TLR4基因敲除对哮喘小鼠气道炎症的影响

Effects of TLR4 knockdown on airway inflammation in asthmatic mice

目的:探讨TLR4基因敲除对哮喘小鼠气道炎症的影响及其意义。方法:利用卵清白蛋白(OVA)腹腔注射、雾化激发野生型(WT)和TLR4基因敲除(TLR4^(−/−))小鼠,分别建立小鼠哮喘模型;观察比较各组小鼠行为变化,HE染色观察肺组织病理切片,ELISA法检测BALF中IL-4、IFN-γ、IL-6、IL-10、IL-17、TGF-β、TSLP,Western blot检测肺组织中GATA-3、T-bet、RORγt、Foxp3及OX40L蛋白水平,RT-PCR法观察GATA-3、T-bet、RORγt、Foxp3 mRNA水平。结果:与野生对照组小鼠相比,野生哮喘组小鼠肺组织及气管周围可见嗜酸性粒细胞、中性粒细胞等大量炎症因子浸润,上层基底膜增厚;BALF中IL-4、IL-6、IL-17、TGF-β、TSLP浓度明显升高,IFN-γ、IL-10浓度显著降低(P<0.01);肺组织中GATA-3、RORγt、OX40L蛋白表达显著增高(P<0.01),T-bet、Foxp3蛋白表达显著降低(P<0.01);GATA-3、RORγt mRNA水平显著上升(P<0.01),T-bet、Foxp3 mRNA水平显著降低(P<0.01)。与野生哮喘组小鼠相比,TLR4基因敲除组哮喘小鼠肺组织炎症程度明显减轻,少见炎症因子浸润;BALF中IL-4、IL-6、IL-17、TGF-β、TSLP浓度明显降低(P<0.01),IFN-γ、IL-10浓度明显升高(P<0.01);肺组织中GATA-3、RORγt、OX40L蛋白表达明显降低(P<0.01),GATA-3、RORγt mRNA水平明显降低(P<0.01),T-bet、Foxp3蛋白表达及mRNA水平明显升高(P<0.01,P<0.05)。结论:TLR4敲除可减轻哮喘模型小鼠气道炎症,其机制可能与TSLP浓度降低,OX40L水平下调,Th1/Th2、Th17/Treg失衡被纠正有关。

Objective:To investigate the effect of TLR4 knockdown on airway inflammation in asthmatic mice.Methods:Using ovalbumin(OVA)intraperitoneal injection and atomization to stimulate wild-type(WT)and TLR4 knockout(TLR4^(−/−))mice to replicate mouse asthma models.Observing the behavior of mice in each group and comparing the pathological sections of lung tissue by HE staining.Detecting the concentrations of IL-4,IFN-γ,IL-6,IL-10,IL-17,TGF-βand TSLP in BALF were detected by ELISA while level of GATA-3,T-bet,RORγt,Foxp3 and OX40L protein in lung tissue were detected by Western blot.mRNA levels of GATA-3,T-BET,RORγt and Foxp3 were observed by RT-PCR.Results:Compared with wild control mice,a large number of inflammatory factors,such as eosinophils and neutrophils,were observed in the lung tissue and around the trachea in the wild asthma group;concentration of IL-4,IL-6,IL-17,TGF-βand TSLP in BALF increased significantly(P<0.01)and the concentration of IFN-γ,IL-10 decreased significantly(P<0.01);GATA-3,RORγt and OX40L protein expressions increased significantly(P<0.01)and T-bet,Foxp3 protein expression decreased significantly(P<0.01);mRNA level of GATA-3,RORγt in lung tissue were significantly increased while T-bet and Foxp3 were decreased.Compared with wild asthma mice,inflammatory degree of lung tissue of asthma mice in TLR4 knockout group is obviously reduced,and inflammatory factor infiltration is rare;IL-4,IL-6,IL-17,TGF-β,TSLP concentration in BALF decreased significantly(P<0.01)and the IFN-γ,IL-10 concentration increased significantly(P<0.01);GATA-3,RORγt,OX40L protein expressions decreased significantly(P<0.01)and the mRNA level of GATA-3 and RORγt were decreased significantly(P<0.01);protein expressions and mRNA level of T-bet,Foxp3 increased significantly(P<0.01,P<0.05).Conclusion:TLR4 knockdown may reduce airway inflammation in asthmatic model mice by reducing TSLP concentrations,downregulating OX40L levels,and correcting Th1/Th2,Th17/Treg imbalances.