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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 study the anti-inflammatory effect of HO-1 on allergic airway inflammation and its effect on T helper (Th ) 1, Th2, Th17 and regulatory T cell (Treg). BALB / c mice were challenged with ovalbumin (OVA) to induce animal models of asthma, which are mainly infiltrated with eosinophils (EOS). HO-1 substrates were also sensitized and challenged Hemin induced high HO-1 expression. The degree of airway inflammation in asthmatic mice was observed by pathological sections of lung tissue, serum IgA-specific IgE level and bronchial alveolar lavage fluid (BALF) inflammatory cells. The lung and spleen were detected by western blot and real-time PCR Tissue HO-1 gene expression and protein levels, lung tissue Th1, Th2, Th17 and Treg secretion of cytokines and specific transcription factor gene expression; flow cytometry analysis of CD4 + T cell subsets. The results showed that the expression of HO-1 in lung and spleen of mice after OVA sensitized and challenged (OVA group) was higher than that of normal group, and the expression of HO-1 protein increased after hemin intervention (OVA + hemin group) Histopathological examination of lung showed that a large number of inflammatory cells infiltrated in OVA group, mainly EOS, with the total number of cells in BALF and EOS number and serum IgA-specific IgE increased, but the above phenomenon was significantly reduced after intervention with heme; OVA group lung tissue But the expression of GATA-3 and RORγt and the levels of IL-4, IL-17A and IL-6 were significantly higher than those of the normal group, but the results of the hemoglobin reversal showed that the expression of T-bet and the level of IFN- The levels of Foxp3, IL-10 and TGF-β in hemin group were significantly higher than those in the other two groups. The results of spleen CD4 + T cell subsets showed that a large number of Th2, Th17 and Th1 and Tregs were slightly increased in OVA group, Significant downregulation of Th2 and Th17 cells significantly increased the proportion of Treg cells, while Th1 showed an upward trend. The results showed that upregulation of HO-1 expression could significantly antagonize EOS-induced airway inflammation by regulating the balance of Th17 / Treg and Th1 / Th2 cells and promoting the secretion of TGF-β and IL-10 to inhibit airway inflammation.