基于p38MAPK/NF-κB信号通路探讨麻黄-苦杏仁药对治疗支气管哮喘大鼠的机制

Exploration of the Study on the Mechanism of Herba Ephedrae-Semen Armeniacae Amarum Drug Pair for the Treatment of Bronchial Asthma in Rats Based on p38MAPK/NF-κB Signaling Pathway

目的探讨麻黄-苦杏仁药对对支气管哮喘大鼠气道炎症的影响,以及对p38MAPK/NF-κB信号通路的调控作用。方法将48只大鼠随机分为正常组、模型组、地塞米松组及麻黄-苦杏仁药对A组(9 g∶6 g)、B组(12 g∶6 g)、C组(9 g∶9 g),每组8只。采用4%卵清蛋白(OVA)致敏复制哮喘大鼠模型,持续造模4周后开始给药,麻黄-苦杏仁药对组(5 g·kg^(-1))、地塞米松组(1 mg·kg^(-1))按相应剂量灌胃给药,正常组及模型组给予等剂量生理盐水,每天1次,持续14 d。检测支气管肺泡灌洗液(BALF)中白细胞介素4(IL-4)、IL-5、IL-6、IL^(-1)3和γ-干扰素(IFN-γ)水平;采用Diff-Quick染色后对BALF进行炎性细胞分类计数;采用免疫组化法检测肺组织p-p38MAPK、NF-κBp65蛋白表达;Western Blot法检测肺组织p38MAPK、p-p38MAPK及NF-κBp65蛋白表达;qRT-PCR法检测肺组织p38MAPK、p-p38MAPK及NF-κBp65 mRNA表达。结果与正常组比较,模型组大鼠BALF中的细胞总数及各炎性细胞数量均显著升高(P<0.01);BALF中的IL-4、IL-5、IL-6、IL^(-1)3水平显著升高(P<0.01),IFN-γ水平显著降低(P<0.01);p-p38MAPK、NF-κBp65蛋白及mRNA表达显著上调(P<0.01)。与模型组比较,麻黄-苦杏仁药对组大鼠BALF中的细胞总数及各炎性细胞数量均显著降低(P<0.01);BALF中的IL-4、IL-5、IL-6、IL^(-1)3水平显著降低(P<0.01),IFN-γ水平显著升高(P<0.01);p-p38MAPK、NF-κBp65蛋白及mRNA表达显著下调(P<0.01)。结论麻黄-苦杏仁药对可能通过抑制p38MAPK/NF-κB信号通路,减少Th2细胞因子生成,减轻哮喘大鼠气道及周围的炎性反应和高气道反应,从而发挥控制哮喘的作用。

Objective To investigate the effects of Herba Ephedrae-Semen Armeniacae Amarum drug pair on airway inflammation in rats with bronchial asthma,and the regulating effect of p38MAPK/NF-κB signaling pathway.Methods Forty-eight rats were randomly divided into the normal group,the model group,the dexamethasone group and the Herba Ephedrae-Semen Armeniacae Amarum drug pair group A(9 g∶6 g),group B(12 g∶6 g)and group C(9 g∶9 g),with 8 rats in each group.The rat model of asthma was replicated by using 4%ovalbumin(OVA)sensitization,and the administration was started after 4 weeks of continuous modeling,with the Herba Ephedrae-Semen Armeniacae Amarum drug pair(5 mg·kg^(-1))and dexamethasone group(1 mg·kg^(-1))administered by gavage at the appropriate doses,and the normal group and model group were given equal dose of saline once daily for consecutive 14 days.The levels of interleukin 4(IL-4),IL-5,IL-6,IL-13 and IFN-γin bronchoalveolar lavage fluid(BALF)were measured;Diff-Quick staining was used to classify and count inflammatory cells in BALF;the protein expression of p-p38MAPK and NF-κBp65 in lung tissue was detected by Western Blot method;the mRNA expression of p38MAPK,p-p38MAPK and NF-κBp65 in lung tissues were detected by qRT-PCR.Results Compared with the normal group,the total number of cells and the number of each inflammatory cell were significantly increased in the BALF of the rats in the model group(P<0.01);the levels of IL-4,IL-5,IL-6 and IL-13 in the BALF were significantly increased(P<0.01)and the level of IFN-γwas significantly decreased(P<0.01);the protein and mRNA expressions of p-p38MAPK,NF-κBp65 were significantly up-regulated(P<0.01).Compared with the model group,the total number of cells and the number of each inflammatory cell in the BALF of The rats in the Herba Ephedrae-Semen Armeniacae Amarum drug pair group were significantly decreased(P<0.01);the levels of IL-4,IL-5,IL-6 and IL-13 in the BALF were significantly decreased(P<0.01),and the level of IFN-γwas significantly increased(P<0.01);the protein and mRNA expressions of p-p38MAPK,NF-κBp65 were significantly down-regulated(P<0.01).Conclusion The Herba Ephedrae-Semen Armeniacae Amarum drug pair may exert asthma control by inhibiting the p38MAPK/NF-κB signaling pathway and reducing Th2 cytokines production to reduce the inflammatory response and high airway response in and around the airways of asthmatic rats.