基于NF-κB信号通路探讨芍药苷对黏蛋白5AC分泌的调控作用

Regulatory effect of paeoniflorin on mucin 5AC secretion based on NF-κB signaling pathway

目的:基于NF-κB信号通路探讨芍药苷调控气道黏蛋白5AC(MUC5AC)合成与分泌的影响及作用机制。方法:体内通过烟熏和慢性感染相结合的方法复制慢性阻塞性肺疾病(COPD)急性加重期大鼠模型,体外通过IL-1β诱导人气道上皮H292细胞建立炎症模型,设置空白组、模型对照组、NF-κB通路抑制剂组(PDTC组)、芍药苷组。采用动物肺功能测量仪检测大鼠肺功能;ELISA检测大鼠支气管肺泡灌洗液(BALF)、血清以及细胞上清中MUC5AC以及炎症因子IL-8、TNF-α表达水平;免疫印迹试验检测细胞P65、核因子κB抑制蛋白(IκB)蛋白表达水平;反转录实时荧光定量聚合酶链式反应(RT-PCR)检测各组细胞MUC5AC mRNA、囊性纤维化跨膜转导调节因子(CFTR)mRNA表达水平。结果:与空白组比较,模型对照组大鼠呼吸频率(f)、呼气峰流值(PEF)、潮气量(VT)、呼吸阻力(Rin)均显示有明显差异有统计学意义(P<0.05);与模型对照组比较,PDTC组、芍药苷组大鼠PEF、VT、Rin有明显改善(P<0.05),PDTC组呼吸频率(f)明显降低(P<0.05);与空白组比较,模型对照组大鼠支气管肺泡灌洗液(BALF)、血清以及细胞上清中MUC5AC、IL-8、TNF-α含量均明显增加(P<0.05),体外模型组细胞中P65蛋白和MUC5AC mRNA表达升高(P<0.05),IκB蛋白降低(P<0.05);与模型组比较,PDTC组、芍药苷组大鼠中BALF、血清及细胞上清中MUC5AC、IL-8、TNF-α含量均明显降低(P<0.05),且PDTC组、芍药苷高低剂量组的细胞中IκB蛋白和MUC5AC mRNA表达升高(P<0.05),P65蛋白和CFTR mRNA表达降低(P<0.05)。结论:芍药苷可改善COPD急性加重期大鼠模型的肺功能,减轻气道炎症反应,其机制与抑制NF-κB信号通路活性,降低气道细胞炎症因子分泌量以及调控MUC5AC mRNA、CTFR mRNA和相关蛋白的表达有关。

Objective:To explore the regulatory effect of paeoniflorin on mucin 5AC(MUC5AC)secretion based on NF-κB signaling pathway.Methods:In vivo experiments used a combination of smoke stimulation and chronic infection to replicate the acute exacerbation of COPD rat model,and in vitro experiments used IL-1βto induce human airway epithelial H292 cells to establish an inflammatory hypersecretion model.The experiments included control group,model group,NF-κB pathway inhibitor(PDTC)group,and paeoniflorin group.The pulmonary function of rats in each group was detected by an animal lung function measuring instrument;the expression level of MUC5AC,TNF-αand the inflammatory factor IL-8 in the bronchoalveolar lavage fluid(BALF),serum and cell supernatant of each group were detected by ELISA.The expression levels of P65 and inhibitor of nuclear factorκB(IκB)in each group were detected by Western blot test;the expression of MUC5AC mRNA and cystic fibrosis transmembrane transduction regulator(CFTR)mRNA were detected by reverse transcription real-time fluorescent quantitative polymerase chain reaction(RT-PCR).Results:Compared with control group,there were significant differences in respiratory frequency(f),peak expiratory flow(PEF),tidal volume(VT)and respiratory resistance(Rin)in model group(P<0.05).Compared with model group,the PEF,VT and Rin of rats in the PDTC group and paeoniflorin group were significantly improved(P<0.05),and at the same time the f in PDTC group was significantly reduced(P<0.05).Compared with control group,the contents of MUC5AC,IL-8 and TNF-αin the bronchoalveolar lavage fluid(BALF),serum and cell supernatant of model group increased significantly(P<0.05),and the expression of P65 protein and MUC5AC mRNA increased significantly(P<0.05).However,the IκB protein decreased significantly(P<0.05).Compared with model group,the contents of MUC5AC,IL-8 and TNF-αin BALF,serum and cell supernatant of rats in paeoniflorin group and PDTC group were reduced significantly(P<0.05),and the expression of IκB protein and CFTR mRNA in PDTC group,high and low dose groups of paeoniflorin increased significantly(P<0.05),and the expression of P65 protein,MUC5AC mRNA decreased significantly(P<0.05).Conclusion:Paeoniflorin can improve pulmonary function and reduce airway inflammation of rats in acute exacerbation of COPD,and the mechanism may be related to inhibiting the activity of NF-κB signaling pathway,reducing the secretion of inflammatory factors in airway cells,and regulating the expressions of MUC5AC mRNA,CTFR mRNA and related proteins.