基于嗜酸细胞活化Eotaxin/CCR3通路探讨疏风通络法治疗支气管哮喘的分子机制

Eosinophil activation is the key mechanism of chronic airway inflammation of bronchial asthma. by inhibiting eosinophil activation may fundamentally improve the disease progression in patients with bronchial asthma. Previous studies that suggested Shufeng Tongluo prescription in the treatment of asthma patients, effectively improve lung function, reduce the onset of asthma, its mechanism is involved in eosinophil activation needs further study. Therefore, this topic proposed Shufeng Tongluo prescription by regulating eosinophil activation Eotaxin/ CCR3 pathway to improve asthma chronic airway inflammation. This study is based on (1) in vitro culture eosinophilic cells as the research object, in serum as a means of intervention. To observe serum of traditional Chinese medicine intervention under the eosinophil secretion EARs of activity, Eotaxin/CCR3 pathway on ERK and p38MAPK phosphorylation level (2) in vitro culture eosinophilic cells as the research object, in serum as a means of intervention. To observe serum of traditional Chinese medicine intervention under eosinophil acid cell adhesion, migration, degranulation. (3) in order to model animals as the research object, taking the traditional Chinese medicine as the intervention method, To observe traditional Chinese medicine intervention under the eosinophil secretion EARs of activity, Eotaxin/CCR3 pathway on ERK and p38MAPK phosphorylation level .To explore the molecular mechanism of the treatment of asthma by Shufeng Tongluo prescription .

嗜酸细胞活化是支气管哮喘气道慢性炎症的关键机制，阻抑嗜酸细胞活化有可能从根本上改善支气管哮喘患者的病情进展。前期研究提示疏风通络法方药治疗哮喘患者，可有效改善肺功能，减少哮喘发作，其作用机制是否涉及嗜酸细胞活化还需进一步探讨，故本课题提出疏风通络法方药通过调控嗜酸细胞活化Eotaxin/CCR3通路改善哮喘气道慢性炎症。本课题研究基于（1）以体外培养嗜酸细胞为研究对象，以含药血清为干预手段，观察中药血清干预下嗜酸细胞分泌EARs活性、Eotaxin/CCR3通路上ERK、P38MAPK磷酸化水平（2）以体外培养嗜酸细胞为研究对象，以含药血清为干预手段，观察中药血清干预下嗜酸细胞粘附、迁移、脱颗粒。（3）以模型动物为研究对象，以中药灌胃为干预手段，观察中药干预下嗜酸细胞分泌EARs活性、Eotaxin/CCR3通路上ERK、P38MAPK磷酸化水平。探讨疏风通络法方药治疗哮喘分子机制。

本项目在细胞—动物层面研究了疏风通络方对哮喘小鼠嗜酸细胞Eotaxin/CCR3通路相关因子的影响，具体包括：1.以 OVA法构建哮喘小鼠模型成功，肺组织切片 HE染色提示气道狭窄、粘液高分泌、嗜酸细胞等炎细胞大量募集；2.以疏风通络方灌胃或通路各途径拮抗剂干预哮喘小鼠，测定小鼠外周血及肺组织 CCL11、CCL24含量，同时以免疫组化及 WB蛋白印记法测定小鼠肺组织 CCR3蛋白含量、p-ERK及总 ERK水平，并以 RT-PCR法测定肺组织CCR3mRNA、CCL11mRNA、CCL24mRNA表达；3.以免疫磁珠分选提取小鼠嗜酸细胞，建立原代培养系进行细胞实验；4.饲喂健康小鼠疏风通络方浓缩液，后提取疏风通络方含药血清；5.在体外条件下以疏风通络方含药血清或各拮抗因子孵育小鼠嗜酸细胞，测定嗜酸细胞脱颗粒 EPO、MBP、EDN、ECP蛋白含量。6.在体外条件下以疏风通络方含药血清或各拮抗因子孵育小鼠嗜酸细胞，测定嗜酸细胞活化粘附、迁移能力；7.在体外条件下以疏风通络方含药血清或各拮抗因子孵育小鼠嗜酸细胞，测定嗜酸细胞ERK磷酸化水平、CCR3蛋白含量及CCR3、p38 MAPK与ERK基因表达。通过本研究发现1.疏风通络方能改善哮喘小鼠模型气道炎症；2.疏风通络方能够降低哮喘小鼠外周血及肺组织中 CCL11、CCL24的含量；3.疏风通络方能够抑制哮喘小鼠肺组织中 CCR3蛋白含量及 ERK磷酸化水平；4.疏风通络方能够降低哮喘小鼠肺组织中 CCR3、CCL11的基因表达水平；5.疏风通络方可抑制体外小鼠嗜酸细胞脱颗粒；6.疏风通络方含药血清可抑制体外小鼠嗜酸细胞粘附与迁移能力；7.疏风通络方含药血清可减少经CCL11孵育激活的体外小鼠嗜酸细胞CCR3含量及ERK磷酸化水平；.8.疏风通络方含药血清可减少经CCL11孵育激活的体外小鼠嗜酸细胞CCR3、p38 MAPK基因表达水平；9.疏风通络方有可能通过抑制 Eotaxin/CCR3信号通路改善哮喘气道炎症。