17β-雌二醇介导的信号通路对急性呼吸窘迫综合征小鼠肺泡上皮钠离子通道的调控研究

摘要

Objective Toinvestigatetheeffectandmechanismof17β-estradiol-mediatedPI3K/AKT/SGK1path-wayonepithelialsodiumchannel(ENaC)regulationinmicewithARDS.Methods 40maleC57B/L6micewererandomlydi-vided into control group,LPS group,E2 group and wortmannin group,with each group 10 mice. The pathological changes in mice lung tissue were evaluated by HE staining;Arterial oxygen tension( PaO2 )changes were measured by the blood gas analy-zer;The lung permeability was measured by W/D;The levels of total protein in BALF were measured by BCA ( bicinchoninic acid);Interleukin-1β( IL-1β),tumor necrosis factor-α( TNF-α)and myeloperoxidase( MPO)activities in the bron-choalveolar lavage fluid ( BALF ) were measured by ELISA;The mRNA transcription levels of ENaC were analyzed by RT-PCR;The protein expression levels of α-,β-,γ-ENaC,p-AKT and p-SGK1 were analyzed by Western blotting method.Results AftermodelingbyinjectingLPS,thelunginjuryscorewas(4.2±0.2)inLPSgroup,(2.7±0.5)inE2 group and(3. 9±0. 3)in wortmannin group,showing statistically significant differences(F=46. 07,P=0. 000). Among the groups,E2 group was lower than LPS group and wortmannin group was higher than E2 group(P<0. 05). The PaO2 among the four groups at different time showed statistically significant differences(F=188. 148,P=0. 000). The W/D,BALF proteinlevel,IL-1β,TNF-α,MPO activity,α-,β-,γ-ENaC mRNA transcription level,α-,β-,γ-ENaC expression and p-AKT and p-SGK1 expression levels showed statistically significant differences(P<0. 05). Compared with the control group,the W/D,BALF protein level,IL-1β,TNF-αlevel and MPO activity in E2 group,LPS group and wortmannin group were increased,while the α-,β-,γ-ENaC mRNA transcription level were decreased(P<0. 05). Compared with the E2 group,the W/D,BALF protein level,IL -1β and TNF -α levels,MPO activity,α -,β -,γ -ENaC expression and p-AKTandp-SGK1expressionlevelsweredecreased(P<0.05).℅onclusion 17β-estradiolcanup-regulateENaCvia the post-transcription mechanism involving rapid activation of PI3K/AKT/SGK1 pathway,thus playing a protective role in alve-olar fluid clearance in mice with ARDS.%目的：探讨17β-雌二醇介导的PI3K/AKT/SGK1信号通路对小鼠急性呼吸窘迫综合征（ ARDS）的保护作用及其对肺泡上皮钠离子通道（ENaC）的调控。方法40只雄性C57B/L6小鼠随机分为对照组（control组）、模型组（ LPS组）、17β-雌二醇治疗组（ E2组）、渥曼青霉素干预组（ wortmannin 组），每组10只。苏木素-伊红（HE）染色观察肺组织病理改变并进行肺损伤评分；血气分析动脉血氧分压（PaO2）变化；肺湿干比（W/D）检测肺水肿的严重程度；BCA法检测肺泡灌洗液（ BALF）中总蛋白含量；酶联免疫吸附试验（ ELIAS）法检测BALF中白介素（ IL）-1β、肿瘤坏死因子α（ TNF-α）水平及髓过氧化物酶（ MPO）活性；反转录聚合酶链式反应（ RT-PCR）法检测各亚基ENaC mRNA表达；Western blotting法检测AKT、SGK1磷酸化水平及各亚基ENaC蛋白表达水平变化。结果小鼠气管内注射脂多糖（LPS）造模后，LPS组肺损伤评分为（4.2±0.2）分，E2组为（2.7±0.5）分，wort-mannin组为（3.9±0.3）分，差异有统计学意义（ F越46.07，P越0.000）；其中E2组较LPS组降低，wortmannin组较E2组升高（P<0.05）。4组不同时间PaO2比较有差异（F越188.148，P越0.000）。4组小鼠肺组织W/D，BALF中总蛋白含量，IL-1β、TNF-α水平，MPO活性，α-、β-、γ-ENaC的mRNA表达水平，α-、β-、γ-ENaC蛋白表达及p-AKT、p-SGK1表达水平比较有差异（P<0.05）；其中与control组比较，LPS组、E2组、wortmannin组W/D、BALF中总蛋白含量、IL-1β、TNF -α水平、MPO 活性升高，α-、β-、γ-ENaC 的 mRNA 表达水平降低（ P <0.05）；与E2组比较，LPS组、wortmannin组W/D、BALF蛋白含量、IL-1β、TNF-α水平、MPO活性升高，α-、β-、γ-ENaC蛋白表达水平及p-AKT、p-SGK1表达水平降低（P<0.05）。结论17β-雌二醇可通过快速激活PI3K/AKT/SGK1信号通路介导的转录后调控机制上调ENaC表达，从而对小鼠ARDS肺水肿发挥保护作用。