降低IRAK磷酸化水平(P<0.05);经LPS刺激,NF-κB p65趋向核内分布,使用siRNA下调TLR-4或IRAK可部分削弱NF-κB p65核内分布(P<0.05);RT-PCR及ELISA结果也显示,经LPS刺激后TLR-4缺陷株MUC5AC mRNA转录水平(0.36±0.05)、分泌量[(0.33±0.04)μg/L],IRAK缺陷株MUC5AC mRNA转录水平(0.48±0.05)、分泌量[(0.42±0.05)μg/L],IRAK激酶抑制剂组MUC5AC mRNA转录水平(0.57±0.07)、分泌量[(0.51±0.06)μg/L]均分别低于野生型BEAS-2B组[0.82±0.09,(0.76±0.09)μg/L;均P<0.05]。结论IRAK作为TLR受体依赖信号通路上游的重要调控蛋白,参与LPS诱导的气道黏液高分泌。
Objective To investigate whether interleukin-1 receptor associated kinase(IRAK)participates the airway mucus hypersecretion induced by lipopolysaccharide(LPS).Methods The expression of Toll-like receptor(TLR)-4 or IRAK was down regulated by the transfection of specific small interfering RNA(siRNA).The transcription level of mucin 5AC(MUC5AC)mRNA as well as the secretion level of MUC5AC protein were judged by reverse transcription polymerase chain reaction(RT-PCR)and enzyme linked immunosorbent assay(ELISA),respectively.The activity of signaling molecules involved in TLR-4 associated pathway,such as the phosphorylated IRAK,nuclear translocation of NF-κB p65,was analyzed by Western blot.Results The level of intracellular phosphorylated IRAK was increased by stimulation of LPS in BEAS-2B cells.However,down-regulation of TLR-4 by siRNA could partially attenuate the additional phosphorylation of IRAK induced by LPS(P<0.05).LPS elicited a nuclear translocation of NF-κB p65 in BEAS-2B cells,which could be partially blocked by the down-regulation of TLR-4 or IRAK.With RT-PCR,an increased expression level of MUC5AC mRNA was discovered in wild-type BEAS-2B cells(0.82±0.09)than TLR-4 defect cells(0.36±0.05),IRAK defect cells(0.48±0.05)and IRAK inhibitor pretreated cells(0.57±0.07)(all P<0.05).Meanwhile,according to ELISA,an increased secretion level of MUC5AC protein was recorded in wild-type BEAS-2B cells[(0.76±0.09)μg/L]than TLR-4 defect cells[(0.33±0.04)μg/L],IRAK defect cells[(0.42±0.05)μg/L]and IRAK inhibitor pretreated cells[(0.51±0.06)μg/L](all P<0.05).Conclusion As an crucial regulator of TLR dependent signaling pathway,IRAK may participate the airway mucus over-synthesis induced by LPS.
作者
何明欣
张华
周向东
尤列·皮尔曼
维克多·科罗索夫
李琪
HE Mingxin;ZHANG Hua;ZHOU Xiangdong;Juliy M Perelman;Victor P Kolosov;LI Qi(Department of Emergency Medicine,First Affiliated Hospital of Hainan Medical University,Haikou,Hainan 570102,P.R.China;Key Laboratory of Emergency and Trauma of Ministry of Education,Hainan Medical University,Haikou,Hainan 571199,P.R.China;Far Eastern Scientific Center of Physiology and Pathology of Respiration,Blagoveschensk 675000,Russia)
出处
《中国呼吸与危重监护杂志》
CAS
CSCD
北大核心
2022年第1期50-54,共5页
Chinese Journal of Respiratory and Critical Care Medicine
基金
海南省创新团队项目820 CXTD448
海南省重点研发项目ZDYF2020223
国家自然科学基金(81860001、82160012、82011530049)。
关键词
白细胞介素-1受体相关激酶
脂多糖
气道黏液高分泌
Interleukin-1 receptor associated kinase
lipopolysaccharide
airway mucus hypersecretion
