柴蜕颗粒通过MAPK/Akt/NF-κB信号通路改善气道炎症治疗咳嗽变异性哮喘的药效及机制研究

To study the efficacy and mechanism of Chaitui granules in the treatment of cough variant asthma by improving airway inflammation through MAPK/Akt/NF-κB signaling pathway

目的基于丝裂原活化蛋白激酶/蛋白激酶B/核转录因子-κB(MAPK/Akt/NF-κB)信号通路探讨柴蜕颗粒改善气道炎症治疗咳嗽变异性哮喘(cough variant asthma,CVA)的药效及作用机制。方法雄性Hartley豚鼠32只,随机分为正常组、模型组、柴蜕颗粒组[5.3 g/(kg·d)]、孟鲁司特钠组[0.001 g/(kg·d)],每组8只。除正常组外,其他各组豚鼠肌注4%OVA溶液0.5 mL,同时腹腔注射2%Al(OH)3凝胶0.2 mL建立CVA模型,致敏14天后雾化吸入1%OVA溶液激发咳嗽症状,同时各给药组连续灌胃14天进行干预。末次给药后,使用小动物肺功能仪检测豚鼠气道高反应性(airway hyperresponsiveness,AHR);血生化分析仪检测外周血、支气管肺泡灌洗液(bronchoalveolar lavage fluid,BALF)中白细胞比例;苏木素—伊红(HE)染色观察肺组织病理学变化;酶联免疫吸附法(enzyme linked immunosorbent assay,ELISA)检测BALF中豚鼠免疫球蛋白E(immunoglobulin E,IgE)、白细胞介素4(interleukin-4,IL-4)、干扰素γ(interferon-γ,IFN-γ)、肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)、白细胞介素10(interleukin-10,IL-10)表达水平;蛋白质免疫印迹法(Western blot,WB)检测肺组织中细胞外信号调节激酶(extracellular signal regulated kinase,ERK)、p-ERK、p38丝裂原活化蛋白激酶(p38 mitogen-activated protein kinase,p38 MAPK)、p-p38 MAPK、丝氨酸/苏氨酸蛋白激酶(serine/threonine protein kinase,Akt)、p-Akt、核因子-κB p65(nuclear factor-κB p65,NF-κB p65)、p-NF-κB p65蛋白表达。结果与正常组比较,模型组豚鼠咳嗽次数、气道阻力、病理评分显著增高(P<0.05,P<0.01),咳嗽潜伏期显著缩短(P<0.05);BALF中白细胞(white blood cell,WBC)总数、嗜酸性粒细胞(eosinophil,EOS)、中性粒细胞(neutrophil,NE)、嗜碱性粒细胞(basophil,BA)总数及比例显著升高(P<0.05,P<0.01);BALF中IgE、IL-4、TNF-α水平显著增高(P<0.05),IFN-γ、IL-10水平显著下降(P<0.05,P<0.01);肺组织中ERK、p38 MAPK蛋白表达量明显降低(P<0.01,P<0.05),p-ERK、p-p38 MAPK、p-Akt、p65 NF-κB、p-p65 NF-κB水平显著增高(P<0.05,P<0.01)。与模型组比较,柴蜕颗粒组和孟鲁司特钠组CVA豚鼠咳嗽次数、气道阻力、病理评分显著降低(P<0.05,P<0.01),咳嗽潜伏期明显延长(P<0.05,P<0.01);外周血及BALF中WBC、EOS、NE、BA总数及比例显著降低(P<0.05,P<0.01);BALF中IgE、IL-4、TNF-α含量明显降低(P<0.05,P<0.01),IFN-γ、IL-10含量明显上升(P<0.05,P<0.01);肺组织中ERK蛋白表达量明显增高,p-ERK、p-p38 MAPK、p-Akt、p65 NF-κB、p-p65 NF-κB蛋白表达量显著下降(P<0.05,P<0.01)。结论柴蜕颗粒可有效改善肺功能和咳嗽敏感性治疗咳嗽变异性哮喘,其机制可能与平衡细胞因子表达,抑制MAPK/Akt/NF-κB信号通路活化作用有关。

Objective To investigate the therapeutic effect and mechanism of Chaitui granules on cough variant asthma(CVA)by improving airway inflammation based on mitogen-activated protein kinase/serine/threonine kinase/nuclear factor-κB(MAPK/Akt/NF-κB)signaling pathway.Methods Thirty-two male Hartley guinea pigs were randomly divided into normal control group,model group,Chaitui granule group[5.3 g/(kg·d)],and montelukast sodium group[0.001 g/(kg·d)],with 8 guinea pigs in each group.Except for the normal group,guinea pigs in other groups were injected intramuscularly with 4%OVA(0.5 mL)and intraperitoneally with 2%Al(OH)3 gel(0.2 mL)to establish the CVA model.After 14 days of sensitization,aerosolized 1%OVA solution was inhaled to induce cough symptoms,and each group was given intragastric administration for 14 days.Airway hyperresponsiveness(AHR)was measured by small animal pulmonary function analyzer after the last administration.The proportion of white blood cells in peripheral blood and bronchoalveolar lavage fluid(BALF)was detected by blood biochemical analyzer.Hematoxylin-eosin(HE)staining was used to observe the pathological changes of lung tissues.The levels of immunoglobulin E(IgE),interleukin-4(IL-4),interferon-γ(IFN-γ),tumor necrosis factor-α(TNF-α)and interleukin-10(IL-10)in BALF were detected by enzyme-linked immunosorbent assay(ELISA).The protein expressions of ERK,p-ERK,p38 MAPK,p-p38 MAPK,Akt,p-Akt,NF-κB p65,and p-NF-κB p65 in lung tissues were detected by Western blot.Results Compared with the normal group,the cough frequency,airway resistance and pathological score of guinea pigs in the model group were significantly increased(P<0.05,P<0.01),and the cough latency was significantly decreased(P<0.05).The total number and proportion of white blood cells,eosinophils(EOS),neutrophils(NE)and basophils(BA)in peripheral blood and BALF were significantly increased(P<0.05,P<0.01).The levels of IgE,IL-4 and TNF-αin BALF were significantly increased(P<0.05),while the levels of IFN-γand IL-10 were significantly decreased(P<0.05,P<0.01).The protein expressions of ERK and p38 MAPK in lung tissues were significantly decreased(P<0.01,P<0.05),and the levels of p-ERK,p-p38 MAPK,p-Akt,p65 NF-κB and p-p65 NF-κB were significantly increased(P<0.05,P<0.01).Compared with the model group,the cough frequency,airway resistance and pathological score of CVA guinea pigs in the Chaitui granule group and montelukast sodium group were significantly decreased(P<0.05,P<0.01),and the cough latency was significantly prolonged(P<0.05,P<0.01).The total number and proportion of white blood cells,eosinophils(EOS),neutrophils(NE)and basophils(BA)in peripheral blood and BALF were significantly decreased(P<0.05,P<0.01).The levels of IgE,IL-4 and TNF-αin BALF were significantly decreased(P<0.05,P<0.01),while the levels of IFN-γand IL-10 were significantly increased(P<0.05,P<0.01).The protein expression of ERK in lung tissues were significantly increased,and the protein expressions of p-ERK,p-p38 MAPK,p-Akt,p65 NF-κB,and p-p65 NF-κB were significantly decreased(P<0.05,P<0.01).Conclusion Chaitui granules can improve lung function and cough state by balancing the expression of cytokines in the treatment of CVA,and its mechanism may be related to the inhibition of the activation of MAPK/Akt/NF-κB signaling pathway.