转录因子RORγt对妊娠期哮喘模型小鼠Th17/Treg平衡的调节作用

Regulatory effect of transcription factors RORγt on the Th17/Treg balances in pregnant asthma mice

目的观察妊娠期哮喘模型小鼠Th17细胞相关细胞因子和维甲酸相关孤儿受体γt(RORγt)的表达变化,探讨地塞米松(DXM)对小鼠气道炎症和Th17细胞分泌功能的影响。方法 30只雌性BALB/c小鼠随机分为健康妊娠(HP)组、妊娠期哮喘(AP)组和地塞米松(DXM)组,每组10只。AP组和DXM组于实验当天(0d)和7、14d时分别给予OVA/Al(OH)3混悬液腹腔注射致敏,HP组仅给予单纯等量生理盐水(NS)。3组小鼠合笼交配至妊娠成功。于21d起,连续7d对AP组和DXM组给予50g/L OVA雾化激发(1次/d,40min/次);DXM组于雾化吸入OVA前30min给予1mg/kgDXM腹腔注射;HP组仅给予单纯等量NS进行雾化激发。各组小鼠于末次激发24h后进行气道反应性检测,结果以相应浓度乙酰胆碱(Mch)激发下增强的呼吸间歇(Penh)值与基线值的百分比[Penh值(%)]来表示;分析支气管肺泡灌洗液(BALF)中白细胞总数及分类计数;取肺组织行病理学观察;采用流式细胞仪检测外周血Th17和Treg细胞的比例并计算Th17/Treg比值;ELISA法测定外周血IL-17、IL-23、IFN-γ和RORγt含量,RT-PCR法检测前述因子在肺组织中的表达,Western blotting检测肺组织中RORγt的表达。结果雌性BALB/c小鼠以OVA致敏后气道反应性增加,BALF中白细胞总数及嗜酸性粒细胞(Eos)、淋巴细胞(Lym)和中性粒细胞(Neu)比例增加,肺组织病理学改变符合哮喘的特征。外周血标本中,AP组Th17细胞显著升高,Treg细胞显著降低(P<0.05),Th17/Treg比值上升。DXM组Th17和Treg细胞变化趋势与AP组相同,Th17/Treg比值高于HP组,但低于AP组(P<0.05)。分别在肺组织和外周血标本中检测蛋白和mRNA表达水平,结果显示,AP组IL-17、IL-23及RORγt含量均高于HP组及DXM组(P<0.05),IFN-γ含量低于HP组及DXM组(P<0.05)。AP组肺组织RORγt蛋白的表达(61.27%±3.11%)明显高于DXM组(37.51%±1.93%,P<0.01)和HP组(22.39%±0.86%,P<0.01),后两者比较差异亦有统计学意义(P<0.01)。相关分析表明,AP组肺组织RORγt蛋白表达量与Eos、Lym和Neu细胞绝对值呈显著正相关(r=0.879、0.759、0.802,P<0.01),与外周血IL-17含量、肺组织IL-17mRNA表达水平亦呈正相关(r=0.696、0.863,P<0.01)。结论 Th17细胞分泌功能及RORγt表达上调参与了妊娠期哮喘的发生发展。DXM干预在改善哮喘症状的同时可降低RORγt表达,使Th17分化和增殖功能受抑。

Objective To observe the expression changes in Th17-related cytokines and retinoid acid receptor-related orphan receptor gamma t（RORγt） in mouse model of asthma during pregnancy,and to explore the effects of dexamethasone（DXM） on Th17 cell secretory function and airway inflammation in mice.Methods Thirty female BALB/c mice were divided into three groups at random（n=10 per group）： healthy pregnancy（HP） group,asthma in pregnancy（AP） group and dexamethasone（DXM） treated group（DXM group）.The mice were treated as follows： those in AP and DXM groups were sensitized with an intraperitoneal injection of chicken ovalbumin-aluminum hydroxide [OVA/A1（OH） 3 ] suspension on days 0,7 and 14.The HP mice were sensitized with normal saline（NS） following the same protocol.All the mice were then bred with male BALB/c male mice.Pregnant mice in AP and DXM groups were exposed to 40-minute of 50g/L OVA aerosol for seven consecutive days starting from day 21.Dexamethasone in a dose of 1mg/kg was injected intraperitoneally in DXM mice 30 minutes before inhaling 50g/L OVA aerosol,while the HP group received NS only.The airway responsiveness to corresponding concentrations of inhaled acetylcholine was recorded 24h after the final OVA challenge.The index of airflow obstruction was expressed as enhanced pause（Penh） and its mean value after each nebulization.The airway tone is further expressed as the Penh（%） after being normalized by the baseline value.Total and differential cell counts in bronchoalveolar lavage fluid（BALF） were carried out in each sample.The histological changes in the lungs were assessed using light microscopy.The propotions of Th17 and Treg cell in mice peripheral blood cells were calculated and the Th17/Treg ratio was determined by flow cytometry.IL-17,IL-23,IFN-γ and RORγt levels in the serum of mice were quantified by using ELISA technique.RT-PCR and Western blotting were performed to determine the levels of IL-17,RORγt,IL-23 and IFN-γ in serum,and RORγt protein in lung tissues respectively.Results After being sensitized by OVA,the increased total and differential cell counts（Eos,Lym and Neu） in BALF,the increased airway hyper-responsiveness and the histological changes of lung tissues were in line with the features of asthma.In AP group,Th17 cells in peripheral blood significantly increased,while the Treg T cells significantly decreased（P0.05）.The increased Th17/Treg ratio suggested that there was an imbalance of Th17/Treg ratio in this group.The change trend of cells in DXM group was the same as that in AP group（P0.01）,while the Th17/Treg ratio in DXM group was higher than that in HP group,but lower than that in AP group（P0.05）.The protein and mRNA expressions of IL17,IL-23 and RORγt in peripheral blood and lung tissues were higher,and IFN-γ level was lower in AP group than in HP and DXM group（P0.05）.RORγt protein expression in the lung tissues was significantly higher in AP group（61.27%±3.11%） than in DXM（37.51%±1.93%） and HP（22.39%±0.86%） groups（P0.01）,and a statistically significant difference was found between the latter two groups（P0.01）.Correlation analysis showed that the expression of RORγt protein was positively correlated with the absolute numbers of eosinophils,lymphocytes and neutrophils（r=0.879,0.759,0.802,P0.01）,and also positively correlated with IL-17 content in peripheral blood and mRNA expression of IL-17（r=0.696,0.863,P0.01） in lung tissues of AP group.Conclusions Up-regulation of Th17 cell secretory function and RORγt expression may be involved in the development and progression of asthma during pregnancy.DXM intervention can lead to improvement of asthma symptoms and inhibition of RORγt production,thus blocking the differentiation and proliferation of Th17 cells.