通过抑制小窝蛋白磷酸化调控Nrf2信号通路可以对呼吸机相关性肺损伤起保护作用  

Regulation of Nrf2 pathway to protect ventilator induce lung injury in vivo via inhibition of caveolin phosphorylation

作　　者：钟荣[1] 肖军[1] 戴春光[1] 于志辉[1] 周吉[1] 

机构地区：[1]桂林医学院附属医院重症医学科,广西壮族自治区桂林541001

出　　处：《中华危重病急救医学》2016年第6期547-552,共6页Chinese Critical Care Medicine

基　　金：基金项目：广西壮族自治区医药卫生经费项目（Z2014323）;广西壮族自治区临床重点专科建设项目[桂卫（2014）13号]

摘　　要：【摘要】目的探讨在体动物抑制小窝蛋白-1（Cav-1）磷酸化是否可有效调节核因子E2相关因子（Nrf2）信号通路及下游效应分子表达，以及是否对呼吸机相关性肺损伤（VILI）起保护作用。方法将90只健康雄性SD大鼠按随机数字表法分为9组，每组10只：假手术组（Sham）仅做气管切开而不通气；保护性通气（PV）1h、2h组；大潮气量（VT）通气（40mL／kg）1h、2h组；酪氨酸蛋白激酶抑制剂PP，或罗格列酮（Rsg）预处理＋大VT通气1h、2h组（PP2或Rsg1h、2h组）。两个预处理组分别于通气前1h腹腔注射PP2（15mg／kg）或灌胃Rsg（5mg／kg）。制模后处死大鼠，收集支气管肺泡灌洗液（BALF），用伊文思蓝（EB）实验检测肺血管通透眭；用酶联免疫吸附试验（ELISA）检测BALF中肿瘤坏死因子-α（TNF—α）、转录激活因子蛋白1（AP-1）、核转录因子-KB（NF—KB）、白细胞介素-8（IL-8）水平。取肺组织，计算肺湿／干质量比值（W／D），光镜下观察肺组织病理学改变；用比色法测定髓过氧化物酶（MPO）活性；用反转录-聚合酶链反应（RT—PCR）检测Nrf2mRNA表达；用蛋白质免疫印迹试验（Western Blot）检测磷酸化小窝蛋白-1酪氨酸残基14（pCav-1-Y14）、Cav-1、过氧化物酶体增殖物激活受体γ（PPARγ）、紧密连接蛋白闭合蛋白-5（elaudin-5）蛋白表达及Nrf2在胞质和胞核中的蛋白表达；用免疫组化法检测PPARγ、claudin-5阳性表达。结果Sham组和PV组肺组织无明显病理学改变，两组各指标均无差异。大VT组肺组织损伤严重，肺W／D比值、EB含量、MPO活性和BALF中TNF—α、AP-1、IL-8、NF—KB水平较Sham组及PV组明显升高，pCav-1-Y14、Cav-1表达均显著高于Sham组及Pv组，PPARγ、elaudin-5表达则显著低于Sham组及PV组，并呈时间依赖性；胞核和胞质Nrt2表达与Sham组和PV组无统计学差异。PP2或Rsg预处理后，肺W／D比值、Objective To investigate whether the inhibition of caveolin-1 （Cav-1） phosphorylation will regulate effectively nuclear factor-erythroid 2-related factor （Nrf2） signal pathway and downstream effector molecules and protest against ventilation induced lung injury （VILI） in an animal model in vivo. Methods Ninety male Sprague-Dawley （SD） rats were randomly divided into nine groups （each n = 10）： sham group in which rats did not receive ventilation but received tracheotomy; lung protective ventilation （PV） for 1 hour or 2 hours group; mechanical ventilation （MV） at high volume tidal （VT, 40 mL/kg） for 1 hour or 2 hours group; protein tyrosine kinase inhibitor PP2 or rosiglitazone （Rsg） pretreatment ± high VT ventilation for 1 hour or 2 hours groups. The two pretreatment groups were given intraperitoneal injection PP2 15 mg/kg or intragastric administration of Rsg 5 mg/kg 1 hour before ventilation respectively. The rats were sacrificed after model reproduction, and bronchoalveolar lavage fluid （BALF） was collected. Pulmonary vascular permeability was measured by Evans blue （EB）. The levels of tumor necrosis factor- α （TNF- α ）, activator protein-1 （AP-1）, nuclear factor-KB （NF-κB）, and interleukin-8 （IL-8） in BALF were determined by enzyme linked immunosorbent assay （ELISA）. Then the lung tissues were collected, the lung wet/dry ratio （W/D） was calculated, the changes in pathology was observed with light microscope, and myeloperoxidase （MPO） activity was determined by colorometric analysis. Nrf2 mRNA was determined by reverse transcription-polymerase chain reaction （RT-PCR）. The expressions of Cav-1 tyrosine residues 14 phosphorylation （pCav-1-Y14）, Cav-1, peroxisome proliferators-activated receptor γ （PPAR γ） and claudin-5 as well as Nrf2 in cytoplasm and nucleus were determined by Western Blot. The positive expressions of PPAR γ and claudin-5 in lung tissues were assayed with immunohistochemistry staining. Results There were

关 键 词：小窝蛋白-1磷酸化 核因子E2相关因子 过氧化物酶体增殖物激活受体Γ 闭合蛋白-5 呼吸机相关性肺损伤 

分 类 号：R563[医药卫生—呼吸系统]