基于糖皮质激素受体介导TGF-β_(1)/Smad3信号轴探讨小青龙汤加减合二陈汤治疗咳嗽变异性哮喘的药效及机制

Based on Glucocorticoid Receptor-Mediated TGF-β_(1)/Smad3 Signal Axis to Explore the Efficacy and Mechanism of Xiaoqinglong Decoction(小青龙汤)and Erchen Decoction(二陈汤)in the Treatment of Cough Variant

目的:观察小青龙汤加减合二陈汤治疗咳嗽变异性哮喘(CVA)的药效,并基于糖皮质激素受体(GR)介导TGF-β_(1)/Smad3信号轴探讨其作用机制。方法:60只SD雄性大鼠通过随机数字表法分为对照组、模型组、布地奈德组、小青龙汤加减合二陈汤低剂量组、小青龙汤加减合二陈汤高剂量组,每组12只。适应性饲养结束后,除对照组外,其余大鼠均采用氢氧化铝和卵清白蛋白(OA)激发联合OA雾化刺激制备大鼠CVA模型,筛选建模成功的大鼠进行药物灌胃治疗,连续给药2周。末次给药1 d后处死大鼠,采用ELISA法检测血清Th1/Th2类细胞因子及支气管肺泡灌流液中LTE-4、NGF水平;HE染色观察支气管-肺组织病理学改变;通过Western blotting法检测支气管-肺组织中TGF-β_(1)、Smad3、GR蛋白表达。结果:CVA大鼠支气管-肺组织出现病理性改变,主要包括支气管管腔变窄、炎性细胞浸润等;血清IL-4、ECP水平均升高,IFN-γ水平降低;支气管肺泡灌流液中LTE-4、NGF水平均升高;支气管-肺组织中TGF-β_(1)、Smad3蛋白表达水平升高,GR蛋白表达水平降低。采用小青龙汤加减合二陈汤治疗后,CVA大鼠支气管-肺组织病理性改变程度明显减轻;与模型组比较,血清IL-4、ECP水平均明显降低,IFN-γ水平明显升高;支气管肺泡灌流液中LTE-4、NGF水平均明显降低;支气管-肺组织中TGF-β_(1)、Smad3蛋白表达水平明显降低,GR蛋白表达水平明显升高。结论:小青龙汤加减合二陈汤可以有效减轻CVA大鼠支气管-肺组织损伤,并抑制机体的炎症反应,其作用机制可能与GR介导TGF-β_(1)/Smad3信号轴有关。

Objective:To observe the efficacy of Xiaoqinglong Decoction and Erchen Decoction in the treatment of cough variant asthma(CVA),and to explore its mechanism of action based on the glucocorticoid receptor(GR)mediated TGF-β_(1)/Smad3 signal axis.Methods:A total of 60 SD male rats were divided into control group,model group,budesonide group,Xiaoqinglong Decoction and Erchen Decoction low-dose group,Xiaoqinglong Decoction and the Erchen Decoction high-dose group by the random number table method,with 12 rats in each group.After the adaptive feeding,except for the control group,the remaining rats were used to prepare the rat CVA model by aluminum hydroxide and oval-protein(OA)stimulation combined with OA atomization stimulation,and the rats with successful modeling were selected for drug gavage treatment,and the rats were continuously given the drug for 2 weeks.The rats were sacrificed 1 d after the last administration,and the levels of serum Th1/Th2 cytokines and LTE-4 and NGF in bronchoalveolar perfusion fluid were detected by ELISA.HE staining was used to observe the histopathological changes of bronchus lung.Western blotting was used to detect the expression of TGF-β_(1),Smad3 and GR protein in broncho-pulmonary tissues.Results:Pathological changes occurred in the bronchial-lung tissue of CVA rats,including narrowing of the bronchial lumen and inflammatory cell infiltration.Compared with the control group,the serum IL-4 and ECP levels were increased,the levels of IFN-γwere decreased,the levels of LTE-4 and NGF in bronchoalveolar perfusion fluid were increased,the expression levels of TGF-β_(1) and Smad3 protein in bronchial-lung tissue were increased,and the expression level of GR protein were decreased in the model group.After treatment with Xiaoqinglong Decoction and Erchen Decoction,the degree of pathological changes in the bronchial-lung tissue of CVA rats was significantly decreased.Compared with the model group,the serum IL-4 and ECP levels were significantly decreased,IFN-γlevels were significantly increased,the LTE-4 and NGF levels in bronchoalveolar perfusion fluid were significantly decreased,the expression levels of TGF-β_(1) and Smad3 protein in bronchial-lung tissue were significantly decreased,and the expression levels of GR protein were significantly increased.Conclusion:Xiaoqinglong Decoction and Erchen Decoction can effectively alleviate bronchial-lung tissue damage in CVA rats and inhibit the body’s inflammatory response.Its mechanism may be related to the GR-mediated TGF-β_(1)/Smad3 signal axis.