大承气汤对过敏性哮喘小鼠肺部炎症及MAPK信号通路的影响
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摘要
目的:观察大承气汤对过敏性哮喘小鼠的肺指数及肺指数抑制率、肺组织形态学,支气管肺泡灌洗液(BALF)炎细胞分类变化以及丝裂原活化蛋白激酶(MAPK)信号通路的影响。方法:40只雌性C57BL/6小鼠随机分为正常组、模型组、地塞米松组(0.005 g·kg~(-1))和大承气汤组(19 g·kg~(-1))。采用卵白蛋白(OVA)致敏加激发的方式建立小鼠过敏性哮喘模型,分别于第0,14天致敏,第21天开始OVA雾化激发,连续7 d,地塞米松组和大承气汤组在雾化激发前1 h进行药物灌胃干预,每只0.2 m L,正常组给予等量生理盐水处理。第28天造模结束后取肺组织,苏木素-伊红(HE)染色观察肺组织形态学,迪夫染色检测肺泡灌洗液炎细胞分类计数,蛋白免疫印迹法(Western blot)检测肺组织MAPK信号通路关键蛋白表达情况。结果:与正常组比较,模型组小鼠肺指数明显升高(P<0.01),肺组织出现炎性病理变化,气道炎细胞渗出严重,以嗜酸性粒细胞为主(P<0.01),且肺组织磷酸化p38 MAPK和细胞外信号调节激酶1/2(ERK1/2)的表达上升;而经过大承气汤治疗后,模型小鼠肺指数升高受到抑制,肺指数抑制率达68.4%,肺组织炎性病理改善明显,炎细胞渗出减轻,且MAPK蛋白磷酸化水平明显降低。结论:基于"肺肠同治"的大承气汤能有效改善过敏性哮喘小鼠的肺部炎症,其机制可能与磷酸化的p38 MAPK和ERK1/2的表达量有关。
Objective: To explore the effect of Da Chengqitang on the lung index,lung index inhibition rate,lung histological morphology,classification changes of inflammatory cells and mitogen-activated protein kinase( MAPK) signal pathway in mice with allergic asthma. Method: Forty female C57BL/6 mice were randomly divided into normal control group,model group,dexamethasone group( 0. 005 g·kg~(-1)) and Da Chengqitang group( 19 g·kg~(-1)). Murine allergic asthma model was established by sensitization and nebulization of ovalbumin( OVA). In brief,asthmatic mice were first sensitized by OVA and Al(OH)_3 mixture ip on day 0 and day 14,and then nebulized by OVA from day 21 to 27. At the same time,each mouse in the dexamethasone and Da Chengqitang groups were intragastrically administered with 0. 2 m L corresponding medicine one hour before the nebulization challenge,while the normal control group was given with the same amount of normal saline. On day 28,pulmonary morphology was detected by htoxylin eosin( HE) staining and inflammatory cells from the brachial alveolar lavage fluid were counted by Diff staining. The expression levels of key proteins in MAPK signaling pathway were detected by Western blot. Result: As compared with the normal control group,the lung indexes were significantly increased in model group( P < 0. 01),showing obvious inflammatory pathological changes,and serious airway inflammation cells exudation( P < 0. 01),with a predominant percentage of eosinophils,moreover,the expression levels of phosphorylated p38 MAPK and extracellular signal-regulated kinase 1/2( ERK1/2) were increased obviously in asthmatic mice. After treatment by Da Chengqitang,lung indexes and pulmonary inflammation were significantly decreased,with an inhibitory rate of 68. 4% for lung indexes,and inflammatory pathology of lung tissues was obviously improved and inflammatory cell exudation was alleviated, with the obviously lower levels of phosphorylated p38 MAPK and ERK1/2 protein. Conclusion: Da Chengqitang based on "Pulmonary Intestinal Treatment" can effectively improve lung inflammation in mice with allergic asthma,which may be related to the expression of phosphorylated p38 MAPK and ERK1/2 protein.
Objective: To explore the effect of Da Chengqitang on the lung index,lung index inhibition rate,lung histological morphology,classification changes of inflammatory cells and mitogen-activated protein kinase( MAPK) signal pathway in mice with allergic asthma. Method: Forty female C57BL/6 mice were randomly divided into normal control group,model group,dexamethasone group( 0. 005 g·kg~(-1)) and Da Chengqitang group( 19 g·kg~(-1)). Murine allergic asthma model was established by sensitization and nebulization of ovalbumin( OVA). In brief,asthmatic mice were first sensitized by OVA and Al(OH)_3 mixture ip on day 0 and day 14,and then nebulized by OVA from day 21 to 27. At the same time,each mouse in the dexamethasone and Da Chengqitang groups were intragastrically administered with 0. 2 m L corresponding medicine one hour before the nebulization challenge,while the normal control group was given with the same amount of normal saline. On day 28,pulmonary morphology was detected by htoxylin eosin( HE) staining and inflammatory cells from the brachial alveolar lavage fluid were counted by Diff staining. The expression levels of key proteins in MAPK signaling pathway were detected by Western blot. Result: As compared with the normal control group,the lung indexes were significantly increased in model group( P < 0. 01),showing obvious inflammatory pathological changes,and serious airway inflammation cells exudation( P < 0. 01),with a predominant percentage of eosinophils,moreover,the expression levels of phosphorylated p38 MAPK and extracellular signal-regulated kinase 1/2( ERK1/2) were increased obviously in asthmatic mice. After treatment by Da Chengqitang,lung indexes and pulmonary inflammation were significantly decreased,with an inhibitory rate of 68. 4% for lung indexes,and inflammatory pathology of lung tissues was obviously improved and inflammatory cell exudation was alleviated, with the obviously lower levels of phosphorylated p38 MAPK and ERK1/2 protein. Conclusion: Da Chengqitang based on "Pulmonary Intestinal Treatment" can effectively improve lung inflammation in mice with allergic asthma,which may be related to the expression of phosphorylated p38 MAPK and ERK1/2 protein.
