基于白细胞介素13/信号传导和转录激活因子6信号通路研究甘草酸二铵对慢性阻塞性肺疾病大鼠气管黏液高分泌的影响

Effects of diammonium glycyrrhizinate on tracheal mucus hypersecretion in rats with COPD based on the IL-13/STAT6 signaling pathway

目的 基于白细胞介素13(IL-13)/信号传导和转录激活因子6(STAT6)信号通路观察甘草酸二铵盐对慢性阻塞性肺疾病(COPD)大鼠气管黏液高分泌的影响。方法 2019年6月至2020年2月,从珠海百试通生物科技有限公司购买健康雄性SD大鼠。将SD雄性大鼠按随机数字表法分为正常组、模型组、盐酸氨溴索组和甘草酸二铵低、中、高剂量组,每组15只。采用香烟烟熏联合脂多糖(LPS)建立COPD大鼠模型,除正常组和模型组注射生理盐水外,其余各组均于尾部静脉注射相应剂量的药物,每天1次,持续14 d。通过检测各组大鼠的肺阻力(RI)、肺顺应性(Cdyn)以及酚红排痰实验反映大鼠肺功能变化情况;ELISA检测肺泡灌洗液中IL-13的水平变化;HE染色反映肺组织的病理学变化;实时定量荧光PCR(qRT-PCR)和Western blotting检测各组肺组织中IL-13、STAT6和黏蛋白5AC(MUC5AC)mRNA及蛋白表达水平。结果 与正常组相比,模型组大鼠Cdyn降低,RI[(2.39±0.30)cmH2O·mL-1·s-1比(0.85±0.11)cmH2O·mL-1·s-1]、排痰量[(1.09±0.14)mg/L比(0.55±0.07)mg/L]、肺泡灌洗液中IL-13[(34.28±5.88)ng/L比(10.36±1.67)ng/L]水平显著升高(P<0.05),肺组织出现病理学损伤,p-STAT6/STAT6[(1.33±0.23)比(0.72±0.14)]水平以及IL-13蛋白[(0.47±0.07)比(0.17±0.03)]和MUC5AC mRNA及蛋白[(0.78±0.15)比(0.15±0.04)]水平均有升高(P<0.05);与模型组相比,甘草酸二铵低、中、高剂量组和盐酸氨溴索组Cdyn增加,RI、排痰量、肺泡灌洗液中IL-13水平显著下降(P<0.05),肺组织损伤程度降低,p-STAT6/STAT6以及IL-13蛋白和MUC5AC mRNA及蛋白水平均有降低(P<0.05);其中甘草酸二铵组作用效果最为明显。结论 甘草酸二铵能抑制COPD模型大鼠IL-13/STAT6信号通路,并可能藉此减轻气管黏液高分泌,改善气管阻塞。

Objective obstructive pulmonary disease(COPD) based on the interleukin-13(IL-13)/signal transducer and activator of transcription 6(STAT6)signaling pathway.Methods From June 2019 to February 2020,healthy male Sprague-Dawley(SD) rats were purchased from Zhuhai BesTest Bio-Tech Co.,Ltd.The rats were divided into the normal group,model group,ambroxol hydrochloride group and diammonium glycyrrhizinate low-dose,medium-dose and high-dose groups according to the random number table method,with 15 rats in each group.The COPD rat model was established by cigarette smoking combined with lipopolysaccharide(LPS).Except for the normal group and the model group,which were injected with normal saline,the other groups were injected with the corresponding dose of drugs through the tail vein once a day for 14 days.The pulmonary resistance(RI),lung compliance(Cdyn) and phenol red sputum excretion test of rats in each group were detected to reflect the changes in pulmonary function.The level of IL-13 in bronchoalveolar lavage fluid(BALF)was detected by enzyme-linked immunosorbent assay(ELISA).The pathological changes in the lung tissue were reflected by HE staining.Quantitative real-time PCR(qRT-PCR) and Western blotting were used to detect the mRNA and protein expression levels of IL-13,STAT6 and mucin 5ac(MUC5AC).Results Compared with the normal group,the rats in the model group had lower Cdyn,RI [(2.39±0.30) cmH2O·mL-1·s-1vs.(0.85±0.11) cmH2O·mL-1·s-1],sputum volume [(1.09±0.14) mg/L vs.(0.55±0.07) mg/L] and IL-13 [(34.28±5.88) ng/L vs.(10.36±1.67) ng/L] levels in BALF were significantly increased(P < 0.05);lung tissue showed pathological damage,pSTAT6/STAT6 [(1.33±0.23) vs.(0.72±0.14)] level and IL-13 protein [(0.47±0.07) vs.(0.17±0.03)] and MUC5AC mRNA and protein[(0.78±0.15) vs.(0.15±0.04)] levels were increased(P < 0.05).Compared with the model group,Cdyn increased,and RI,sputum volume,and IL-13 levels in bronchoalveolar lavage fluid decreased significantly in the low-,medium-and high-dose groups of diammonium glycyrrhizinate and ambroxol hydrochloride group(P < 0.05).The degree of lung tissue injury was reduced,and the levels of pSTAT6/STAT6,IL-13 protein and MUC5AC mRNA and protein were decreased(P < 0.05),and the effect of diammonium glycyrrhizinate group was the most obvious.Conclusions Diammonium glycyrrhizinate can inhibit the IL-13/STAT6 signaling pathway in COPD model rats,and may reduce tracheal mucus hypersecretion and improve tracheal obstruction.