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

【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、EB含量、MPO活性和BALF中TNF—α、AP-1、IL-8、NF—κB水平均较大VT组明显降低；肺组织Nrf2mRNA表达明显增加；胞核内Nrf2蛋白表达较大VT组明显上调[核内Nrf2蛋白（灰度值）：1h为0．61±0．06、0．56±0．06比0．31±0．02，2h为0．38±0．06、0．43±0．07比0．22±0．03，均P〈0．05]，各组胞质内Nrf2蛋白表达无差异。PP2预处理组pCav-1-Y14表达明显低于大VT组（灰度值：1h为0．89±0．04比1．48±0．02，2h为0．86±0．02比1．31±0．01，均P〈0．05）；PP，或Rsg预处理组PPARl、elaudin-5的蛋白表达均明显高于大VT组[PPARγ（灰度值）：1h为0．34±0．07、0．42±0．13比0．17±0．07，2h为0．38±0．09、0．33±0．07比0．16±0．03；claudin-5（灰度值）：1h为0．33±0．05、0．38±0．07比0．14±0．03，2h为0．30±0．06、0．31±0．04比0．17±0．04；均P〈0．05]。结论抑制Cav-1-Y14磷酸化可增加Nrf2的核内表达及其效应分子PPAR1、elaudin-5的表达，从而减轻肺组织炎症及降低毛细血管通透性。

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 no obvious pathological changes in the lung tissue in sham group and PV groups, and there were no significant differences in all the parameters between the two groups either. However, the injury in lung tissue was severe in the high VT groups in which W/D ratio, EB contents, MPO activity, and TNF- α, AP-1, IL-8, NF-κB levels in BALF as well as the protein expressions of Cav-1 and pCav-1-Y14 were significantly higher than those of sham group and PV groups, and the protein expressions of PPAR γ and claudin-5 were significant lower than those of sham group and PV groups with a dose-dependent manner; but Nrf2 expressions in cytoplasm and nucleus did not show a statistical increase. After pretreatment of PP2 or Rsg, W/D ratio, MPO activity, EB contents, TNF- α, AP-1, IL-8, and NF-κB in BALF were significantly decreased as compared with those of high VT group, and RT-PCR showed significant up-regulation of Nrf2 mRNA in lung tissues too. Moreover, there was a statistically significant increase in expressed Nrf2 proteins in nucleus in PP2 or Rsg groups as compared with those of high VT groups [Nrf2 in nucleus （gray value）： 0.61 ± 0.06, 0.56 ± 0.06 vs. 0.31 ± 0.02 at 1 hour, 0.38 ± 0.06, 0.43 ± 0.07 vs. 0.22 ± 0.03 at 2 hours; all P 〈 0.05], but no significant difference was found in the expression of Nrf2 protein in the cytoplasm among all groups. The protein expressions of pCav-1-Y14 in PP2 pretreatment groups were significantly lower than those of high VT groups （gray value： 0.89 ± 0.04 vs. 1.48 ± 0.02 at 1 hour, 0.86 ± 0.02 vs. 1.31 ± 0.01 at 2 hours; both P 〈 0.05）; but expressed PPAR γ proteins and expressed claudin-5 proteins in PP2 or Rsg pretreatment groups were significantly higher than those of high VT groups [PPAR γ （gray value）： 0.34 ± 0.07, 0.42 ± 0.13 vs. 0.17 ± 0.07 at 1 hour, 0.38 ± 0.09, 0.33 ± 0.07 vs. 0.16 ± 0.03 at 2 hours; claudin-5 （gray value）： 0.33±0.05, 0.38±0.07 vs. 0.14±0.03 at 1 hour; 0.30±0.06, 0.31±0.04 vs. 0.17±0.04 at 2 hours; all P 〈 0.05]. Conclusions The inhibition of Cav-1-Y14 phosphorylation can increase the expression of Nrf2 in the nucleus, then result in an increase in the protein expressions of PPAR γ and claudin-5 of its effector molecules. This effect can reduce the inflammation and capillary permeability of lung tissue in the model of VILI.