引文
[1]张楠,王达,隋博文.刘建秋教授应用“肺与大肠相表里”理论从肠治肺经验总结[J].中国中医急症,2015,24(3):436-438.
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[11]白亚辉,陈辉,杨宇.肺肠合治法对哮喘小鼠血清总Ig E影响的实验研究[J].中华中医药学刊,2007,25(6):1131-1133.
[12]徐云,李宇航,郑丰杰,等.“通腑”对卵清蛋白致敏支气管哮喘小鼠模型的影响[J].辽宁中医杂志,2012,39(3):553-555.
[13]Oeser K,Maxeiner J,Symowski C,et al.T cells are the critical source of IL-4/IL-13 in a mouse model of allergic asthma[J].Allergy,2015,70(11):1440-1449.
[14]LIU J,ZHANG M,NIU C,et al.Dexamethasone inhibits repair of human airway epithelial cells mediated by glucocorticoid-induced leucine zipper(GILZ)[J].PLo S One,2013,8(4):e60705.
[15]徐凤,肖韩艳,周淑芬,等.柴朴汤对哮喘模型大鼠气道炎症及ERK/p38 MAPK信号通路的影响[J].中国实验方剂学杂志,2018,24(2):104-109.
[2]毛峥嵘.大承气汤联合肺复张治疗肺外源性急性呼吸窘迫综合征临床研究[J].辽宁中医杂志,2009,36(6):974-976.
[3]郑丰杰,李宇航,许红,等.通腑对哮喘模型小鼠肺及肠组织VIP、TFF3及NKA含量的影响[J].中华中医药杂志,2012,27(8):2023-2027.
[4]钟大玲,吴佳佳,李根茂,等.大承气汤对过敏性哮喘小鼠肺组织形态及Ig E水平的影响[J].中国中医药信息杂志,2016,23(7):58-60.
[5]Reddy A T,Lakshmi S P,Reddy R C.Murine model of allergen induced asthma[J].J Vis Exp,2012,63(63):e3771.
[6]李鸿涛,高思华,王柳青,等.藏象学说中“肺与大肠相表里”内涵及其在温病辨治中的运用[J].中医杂志,2011,52(4):271-273.
[7]王永强,孟红丽.浅谈“肺肠同治”在喘症治疗中的作用[J].陕西中医学院学报,2012,35(2):11-13.
[8]Simon K,Talley N J,Hansbro P M.Pulmonary-intestinal cross-talk in mucosal inflammatory disease[J].Mucosal Immunol,2012,5(1):7-18.
[9]陈银芳,于小娟,刘新辉,等.差异蛋白质组学法探索大承气汤优化方治疗便秘小鼠的生物学基础[J].中国实验方剂学杂志,2017,23(14):93-99.
[10]易剑锋,叶蓁蓁,潘海邦,等.醋甘遂、复方大承气汤、生长抑素联合治疗麻痹性肠梗阻[J].中国实验方剂学杂志,2015,21(10):182-186.
[11]白亚辉,陈辉,杨宇.肺肠合治法对哮喘小鼠血清总Ig E影响的实验研究[J].中华中医药学刊,2007,25(6):1131-1133.
[12]徐云,李宇航,郑丰杰,等.“通腑”对卵清蛋白致敏支气管哮喘小鼠模型的影响[J].辽宁中医杂志,2012,39(3):553-555.
[13]Oeser K,Maxeiner J,Symowski C,et al.T cells are the critical source of IL-4/IL-13 in a mouse model of allergic asthma[J].Allergy,2015,70(11):1440-1449.
[14]LIU J,ZHANG M,NIU C,et al.Dexamethasone inhibits repair of human airway epithelial cells mediated by glucocorticoid-induced leucine zipper(GILZ)[J].PLo S One,2013,8(4):e60705.
[15]徐凤,肖韩艳,周淑芬,等.柴朴汤对哮喘模型大鼠气道炎症及ERK/p38 MAPK信号通路的影响[J].中国实验方剂学杂志,2018,24(2):104-109.