基于TLR2/NF-κB信号通路探讨平喘方及拆方改善哮喘小鼠气道重塑作用机制

Discussion on Mechanism of Pingchuan Prescription and Decomposed Prescription for Improving Airway Remodeling in Asthmatic Mice through TLR2/NF-κB Signaling Pathway

目的基于TLR2/NF-κB信号通路研究平喘方及拆方改善卵清蛋白诱导的哮喘小鼠气道重塑的作用机制。方法将75只SPF级BALB/c雄性小鼠随机分为空白组、模型组、地塞米松组、平喘方组和平喘祛瘀方组,每组15只。除空白组外,其余组小鼠于第1日和第14日采用卵清蛋白联合氢氧化铝凝胶腹腔注射致敏,第21~27日连续予5%卵清蛋白雾化激发哮喘,每次雾化激发1 h后灌胃相应药液,连续7 d,空白组和模型组予等量蒸馏水灌胃。末次给药24 h后取材,HE染色观察小鼠肺组织病理变化,Masson染色观察肺组织支气管周围胶原纤维沉积,RT-qPCR检测肺组织平滑肌肌球蛋白重链(SM-MHC)、钙调蛋白(Calponin)、α-平滑肌肌动蛋白(α-SMA)、Toll样受体2(TLR2)、核因子(NF)-κB抑制因子α(IκBα)、NF-κBp65 mRNA表达,Western blot检测肺组织纤维连接蛋白1(FN1)、E钙黏蛋白(E-cadherin)、Ⅰ型胶原(CollagenⅠ)、TLR2、IκBα、NF-κBp65蛋白表达。结果与空白组比较,模型组小鼠支气管管腔狭窄,炎性浸润明显,肺间质可见大量胶原纤维沉积,肺组织SM-MHC、Calponin、IκBαmRNA表达显著降低(P<0.01),α-SMA、TLR2、NF-κBp65 mRNA表达显著升高(P<0.01),FN1、CollagenⅠ、TLR2、NF-κBp65蛋白表达显著升高(P<0.01),E-cadherin、IκBα蛋白表达显著降低(P<0.01);与模型组比较,地塞米松组、平喘方组及平喘祛瘀方组小鼠肺组织病理损伤减轻,炎性浸润及胶原纤维沉积减少,肺组织SM-MHC、Calponin、IκBαmRNA表达显著升高(P<0.01,P<0.05),α-SMA、TLR2、NF-κBp65 mRNA表达显著降低(P<0.01,P<0.05),FN1、CollagenⅠ、TLR2、NF-κBp65蛋白表达显著降低(P<0.01,P<0.05),E-cadherin蛋白表达显著升高(P<0.01),地塞米松组IκBα蛋白表达显著升高(P<0.01)。结论平喘方及拆方能明显改善哮喘小鼠气道重塑,其机制可能与抑制TLR2/NF-κB信号通路相关。

Objective To study the mechanism of Pingchuan Prescription and decomposed prescription for improving airway remodeling in asthmatic mice induced by ovalbumin through TLR2/NF-κB signaling pathway.Methods Totally 75 SPF BALB/c male mice were randomly divided into blank group,model group,dexamethasone group,Pingchuan Prescription group and Pingchuan Quyu Prescription group.Except the blank group,the other groups were sensitized with ovalbumin combined with aluminum hydroxide gel on 1st and 14th day,and then challenged with 5%ovalbumin by aerosol continuously from 21st to 27th day,1 hour after each nebulization stimulation,gavage administration for 7 consecutive days.The blank group and the model group were given equal amounts of distilled water by gavage.The mice were killed 24 hours after the last dose.HE staining was used to observe the histopathological changes in lung tissue,Masson staining was used to observe the deposition of collagen fibers around the bronchus.The mRNA expression of SM-MHC,Calponin,α-SMA,TLR2,IκBαand NF-κBp65 in lung tissue were detected by RT-qPCR.The protein expression of FN1,E-cadherin,CollagenⅠ,TLR2,IκBαand NFκBp65 in lung tissue were detected by Western blot.Results Compared with the blank group,the bronchial lumen was narrowed of the mice in model group,the infiltration of inflammatory material was obvious,and a large number of collagen fibers were seen in interstitium of the lung,the expression of SM-MHC,Calponin,IκBαmRNA in lung tissue were significantly decreased(P<0.01),the expression ofα-SMA,TLR2,NFκBp65 mRNA significantly increased(P<0.01),the expression of FN1,CollagenⅠ,TLR2,NF-κBp65 protein significantly increased(P<0.01),the expression of E-cadherin and IκBαprotein significantly decreased(P<0.01).Compared with the model group,the pathological damage of lung tissue in dexamethasone group,Pingchuan Prescription group,and Pingchuan Quyu Prescription group were reduced,the inflammatory infiltration and collagen fiber deposition were reduced,the expression of SM-MHC,Calponin,IκBαmRNA in lung tissue significantly increased(P<0.01,P<0.05),the expression ofα-SMA,TLR2,NFκBp65 mRNA significantly decreased(P<0.01,P<0.05),the expression of FN1,CollagenⅠ,TLR2,NFκBp65 protein significantly decreased(P<0.01,P<0.05),E-cadherin protein expression significantly increased(P<0.01),and IκBαprotein expression in dexamethasone group significantly increased(P<0.01).Conclusion Pingchuan Prescription and decomposed prescription can significantly improve airway remodeling in asthmatic mice,and the mechanism may be related to the inhibition of TLR2/NFκB signaling pathway.