摘要
目的 探讨固本防哮饮防治支气管哮喘缓解期慢性气道炎症的可能作用机制.方法 36只雌性Balb/c小鼠随机分为正常组,模型组,固本防哮饮低、中、高剂量组,孟鲁司特钠组,每组6只.除正常组外,其余各组小鼠采用卵白蛋白联合呼吸道合胞病毒诱导建立支气管哮喘缓解期小鼠模型.造模后固本防哮饮低、中、高剂量组分别给予固本防哮饮12、24、36 g/(kg·d)灌胃,孟鲁司特钠组给予孟鲁司特钠颗粒2.6 mg/(kg·d)灌胃,正常组、模型组小鼠给予双蒸水20 ml/(kg·d)灌胃,均每天1次,共28天.检测小鼠肺组织白细胞介素4(IL-4)、白细胞介素5(IL-5)水平;HE染色观察肺组织病理并进行炎症评分;DHE染色观察肺组织活性氧(ROS)水平;检测肺组织线粒体呼吸链复合物Ⅰ、Ⅱ、Ⅲ、Ⅳ、V活性;检测血清超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、丙二醛(MDA)及三磷酸腺苷(ATP)水平;Western blot法检测小鼠肺组织中磷酸化腺苷单磷酸活化蛋白激酶(p-AMPK)、核因子E2相关因子2(Nrf2)、血红素加氧酶1(HO-1)及cAMP应答元件结合蛋白(CREB)蛋白表达水平.结果 与正常组比较,模型组小鼠肺组织病理结果显示气道周围炎症细胞增多,炎症评分升高;DHE染色显示ROS水平升高,肺组织IL-4、IL-5水平升高;血清SOD、CAT及ATP水平降低,MDA水平升高;肺组织线粒体呼吸链复合物Ⅰ、Ⅱ、Ⅲ、Ⅳ、V活性降低,p-AMPK、Nrf2、CREB蛋白表达降低(P＜0.05).与模型组比较,固本防哮饮各剂量组及孟鲁司特钠组小鼠肺组织炎症细胞减少,炎症评分降低;肺组织ROS及IL-4、IL-5水平降低;血清CAT及ATP水平升高,MDA水平降低;肺组织线粒体呼吸链复合物Ⅰ、Ⅱ活性及CREB蛋白表达升高(P＜0.05).与固本防哮饮高剂量组比较,固本防哮饮中剂量组MDA水平降低(P＜0.05).固本防哮饮中剂量组肺组织线粒体呼吸链复合物V活性较孟鲁司特钠组升高,固本防哮饮中、高剂量组肺组织线粒体呼吸链复合物Ⅳ活性较固本防哮饮低剂量组升高(P＜0.05).结论 固本防哮饮能够抑制支气管哮喘缓解期模型小鼠肺组织氧化应激,改善线粒体功能,缓解慢性气道炎症,其作用机制可能与激活肺组织AMPK/Nrf2/HO-1信号通路有关.
Abstract
Objective To investigate the possible mechanism of Guben Fangxiao Beverage(固本防哮饮)for the prevention and treatment of chronic airway inflammation during asthma remission.Methods Thirty-six female Balb/c mice were randomly divided into normal group,model group,low-,medium-,and high-dose of Guben Fangxiao Beverage group and montelukast sodium group,with 6 mice in each group.Except for the normal group,ovalbumin and respiratory syncytial virus were used in other groups to establish a mouse model of bronchial asthma in remission stage.After molding,the low-,medium-,and high-dose groups of Guben Fangxiao Beverage were respectively given 12,24,and 36 g/(kg·d),the montelukast sodium group was given montelukast sodium granule 2.6 mg/(kg·d),and the mice in the normal group and model group were given 20 ml of double-distilled water,all by gavage,once a day for 28 days.The levels of interleukin 4(IL-4)and interleukin 5(IL-5)in the lung tissue of mice were detected;HE staining was used to observe the pathology of the lung tissue and to score the inflammation;DHE staining was used to observe the level of reactive oxygen species(ROS)in the lung tissue,and the activities of mitochondrial respi-ratory chain complexes Ⅰ,Ⅱ,Ⅲ,Ⅳ,and Ⅴ in the lung tissue were detected;the levels of serum superoxide dis-mutase(SOD),catalase(CAT),malondialdehyde(MDA)and adenosine triphosphate(ATP)were detected;the protein expression levels of phosphorylated adenosine monophosphate-activated protein kinase(p-AMPK),nuclear factor erythroid 2-related factor 2(Nrf2),haem oxygenase 1(HO-1)and cAMP responsive element binding protein(CREB)in the lung tissues of the model group were detected by Western blot.Results Compared with the normal group,the histopathological results of the lungs of mice in the model group showed an increase in inflammatory cells around the airways and an increase in inflammatory score;DHE staining showed an increase in the level of ROS,and an increase in the levels of IL-4 and IL-5 in the lung tissues;the levels of serum SOD,CAT,and ATP were reduced,and the level of MDA was elevated;the activities of the mitochondrial respiratory chain complexes Ⅰ,Ⅱ,Ⅲ,Ⅳ,and Ⅴ of the lung tissues were reduced,and the activities of p-AMPK,Nrf2,CREB protein expression decreased(P＜0.05).Compared with the model group,the lung tissue inflammatory cells and inflammation scores of mice in each Guben Fangxiao Beverage dose group and montelukast sodium group were reduced;the levels of ROS,IL-4 and IL-5 in the lung tissue were reduced;the levels of CAT and ATP in the serum increased,and the content of MDA was reduced;and the activities of mitochondrial respiratory chain complexes Ⅰ and Ⅱ,as well as the expression of CREB protein,were elevated in the lung tissue(P＜0.05).Compared with the high-dose group,the MDA level of the medium-dose Guben Fangxiao Beverage group decreased(P＜0.05).The activity of mitochondrial respiratory chain complex Ⅴ in the medium-dose group was higher than that in the montelukast sodium group,and the activity of mitochondrial respiratory chain complex Ⅳ in the medium-and high-dose groups was higher than that in the low-dose group(P＜0.05).Conclusion Guben Fangxiao Beverage can inhibit oxidative stress and improve mitochondrial function to relieve chronic airway inflammation in bronchial asthma model mice during asthma remission,and its mechanism may be related to the activation of AMPK/Nrf2/HO-1 signaling pathway.
基金项目
国家自然科学基金(82074489)
江苏省中医院高峰学术人才培养项目(Y2021rc21)
江苏高校优势学科建设工程()
维普
万方数据