基于上皮细胞-间充质转化的搜风愈喘方调控哮喘大鼠气道重塑的机制研究

Mechanism Study of Soufeng Yuchuan Formula on Regulating Airway Remodeling in Asthmatic Rats Based on Epithelial-Mesenchymal Transformation

目的探讨搜风愈喘方通过调控上皮细胞-间充质转化(EMT)抑制哮喘气道重塑的作用机制。方法将60只大鼠随机分为正常对照组、模型对照组、地塞米松组及搜风愈喘方高、中、低剂量组,每组10只。除正常对照组外,其余各组均采用卵清白蛋白(OVA)注射和雾化复制哮喘大鼠模型。各组大鼠给予相应药物灌胃21 d后处死大鼠,苏木素-伊红(HE)染色,观察肺组织病理形态学变化;透射电镜观察大鼠肺组织中上皮细胞超微结构情况;酶联免疫吸附测定法(ELISA)检测血清中的转化生长因子β1(TGF-β1)、白细胞介素17(IL-17)的含量;实时荧光定量聚合酶链式反应(RT-PCR)法检测肺组织匀浆中TGF-β1、α-平滑肌肌动蛋白(α-SMA)、E-钙黏蛋白(E-cadherin)和N-钙黏蛋白(N-cadherin)的mRNA表达。蛋白免疫印迹法(Western Blot)检测肺组织中TGF-β1、α-SMA、E-cadherin、N-cadherin蛋白表达水平。结果与正常对照组比较,模型对照组大鼠血清中TGF-β1、IL-17含量明显升高(P<0.01),肺组织中TGF-β1、α-SMA和N-cadherin mRNA和蛋白表达明显上调(P<0.01),E-cadherin mRNA和蛋白表达明显下调(P<0.01);HE染色显示气道壁及肺泡壁增厚,视野内可见大量炎症细胞浸润,炎症评分明显升高(P<0.01);透射电镜显示肺组织中上皮细胞水肿程度严重,胞内基质局部溶解、变淡,细胞器肿胀明显。与模型对照组比较,地塞米松组和搜风愈喘方各剂量组大鼠血清中TGF-β1、IL-17含量明显降低(P<0.05,P<0.01),TGF-β1、α-SMA和N-cadherin mRNA和蛋白表达明显下调(P<0.05,P<0.01),E-cadherin mRNA和蛋白表达明显上调(P<0.05,P<0.01);肺组织炎症细胞浸润减轻,支气管壁及肺泡壁增厚程度减轻,地塞米松组和搜风愈喘方高剂量组炎症评分降低(P<0.05);超微结构显示上皮细胞水肿程度明显减轻,细胞膜完整,胞内基质均匀,细胞器大多轻微肿胀。结论搜风愈喘方可降低TGF-β1水平,从而改善肺组织EMT,达到防治哮喘气道重塑的目的。

Objective To explore the mechanism of Suofeng Yuchuan Formula on inhibiting airway remodeling in asthma by regulating epithelial-mesenchymal transformation(EMT).Methods Sixty rats were randomly divided into normal control group,model control group,Dexamethasone group,high-,medium-and low-dose groups of Soufeng Yuchuan Formula,with 10 rats in each group.In addition to the normal control group,the other groups were injected and inhaled with ovalbumin(OVA)to replicate the asthma rat model.The rats in each group were killed after 21 days of administration of the corresponding drug.Hematoxylin-eosin(HE)staining was used to observe the pathological changes of lung tissue.The ultrastructure of epithelial cells in rat lung was observed by transmission electron microscope.The contents of transforming growth factorβ1(TGF-β1)and interleukin-17(IL-17)in serum were detected by enzyme-linked immunosorbent assay(ELISA).Real-time fluorescence quantitative polymerase chain reaction(RT-PCR)was used to detect mRNA expressions of TGF-β1,α-smooth muscle actin(α-SMA),E⁃cadherin and N⁃cadherin in lung homogenate.Protein expression levels of TGF-β1,α-SMA,Ecadherin and N-cadherin in lung tissue were detected by Western Blot.Results Compared with the normal control group,the serum contents of TGF-β1 and IL-17 in the model control group were significantly increased(P<0.01).The mRNA and protein expressions of TGF-β1,α-SMA and N-cadherin in lung tissues were significantly up-regulated(P<0.01),while the mRNA and protein expressions of E-cadherin were significantly down-regulated(P<0.01).HE staining showed thickening of airway wall and alveolar wall,and a large number of inflammatory cells infiltrated in the visual field,and the inflammation score was significantly increased(P<0.01).Transmission electron microscopy showed that the epithelial cells of lung tissue had severe edema.Local dissolution and thinning of intracellular matrix,and obvious swelling of organelles were observed.Compared with model control group,the contents of TGF-β1 and IL-17 in serum of dexamethasone group and all doses of Soufeng Yuchuan Formula groups were significantly decreased(P<0.05,P<0.01).The mRNA and protein expressions of TGF-β1,α-SMA and Ncadherin were significantly down-regulated(P<0.05,P<0.01),while the mRNA and protein expressions of Ecadherin were significantly up-regulated(P<0.05,P<0.01).The inflammatory cell infiltration of lung tissue and thickening of bronchial wall and alveolar wall were found to be reduced.The inflammation score of dexamethasone group and high-dose group of Suofeng Yuchuan Formula was reduced(P<0.05).Ultrastructure showed that the degree of edema of epithelial cells was significantly reduced,the cell membrane was intact,the intracellular matrix was uniform,and most of the organelles were slightly swollen.Conclusion Soufeng Yuchuan Formula can reduce the level of TGF-β1,thereby improve EMT in lung tissue,and achieve the purpose of preventing and treating airway remodeling in asthma.