摘要
目的:探讨搜风愈喘方调控转化生长因子-β1及其信号转导蛋白Smad(TGF-β1/Smad)信号通路干预哮喘大鼠气道重塑的作用机制。方法:将32只SPF级SD雄性大鼠随机分为正常对照组、模型对照组、地塞米松0.000125 g/kg组和搜风愈喘方11.4 g/kg组,除正常对照组外,其余大鼠均制备慢性哮喘模型,造模成功后各组分别予蒸馏水和对应药物灌胃,连续21 d。观察各组一般情况;ELISA法检测大鼠支气管肺泡灌洗液(BALF)中肺组织转化生长因子-β1(TGF-β1)、血管内皮生长因子(VEGF)和白细胞介素-13(IL-13)含量;HE染色、PAS染色和Masson染色观察大鼠肺组织病理改变、杯状细胞分泌黏液及气道胶原沉积情况;记录支气管管腔内周长(Pi)、气道管壁面积(WAt)、气道平滑肌层面积(WAm)、观察大鼠气道重塑情况;透射电镜观察大鼠肺组织中上皮细胞和平滑肌细胞情况;Real-time PCR法检测大鼠肺组织Tgfb1、Smad2、Smad3、α-平滑肌肌动蛋白(α-smooth muscle actin,Asma)、Vegf和Il13 mRNA表达;Western blot法检测大鼠肺组织TGF-β1、Smad2、Smad3、α-SMA蛋白表达。结果:与正常对照组比较,模型对照组大鼠存在呼吸深快、烦躁喘促等症状且BALF中TGF-β1、VEGF和IL-13含量显著升高(P<0.01);肺组织中广泛可见炎性细胞浸润、杯状细胞增生以及胶原纤维沉积;气道重塑相关指标显著升高(P<0.01);肺组织超微结构显示肺组织中上皮细胞和平滑肌细胞水肿程度严重,胞内基质溶解、变淡,胶原纤维排列紊乱,细胞器和线粒体肿胀;Tgfb1、Smad2、Smad3、Asma、Vegf和Il13 mRNA相对表达显著上调(P<0.01);TGF-β1、Smad2、Smad3、α-SMA蛋白表达显著上调(P<0.01);与模型对照组比较,地塞米松0.000125 g/kg组和搜风愈喘方11.4 g/kg组大鼠呼吸平缓,精神活跃;大鼠BALF中TGF-β1、VEGF、IL-13含量显著降低(P<0.01),支气管周围炎性细胞浸润程度减轻、杯状细胞增生不明显且胶原纤维沉积情况明显改善,气道重塑相关指标明显降低(P<0.05或P<0.01);超微结构显示肺组织上皮细胞和平滑肌细胞水肿程度明显减轻,胞内基质均匀,线粒体未见明显肿胀;Tgfb1、Smad2、Smad3、Asma、Vegf和Il13 mRNA表达下调(P<0.05或P<0.01);TGF-β1、Smad2、Smad3、α-SMA蛋白表达明显下调(P<0.05或P<0.01)。结论:搜风愈喘方可通过减少细胞外基质合成、抑制平滑肌细胞增生和上皮间质转化(EMT),干预哮喘大鼠气道重塑,降低TGF-β1、VEGF和IL-13含量,且与抑制TGF-β1/Smad2/3信号通路传导,下调TGF-β1、Smad2、Smad3、α-SMA表达相关。
Objective:To explore the mechanism of Soufeng Yuchuan(搜风愈喘)Recipe against airway remodeling in asthmatic rats by regulating the transforming growth factorβ1 and its signal transduction protein Smad(TGF-β1/Smad)signaling pathway.Methods:Thirty two male SPF SD rats were randomly divided into normal control group,model group,0.000125 g/kg dexamethasone group and 11.4 g/kg Soufeng Yuchuan Recipe group.Except for the normal control group,chronic asthma model was prepared among the other rats.After successful modeling,each group was orally administrated with distilled water or corresponding drugs for 21 consecutive days,and then the general situation of rats was observed.The levels of TGF-β1,vascular endothelial growth factor(VEGF)and interleukin-13(IL-13)in bronchoalveolar lavage fluid(BALF)were determined by enzyme-linked immunosorbent assay(ELISA).Hematoxylin-eosin(HE)staining,pereonic acid-Schifff's(PAS)staining and Masson staining were used to detect the pathological changes of lung tissue,the secretion of mucus by goblet cells and the deposition of collagen in airway of rats.The internal perimeter of bronchial lumen(Pi),total airway wall area(WAt)and airway smooth muscle area(WAm)were recorded to observe the airway remodeling of rats.The epithelial cells and smooth muscle cells in lung tissue of rats were observed by transmission electron microscope.The mRNA expressions of Tgf-β1,Smad2,Smad3,α-smooth muscle actin(α-Sma),Vegf and Il-13 in lung tissue of rats were detected by real-time quantitative polymerase chain reaction(Real-time PCR),while the protein expressions of