血红素加氧酶-1调节CD4^+T细胞亚群拮抗过敏性哮喘的实验研究

Heme oxygenase-1 alleviates allergic airway inflammation by regulating CD4^+T subsets

制备哮喘小鼠模型,干预血红素加氧酶-1(heme oxygenase-1,HO-1)表达,探讨HO-1在过敏性气道炎症中抗炎及其对T辅助细胞(T helper,Th)1、Th2、Th17和调节性T细胞(regulatory T cell,Treg)的调节作用。用卵清蛋白(ovalbumin,OVA)致敏、激发BALB/c小鼠制备以嗜酸性粒细胞(eosinophil,EOS)浸润为主的哮喘动物模型,并在致敏、激发中给予HO-1底物氯化高铁血红素(hemin)诱导HO-1高表达。经肺脏组织病理切片,血清OVA特异性IgE水平,肺泡灌洗液(bronchial alveolar lavage fluid,BALF)炎症细胞分类计数观察哮喘小鼠气道炎症程度;western blot和real-time PCR分别检测肺脏和脾脏组织HO-1基因表达和蛋白量,肺脏组织Th1、Th2、Th17及Treg分泌的细胞因子及特定转录因子基因表达;流式细胞术分析脾脏CD4+T细胞亚群。实验结果显示,OVA致敏、激发后(OVA组),小鼠肺脏和脾脏HO-1表达较正常组增高,血红素干预后(OVA+hemin组),HO-1蛋白表达则进一步升高;肺脏病理组织学显示OVA组见大量炎症细胞浸润,以EOS为主,伴BALF中细胞总数和EOS数及血清OVA特异性IgE增加,但经血红素干预后,上述现象明显减轻;OVA组肺组织中T-bet表达及IFN-γ水平降低;但GATA-3和RORγt表达及IL-4、IL-17A和IL-6水平较正常组显著增高,而血红素能逆转该结果;进一步显示OVA+hemin组Foxp3表达和IL-10及TGF-β水平较其余两组显著增高;脾脏CD4+T细胞亚群结果显示OVA组可见大量Th2,Th17略有增多,Th1和Treg则略降低,血红素干预明显下调Th2和Th17细胞比例,显著提升Treg细胞比例,而Th1则呈现升高趋势。结果表明,上调HO-1表达能显著拮抗EOS性气道炎症,该作用是通过调节Th17/Treg和Th1/Th2细胞平衡,促进TGF-β和IL-10分泌以抑制气道炎症。

To explore the role of heme oxygenase-1 （HO-1） in alleviating allergic airway inflammation and regulating Th cells （T helper cells, Th） 1, Th2, Thl7 and regulatory T cells （Tregs） in a mouse model of asthma, BALB/c mice were sensitized and challenged with ovalbumin （OVA） to induce eosinophilie airway inflammation. Hemin, the substrate of HO-1, was admin- istered to up regulate HO-1 expression. The analysis of pulmonary histopathology, serum OVA-specific IgE level and the in- flammatory cell differential in bronchial alveolar lavage fluid （BALF） were detected to evaluate the airway inflammation. H（Yl protein expression in the lung and the spleen was detected by Western blot and real-time PCR. CD4＋ T cell subsets were ana- lyzed by flow cytometry. The mRNA levels of eytokines and specific transcription factors associated with Thl, Th2, Thl7 and Treg cells in the lung were measured by real-time PCR. The results showed that the expression of HO-1 in the lung and spleen was increased in OVA group compared with the control group, which was further elevated by heroin treatment （OVA＋ Hemin group）. The pulmonary histopathology revealed that there were obvious inflammatory cell infiltrates, consisting mainly of eo sinophils （EOS） in OVA group. Meanwhile, the total number of cells in BALF and the level of serum OVA-specific IgE were elevated. These OVA-induced changes were markedly reduced by hemin treatment. In lung tissues, the mRNA expressions of T-bet and IFN-7 were down-regulated, while the mRNA expressions of GATA-3, RORγt, IL-4, IL-J7A and IL-6 were much higher in OVA group than those in the control group. Also, administration of hemin could reverse the above results. Further- more, the mRNA express~.ons of Foxp3, IL-10 and TGF-~ were significantly higher in heroin＋OVA group compared with the other two groups. CD4＋ T cell subsets in the spleen showed that a mass of Th2 cells were observed in OVA group accompanied by a slight increase in Thl 7 cells and a decrease in Thl and Treg ceils. Heroin treatment significantly reduced the percentages of Th2 and Th17 cells and promoted percentage of Treg cells. The percentage of Thl cells presented an increasing trend in he- min＋OVA group. Our data demonstrate that induction of HO-1 might alleviate eosinophilic airway inflammation by regulating the balance of Th1T/Treg and Th1/Th2 cells as well as promote TGF-β and IL-10 production to suppress airway inflammation in asthma.