固本防哮饮对IL-4联合IL-13诱导的人支气管上皮细胞中CGRP/ILC2及GABA表达的影响  

Guben Fangxiao decoction Regulates Expression of CGRP/ILC2 and GABA in Human Bronchial Epithelial Cells Stimulated with IL-4 Combined with IL-13

作　　者：欧炜莹 袁雪晶[1] 纪建建 OU Weiying;YUAN Xuejing;JI Jianjian(Affiliated Hospital of Nanjing University of Chinese Medicine,Nanjing 210029,China;The First Clinical Medical College of Nanjing University of Chinese Medicine,Nanjing 210023,China)

机构地区：[1]南京中医药大学附属医院,南京210029 [2]南京中医药大学第一临床医学院,南京210023

出　　处：《中国实验方剂学杂志》2025年第2期93-100,共8页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：第五批全国中医临床优秀人才研修项目(国中医药人教函〔2022〕1号);第七批全国老中医药专家学术经验继承工作项目(国中医药人教函〔2022〕76号);江苏省中医药管理局科技发展计划重点项目(ZD202308);江苏省中医院科主任提升专项(Y2021ZR23)。

摘　　要：目的:观察固本防哮饮含药血清对白细胞介素-4(IL-4)和白细胞介素-13(IL-13)刺激的人支气管上皮细胞(BEAS-2B)中降钙素相关基因肽(CGRP)、γ-氨基丁酸(GABA)及Ⅱ型固有淋巴细胞(ILC2)相关转录因子、细胞因子的表达,探究其改善支气管上皮细胞Ⅱ型炎症的可能机制。方法:用含有高(20%)、中(15%)、低(10%)体积分数的固本防哮饮含药血清及孟鲁斯特钠(10%)含药血清对IL-4和IL-13联合刺激的BEAS-2B细胞进行干预,实时荧光定量聚合酶链式反应(Realtime PCR)检测各组细胞中CGRP、GABA、黏蛋白(MUC)5AC、胸腺基质淋巴细胞生成素(TSLP)、白细胞介素-25(IL-25)、白细胞介素-33(IL-33)、白细胞介素-5(IL-5)、GATA结合蛋白3(GATA3)、B细胞淋巴瘤-11B(Bcl-11B)mRNA表达;蛋白免疫印迹法(Western blot)检测各组细胞CGRP、MUC5AC蛋白相对表达;免疫荧光技术检测CGRP和GABA在细胞中的相对表达;酶联免疫吸附测定法(ELISA)检测培养基上清和细胞上清中IL-33、MUC5AC、IL-5的蛋白含量;过碘酸-雪夫反应(PAS)染色检测黏蛋白的分泌情况。结果:IL-4联合IL-13刺激细胞后,与正常组比较,各给药组CGRP、GABA、MUC5AC、TSLP、IL-25、IL-33 mRNA表达量明显上升(P<0.05,P<0.01);与模型组比较,固本防哮饮含药血清各剂量组可显著降低造模后细胞中CGRP、GABA、MUC5AC、IL-5、GATA3、Bcl-11B mRNA表达(P<0.05,P<0.01),并表现出剂量依赖性。与正常组比较,模型组CGRP、GABA、MUC5AC、IL-33和IL-5蛋白表达明显增加(P<0.05,P<0.01);与模型组比较,固本防哮饮含药血清各剂量组细胞中CGRP、GABA、MUC5AC、IL-33和IL-5的蛋白表达明显降低(P<0.05,P<0.01)。与正常组比较,模型组CGRP和GABA的平均荧光强度显著升高(P<0.01);与模型组比较,固本防哮饮含药血清各剂量组中CGRP和GABA的平均荧光强度显著降低(P<0.01)。与正常组比较,模型组中黏蛋白分泌增多;与模型组比较,固本防哮饮含药血清各剂量组中黏Objective:To observe the effects of Guben Fangxiao decoction-containing serum on the expression of transcription factors and cytokines associated with calcitonin gene-related peptide(CGRP),γ-aminobutyric acid(GABA),and type Ⅱ innate lymphoid cells(ILC2)in human bronchial epithelial cells(BEAS-2B)stimulated with interleukin-4(IL-4)combined with interleukin-13(IL-13),and explore the possible mechanism of Guben Fangxiao decoction-containing serum in alleviating type Ⅱ inflammation of bronchial epithelial cells.Methods:BEAS-2B cells stimulated with IL-4 combined with IL-13 were treated with the sera containing high(20%),medium(15%),and low(10%)doses of Guben Fangxiao decoction and the montelukast-containing serum(10%).Real-time PCR was used to measure the mRNA levels of CGRP,GABA,mucin 5AC(MUC5AC),thymic stromal lymphopoietin(TSLP),interleukin(IL)-25,IL-33,IL-5,GATA-binding protein 3(GATA3),and B cell lymphoma/leukemia 11B(Bcl-11B)in each group.Western blot was employed to determine the relative protein levels of CGRP and MUC5AC.The immunofluorescence assay was employed to observe the relative expression of CGRP and GABA in the cells.Enzyme-linked immunosorbent assay was used to quantify the protein levels of IL-33,MUC5AC,and IL-5 in the culture and cell supernatants.Periodic acid-Schiff staining was performed to assess mucin secretion.Results:Compared with the control group,the model group showed up-regulated mRNA levels of CGRP,GABA,MUC5AC,TSLP,IL-25,and IL-33(P<0.05,P<0.01).Compared with the normal group,the Guben Fangxiao decoction-containing serum down-regulated the mRNA levels of CGRP,GABA,MUC5AC,IL-5,GATA3,and Bcl-11B(P<0.05,P<0.01)in a dose-dependent manner.Compared with the normal group,the model group showed up-regulated protein levels of CGRP,GABA,MUC5AC,IL-33,and IL-5(P<0.05,P<0.01),which were down-regulated by the Guben Fangxiao decoction-containing serum(P<0.05,P<0.01).Compared with the normal group,the model group showed enhanced average fluorescence intensity of CGRP and GABA(P<0.01),which was we

关 键 词：支气管哮喘 固本防哮饮 降钙素相关基因肽 Γ-氨基丁酸 Ⅱ型固有淋巴细胞 

分 类 号：R255[医药卫生—中医内科学] R285[医药卫生—中医学] R562