黄芩苷通过NF-κB信号对气道上皮黏液调节因子MUC5AC和CFTR表达的影响

Effects of baicalin on expression of mucin 5AC and cystic fibrosis transmembrane conductance regulator in airway epithelium through NF-κB signaling pathway

目的基于核因子κB(nuclear factor kappa B,NF-κB)信号探讨黄芩苷调控气道上皮黏蛋白5AC(mucin 5AC,MUC5AC)和囊性纤维化跨膜传导调节因子(cystic fibrosis transmembrane conductance regulator,CFTR)的表达以改善病理性气道黏液的机制。方法采用MTT法检测不同浓度黄芩苷对H292细胞(人肺癌细胞)活性的影响并确定用于后续实验的黄芩苷浓度;将细胞分为空白对照组、模型组、NF-κB抑制剂组(PDTC组,100μmol/L)和黄芩苷组(200μg/mL)。采用IL-1β诱导H292细胞建立体外黏液高分泌炎症细胞模型,再以相应浓度的药物干预24 h后,采用ELISA方法检测各组细胞上清中MUC5AC、IL-8、TNF-α的含量,检测各组细胞中MUC5AC和CFTR蛋白的含量;采用实时荧光定量PCR检测各组细胞中MUC5AC和CFTR mRNA的表达;采用Western blot检测各组细胞中p65和IκB的表达。结果MTT检测结果显示,当黄芩苷浓度为200μg/mL时,细胞活性在80%以上,可作为后续实验的安全浓度。与空白对照组比较,模型组细胞培养上清及细胞质中MUC5AC分泌水平及IL-8、TNF-α、p65、MUC5AC mRNA表达均明显升高(P<0.05),IκB蛋白表达、CFTR蛋白含量、CFTR mRNA的表达显著降低(P<0.05);与模型组比较,PDTC组细胞培养上清中MUC5AC分泌水平,IL-8、TNF-α、p65蛋白表达及MUC5AC mRNA表达显著降低(P<0.05),细胞质中MUC5AC水平、CFTR蛋白含量、IκB蛋白表达、CFTR mRNA的表达显著升高(P<0.05);与模型组比较,黄芩苷组细胞培养上清中MUC5AC分泌水平、IL-8和TNF-α分泌水平、p65蛋白表达降低(P<0.01),CFTR蛋白含量、IκB蛋白表达、CFTR mRNA表达均显著增加(P<0.01),而MUC5AC mRNA表达差异无统计学意义。结论黄芩苷通过抑制NF-κB活性,减少MUC5AC的分泌,增加CFTR的表达,减轻气道炎症。

ObjectiveTo investigate the mechanism of baicalin in improving pathological airway mucus by regulating the expression of airway epithelial mucus regulators mucin 5AC(MUC5AC)and cystic fibrosis transmembrane conductance regulator(CFTR)through nuclear factorκB(NF-κB)signaling pathway.MethodsMTT assay was used to detect the effects of different concentrations of baicalin on the viability of human lung cancer H292 cells in order to determine the proper dose of baicalin for subsequent experiments.After H292 cells were induced by IL-1βto establish in vitro inflammatory cell model of mucus hypersecretion,the cells were divided into blank control group,model group,NF-κB inhibitor group(PDTC group,100μmol/L)and baicalin group(200μg/mL).After 24 h of drug intervention,the contents of MUC5AC,IL-8,and TNF-αin the culture supernatant and the levels of MUC5AC and CFTR in the cytoplasm of each group were detected by ELISA.Real-time fluorescent quantitative PCR was adopted to detect the mRNA expression of MUC5AC and CFTR.Western blotting was performed to test the expression of p65 and IκB(inhibitor of NF-κB).ResultsThe results of MTT assay showed that 200μg/mL was a safe concentration of baicalin for subsequent experiments,with cell viability larger than 80%.As compared with the blank control group,the protein expression of MUC5AC,IL-8,TNF-α,p65 and the mRNA level of MUC5AC in culture supernatant and cytoplasm were all significantly increased in the model group(P<0.05),while the expression of IκB and CFTR proteins as well as the CFTR mRNA level were remarkably decreased(P<0.05).When compared with the model group,the PDTC group presented lower level of MUC5AC secretion,declined protein levels of IL-8,TNF-αand p65,as well as MUC5AC mRNA in the culture supernatant(P<0.05),while higher protein expression of MUC5AC,CFTR and IκB in the cytoplasm,along with elevated mRNA level of CFTR(P<0.05).In terms of the baicalin group,the secretion levels of MUC5AC,IL-8 and TNF-αwere greatly diminished in culture supernatant(P<0.01)as compared with the model group,whereas the expression of IκB as well as the protein and mRNA levels of CFTR notably improved(P<0.01),and there was no significant difference in MUC5AC mRNA expression.ConclusionBaicalin reduces MUC5AC secretion and increases CFTR expression by inhibiting the activity of NF-κB,and thus attenuates airway inflammation.