麻杏石甘汤对哮喘模型大鼠气道损伤及EGFR表达的影响

Effect of Maxing Shigantang on Airway Injury and EGFR Expression in Asthma Model Rats

目的:探讨麻杏石甘汤调节表皮生长因子受体(EGFR)表达、减轻大鼠气道损伤的作用,并找出其治疗哮喘较优的配伍比例。方法:SD雄性大鼠随机分为正常组、模型组、地塞米松组(5×10^(-4)g·kg^(-1))及麻杏石甘汤1∶0.5、1∶1、1∶2组(中药A、B、C组,剂量均为10 g·kg^(-1)),每组8只,除正常组外,其余各组用2%氯化乙酰胆碱与0.4%磷酸组胺雾化复制哮喘模型,造模前1 h,正常组和模型组大鼠灌服等体积生理盐水,其余各给药组灌服等体积相应药物,1次/d,连续7 d。第7天造模并记录引喘潜伏期后即刻麻醉,取动脉血和气管组织。酶联免疫吸附测定法(ELISA)检测血清中白细胞介素-2(IL-2)、白细胞介素-4(IL-4)和肿瘤坏死因子-α(TNF-α)水平,病理切片观察各组气管组织病理形态变化及上皮细胞超微结构,原位末端标记法(TUNEL)检测上皮细胞凋亡,原位杂交和蛋白免疫印迹法(Western blot)分别检测EGFR mRNA和蛋白表达。结果:与模型组比较,中药A、B、C组均可延长哮喘大鼠引喘潜伏期(P<0.05,P<0.01)。与正常组比较,模型组大鼠IL-2水平显著降低(P<0.01),IL-4和TNF-α水平明显升高(P<0.05,P<0.01),气道病理学评分、蓝色胶原面积和气道上皮细胞凋亡指数均显著升高(P<0.01),气管组织EGFR mRNA和蛋白的表达均显著增高(P<0.01);与模型组比较,中药A组IL-2水平明显升高(P<0.05);中药A、B组IL-4水平明显降低(P<0.05,P<0.01);中药A、B、C组TNF-α水平均显著降低(P<0.01);中药A、B、C组对气管组织病理改变有不同程度的修复作用(P<0.05),中药A组可以抑制气管组织纤维化(P<0.05),且中药A组对气道上皮细胞超微结构变化的修复作用最为显著;中药A、B、C组可明显抑制气道上皮细胞的凋亡(P<0.05);中药A、B、C组均可明显抑制EGFR mRNA的过表达(P<0.05,P<0.01),中药B、C组可显著抑制气道上皮组织EGFR蛋白上调(P<0.05,P<0.01)。结论:麻杏石甘汤可以抑制哮喘模型大鼠气道上皮细胞凋亡及其结构的异常改变,减轻气道损伤,降低气管组织中EGFR的表达。麻杏石甘汤减轻哮喘大鼠气道上皮损伤的较优配伍组是中药A组,其比例为麻黄-苦杏仁-甘草-石膏1∶0.5∶4∶1。

Objective:To explore the optimal formula of Maxing Shigantang in regulating epidermal growth factor receptor(EGFR)expression and alleviating airway injury in asthmatic rats and to reveal the underlying mechanism.Method:SD male rats were randomly divided into normal group,model group,dexamethasone group(5×10^(-4)g·kg^(-1))and Maxing Shigantang 1∶0.5,1∶1,1∶2 groups(group A,B,C,10 g·kg^(-1)),with 8 rats in each group.The other groups except the normal group received nebulization of 2%acetylcholine chloride and 0.4%histamine phosphate for the modeling of asthma.One hour before modeling,the normal group and the model group were given the same amount of normal saline,and the other groups were given the same amount of corresponding drugs,once a day for 7 days.On the 7th day,the model was established and the incubation period of asthma was recorded.The rats were then immediately anesthetized,and arterial blood and tracheal tissue were collected.Enzyme-linked immunosorbent assay(ELISA)was employed to detect the levels of interleukin-2(IL-2),interleukin-4(IL-4),and tumor necrosis factor-α(TNF-α)in serum.Pathological sections were prepared for the observation of the pathological changes of tracheal tissues and the ultrastructure of epithelial cells in each group.Terminal-deoxynucleotidyl transferase-mediated nick-end labeling(TUNEL)was adopted to detect epithelial cell apoptosis,and in situ hybridization and Western blot were employed to determine the mRNA and protein levels of epidermal growth factor receptor(EGFR),respectively.Result:Compared with the model group,groups A,B and C prolonged the incubation period of asthma(P<0.05,P<0.01).Compared with the control group,the model group showed declined IL-2 level(P<0.01),risen IL-4 and TNF-αlevels(P<0.05,P<0.01),increased airway pathology score,collagen volume fraction,and airway epithelial cell apoptosis index(P<0.01),and up-regulated mRNA and protein levels of EGFR in trachea tissue(P<0.01).Compared with the model group,group A showed increased IL-2 level(P<0.05)and declined IL-4(P<0.05,P<0.01)level,and group B showed declined IL-4 level(P<0.05).The level of TNF-αin groups A,B,and C declined compared with that in the model group(P<0.01).Maxing Shigantang repaired the tracheal tissue to different degrees(P<0.05).Among the three groups,group A inhibited tracheal fibrosis(P<0.05)and had the most significant effect of repairing the ultrastructural changes of airway epithelial cells.Groups A,B and C all inhibited the apoptosis of airway epithelial cells(P<0.05).All the three groups inhibited the up-regulation of EGFR mRNA level(P<0.05,P<0.01),and groups B and C inhibited the up-regulation of EGFR protein level(P<0.05,P<0.01).Conclusion:Maxing Shigantang can inhibit the abnormal changes of airway epithelial structure,alleviate airway injury,and can down-regulate the expression of EGFR in the tracheal tissue of asthma model rats.In this study,the optimal compatibility of Maxing Shigantang to repair airway epithelial injury in asthmatic rats was group A,with the Ephedrae Herba-Armeniacae Semen Amarum-Glycyrrhizae Radix et Rhizoma-Gypsum Fibrosum ratio of 1∶0.5∶4∶1.