TGF-β1,Smad2,Smad3 andα-SMA were detected by Western blot.Results:Compared with the normal control group,the model group had symptoms such as deep rapid breathing,irritability and shortness of breath,and increased TGF-β1,VEGF and IL-13 in the BALF(P0.01);inflammatory cell infiltration,goblet cell hyperplasia and collagen fiber deposition were widely observed in lung tissue;the related indexes of airway remodeling were elevated(P0.01);the ultrastructure of lung tissue indicated severe edema of epithelial cells and smooth muscle cells in lung tissue with degraded and weakened intracellular matrix,disordered collagen fibers,and swollen organelles and mitochondria;there was an up-regulation in the relative mRNA expressions of Tgf-β1,Smad2,Smad3,α-Sma,Vegf and Il-13(P0.01)as well as in the protein expressions of TGF-β1,Smad2,Smad3 and α-SMA(P0.01).Compared with the model group,the 0.000125 g/kg dexamethasone group and 11.4 g/kg Soufeng Yuchuan Recipe group presented gentle breathing and active condition,and decreased TGF-β1,VEGF and IL-13 in BALF(P0.01);there was alleviated infiltration degree of inflammatory cells around bronchus,indistinct goblet cell hyperplasia and improved collagen fiber deposition,and the related indexes of airway remodeling were lowered(P0.05 or P0.01);the ultrastructure of lung tissue revealed that epithelial cells and smooth muscle cells had reduced edema with uniform intracellular matrix,and there was no obvious swelling of mitochondria;the mRNA expressions of Tgf-β1,Smad2,Smad3,α-Sma,Vegf and Il-13 were down regulated(P0.05 or P0.01),and the protein expressions of TGF-β1,Smad2,Smad3 andα-SMA were also down regulated(P0.05 or P0.01).Conclusion:Soufeng Yuchuan Recipe intervenes in airway remodeling in asthmatic rats by reducing extracellular matrix synthesis,inhibiting smooth muscle cell hyperplasia and epithelial-mesenchymal transition(EMT).Moreover,it reduced the contents of TGF-β1,VEGF and IL-13,and the effect of Soufeng Yuchuan recipe against airway remodeling is correlated with the inhibition of TGF-β1/Smad2/3 signaling pathway and the down regulation of TGF-β1,Smad2,Smad3,α-SMA.
作者
郭旭冉
郑海涛
陈晨
李牧瑶
张宇婧
梁磊
闫永彬
GUO Xuran;ZHENG Haitao;CHEN Chen;LI Muyao;ZHANG Yujng;LIANG Lei;YAN Yongbin(School of Pediatrics,Henan University of Chinese Medicine,Zhengzhou 450000;The First Affiliated Hospital of Henan University of Chinese Medicine,Zhengzhou 450000)
出处
《中药药理与临床》
CAS
CSCD
北大核心
2023年第7期14-20,共7页
Pharmacology and Clinics of Chinese Materia Medica
基金
国家自然科学基金面上项目(编号:81973903、82174438)
河南省中医药科学研究专项重点课题(编号:20-21ZY1002)
河南中医药大学2021年度研究生科研创新项目(编号:2021KYCX048)。
关键词
搜风愈喘方
哮喘
气道重塑
转化生长因子-β1/Smad2/3信号通路
Α-平滑肌肌动蛋白
上皮-间质转化
Soufeng Yuchuan(搜风愈喘)Recipe
Asthma
Airway remodeling
Transforming growth factorβ1(TCF-β1)/signal transduction protein(Smad)2/3 signaling pathway
α-smooth muscle actin
Epithelial-mesenchymal transition
