摘要
目的明确热休克转录因子1(HSF1)是否通过调控大鼠肺泡巨噬细胞(AM)中NOD样受体蛋白3(NLRP3)炎症小体活性改善脓毒症诱导的急性肺损伤(ALI)。方法采用随机数字表法将24只SPF级SD大鼠分为对照组、脂多糖(LPS)组、过表达空载+LPS组、过表达HSF1+LPS组,每组6只。采用腹腔注射LPS 5 mg/kg制备脓毒症诱导的ALI大鼠模型;对照组给予等量生理盐水。过表达空载+LPS组和过表达HSF1+LPS组分别于制模前经气管内滴注过表达空载腺病毒或过表达HSF1腺病毒100μL;对照组和LPS组滴注等量生理盐水。制模后6 h取颈动脉血,测定氧合指数(PaO2/FiO2);取肺组织,苏木素-伊红(HE)染色后光镜下观察肺组织病理学改变,测定肺组织湿/干质量(W/D)比值,采用免疫组化法检测巨噬细胞特异性标志抗体CD68的阳性表达,采用蛋白质免疫印迹试验(Western blotting)检测HSF1和NLRP3的蛋白表达水平;收集支气管肺泡灌洗液(BALF),采用酶联免疫吸附试验(ELISA)检测白细胞介素(IL-1β、IL-18)的水平。另取正常大鼠BALF,分离原代AM培养并分为4组。空白对照组AM正常培养,不给予任何处理;LPS组采用1 mg/L的LPS处理AM 24 h制备LPS攻击模型;过表达空载+LPS组和过表达HSF1+LPS组分别用空载质粒或过表达HSF1质粒转染AM 48 h后,再用1 mg/L的LPS处理AM。采用细胞计数试剂盒-8(CCK-8)检测细胞活性,采用Western blotting检测AM中HSF1和NLRP3的蛋白表达水平,采用ELISA法测定AM培养液中IL-1β和IL-18含量。结果与对照组比较,LPS组大鼠肺组织结构损伤严重,肺泡腔及肺间质充血水肿,肺泡壁明显增厚及断裂,并伴有大量炎症细胞浸润,且肺W/D比值升高,巨噬细胞增加,PaO2/FiO2降低,肺组织和AM中HSF1蛋白表达降低、NLRP3蛋白表达升高,BALF和AM培养液中IL-1β、IL-18表达水平升高。与LPS组比较,过表达HSF1+LPS组大鼠肺损伤程度得到明显改善,且肺W/D比值显著降低(4.76±0.16比6.93±0.33,P<0.
Objective To determine whether heat shock transcription factor 1(HSF1)ameliorates sepsis induced acute lung injury(ALI)by regulating NOD-like receptor protein 3(NLRP3)inflammasome activity in rat alveolar macrophages(AM).Methods Twenty-four SPF Sprague-Dawley(SD)rats were divided into control group,lipopolysaccharide(LPS)group,overexpression empty vector+LPS group,and overexpression HSF1+LPS group by random number table method,with 6 rats in each group.The rat model of sepsis-induced ALI was reproduced by intraperitoneal injection of LPS(5 mg/kg);the rats in the control group were given the same volume of normal saline.The rats in the overexpression empty vector+LPS group and the overexpression HSF1+LPS group were instilled with 100μL of overexpressed empty vector adenovirus or overexpressed HSF1 adenovirus through the trachea,respectively;the rats in the control group and LPS group were instilled with an equal volume of normal saline at the same time.At 6 hours after the model was reproduced,carotid blood was collected to determine the oxygenation index(PaO2/FiO2);lung tissue was obtained,and hematoxylin-eosin(HE)staining was performed to observe the pathological changes of lung tissue under a light microscope.Lung tissue wet/dry ratio(W/D)was determined.Immunohistochemistry was used to detect the positive expression of macrophage-specific marker antibody CD68.Western blotting was used to detect the protein expressions of HSF1 and NLRP3.Bronchoalveolar lavage fluid(BALF)was collected,and the levels of interleukins(IL-1β,IL-18)were detected by enzyme-linked immunosorbent assay(ELISA).In addition,BALF of normal rats was collected,and primary AM was isolated,cultured and divided into four groups.The AM in the blank control group was cultured normally without any treatment;the LPS group was treated with 1 mg/L LPS for 24 hours to reproduced the LPS stimulation model;AM in the overexpression empty vector+LPS group and the overexpression HSF1+LPS group were transfected with empty vector plasmid or overexpressed HSF1
作者
金灿
张伟
Jin Can;Zhang Wei(Department of Critical Care Medicine,Affiliated Hospital of Zunyi Medical University,Zunyi 563000,Guizhou,China)
出处
《中华危重病急救医学》
CAS
CSCD
北大核心
2022年第11期1167-1172,共6页
Chinese Critical Care Medicine
基金
贵州省科技计划项目(2020-1Z061)。
