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(Real-time 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蛋白的分泌减少.结论:固本防哮饮通过减轻Ⅱ型免疫性气道炎症和减少气道黏液分泌,达到防治支气管哮喘的目的,其可能的机制是固本防哮饮降低了 CGRP、GABA、MUC5AC及ILC2相关转录因子的表达.
Abstract
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 weakened by the Guben Fangxiao decoction-containing serum(P＜0.01).Compared with the normal group,the model group showed increased secretion of mucin,which was reduced by the Guben Fangxiao decoction-containing serum.Conclusion:Guben Fangxiao decoction can treat bronchial asthma by alleviating type Ⅱ immune airway inflammation and reducing airway mucus secretion,which is achieved by regulating the expression of CGRP,GABA,MUC5AC,and ILC2-related transcription factors.
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