目的 观察T细胞特异转录因子T-bet/GATA-3在支气管哮喘中失衡表达,探讨黄芪和地塞米松对其不同的调节作用.方法 40只雄性SPF级SD大鼠随机分为对照组、哮喘组、地塞米松组和黄芪组,20只雄性SPF级致敏SD大鼠脾脏中分离获得CD4+T细胞,苏木精·伊红染色观察气道病理改变;酶联免疫吸附试验(ELISA)检测大鼠血清中IL-4、IL-5、IFN-γ含量;逆转录-聚合酶链反应(RT-PCR)检测IL-4、IL-5、IFN-γ、T-bet和GATA-3 mRNA表达;Western blot法检测T-bet和GATA-3蛋白表达.结果 肺组织中嗜酸粒细胞(EOS)、淋巴细胞、管壁面积/支气管管腔内周长(WA/Pi)和支气管平滑肌面积/支气管管腔内周长(ASM/Pi)哮喘组比对照组明显增多或增厚(P均<0.01),地塞米松组和黄芪组明显低于哮喘组(P均<0.01);血清中IL-4和IL-5含量,哮喘组高于对照组(P均<0.01),地塞米松组和黄芪组均低于哮喘组(P均<0.01),血清中IFN-γ含量哮喘组远低于对照组(P<0.01),地塞米松组低于对照组(P<0.01),但黄芪组明显高于哮喘组(P<0.01).在大鼠肺组织和致敏大鼠脾CD4+ T细胞中,IFN-γ mRNA、T-bet mRNA和蛋白表达哮喘组明显低于对照组(P均<0.01),地塞米松组与哮喘组相似(P>0.05),黄芪组与对照组相似,但明显高于哮喘组(P<0.01);IL-4和IL-5 mRNA、GATA-3 mRNA和蛋白表达,哮喘组异常高于对照组(P<0.01),地塞米松组和黄芪组均明显低于哮喘组(P<0.01);肺组织中T-bet/GATA-3蛋白表达量比值,哮喘组明显低于对照组(P<0.01),地塞米松组与哮喘组相近(P>0.05),黄芪组与对照组的比值相近(P>0.05),明显高于哮喘组和地塞米松组(P均(0.01).结论 T细胞特异转录因子T-bet/GATA-3在哮喘中失衡表达,黄芪抑制哮喘气道炎症可能通过双向调节T-bet和GATA-3平衡来实现,地塞米松抑制GATA-3表达,不能增加T-bet表达,其抑制哮喘气道炎症并非通过调节转录因子T-bet和GATA-3平衡实现.%Objective To investigate imbalanced T cell-specific transcription factors T-bet and GATA-3 in asthmatic rats.and to identify the difierent effects of astragalus and dexamethasone ou them.Methods Forty male SD rats were randomly divided into a control group,an asthmatic group,a dexamethasone group,and an astragalus group,and were observed the change of airway histology.Two weeks after immunization,CD4+ T cells were obtalned from singled-cell suspension of spleen(after lysis of RBCs).The concentrations of IL-4,IL-5,IFN-γ in sernm were measured by ELISA.The mRNA expressions of IL-4,IL-5,IFN-γ,T-bet and GATA-3 in the lungs and CD4+ T cells were detected by RT-PCR.and the protein expressions of T-bet and GATA-3 in the lungs and CD4+ T cells were detected by Western blot respectively.Results In the asthmatic group,the numbers of eosinophils and lymphocytes.the thicknesses of WA/Pi and ASM/Pi were(25.70±2.50)/mm2,(44.80±5.69)/mm2,(12.15±1.47)mm,(6.82±0.91)mm,and those of the control group were(2.40±1.27)/mm2,(8.90±1.97)/mm2,(6.49±1.57)mm,(2.75±0.62)mm,and thore of the dexamethasone group were(6.70±1.89)/mm2,(15.80±3.12)/mm2,(8.02±1.35)mm,(3.38±0.65)mm,and those ofthe astragalus group were(11.20±2.97)/mm2,(19.80±4.59)/mm2,(8.86±0.96)mm,(3.96±0.61)mm.There were significant differences between the asthmatic group and the control group.the asthmatic group and the dexamethasone group,and the astragalus group (P<0.01).In the asthmatic group,the concentrations of IL-4,IL-5,IFN-γ in serum were (35.23±8.02) pg/ml,(32.03±3.99) pg/ml,(16.73±5.15) pg/ml,and those of the control group were (14.48±1.68) pg/ml,(14.23±0.97) pg/ml,(64.55±14.04) pg/ml,and those of the dexamethasone group were (14.97±1.09) pg/ml,(16.13±2.36) pg/ml,(16.69±4.05) pg/ml,those of the astragalus group were(15.54±1.35) pg/ml,(15.99±2.73) pg/ml,(52.15±18.65)pg/ml,there were significant differences among four groups(P<0.01).In the asthmatic group,the expressions of IFN-γ mRNA,T-bet mRNA and protein were significantly decreased compared with those of the control group (P <0.01) in the lungs and CD4+ T cells.There was no difference between the asthmatic group and dexamethasone group in the expressions of T-bet mRNA and protein in the lungs and CD4+ T cells (P>0.05).In the astragalas group,the expressions of T-bet mRNA and protein were increased compared with those of the asthmatic group (P<0.01).There was no difference between the control group and astragalus group(P>0.05).In the asthmatic group,the levels of IL-4 and IL-5 mRNA,GATA-3 mRNA and protein expressions were significantly increased compared with those of the control group (P<0.01) in the lungs and CD4+ T cells.In the dexamethasone group,the levels of IL-4 and IL-5 mRNA,GATA-3 mRNA and protein expressions were significantly decreased compared with those of the asthmatic group in the lungs and CD4+ T cells(P<0.01).In the astragalus group,the levels of IL-4 and IL-5 mRNA,GATA-3 mRNA and protein expressions were significantly increased compared with those of the asthmatic group(P<0.01),but there was no difference between the astragalus group and control group(P>0.05).In the control group,the ratios of protein expressions of T-bet and GATA-3 were 0.80±0.53,significantly increased compared with the asthmatic group(0.07±0.05,P<0.01).In the dexamethasone group,the ratios of protein expressions of T-bet and GATA-3 were 0.14±0.19,significantly decreased compared with the control group (P<0.01),but there was no difference between the dexamethasone group and asthmatic group(P>0.05).In the astragalus group,the ratios of protein expressions of T-bet and GATA-3 were 0.58±0.29,significantly increased compared with those of the asthmatic group(P<0.01),but there was no difference between the astragalus group and control group (P>0.05).Conclusion Astragalus membranaceus treatment inhibits OVA-induced airway inflammation by modulating key master switches GATA-3 and T-bet that results in committing in T helper cell to a Th1 phenotype.Dexamethasone inhibits the asthmatic airway inflammation without lating the balance of transcription factors T-bet and GATA-3.
展开▼
