雷公藤甲素通过CXCL10/CXCR3轴对支气管哮喘小鼠气道炎症的影响研究

Effect of Triptolide on Airway Inflammation in Asthmatic Mice via CXCL10/CXCR3 Axis

目的探讨雷公藤甲素通过CXCL10/CXCR3轴对支气管哮喘(以下简称为哮喘)小鼠气道炎症的影响,为临床应用雷公藤甲素治疗哮喘提供理论依据。方法2020年7—12月选取36只雌性SPF级BALB/c小鼠,采用简单随机法将其均分为对照组、哮喘模型组和雷公藤甲素组,每组12只。哮喘模型组和雷公藤甲素组采用卵清蛋白腹腔注射致敏及雾化激发构建哮喘小鼠模型,对照组小鼠腹腔注射0.9%氯化钠溶液;每次雾化前1 h,雷公藤甲素组腹腔注射0.2 ml雷公藤甲素(40μg/kg),哮喘模型组和对照组则腹腔注射0.2 ml 0.9%氯化钠溶液,连续干预14 d。干预后观察三组小鼠行为学表现;最后一次雾化后24 h内采用颈椎脱臼法处死小鼠,取三组小鼠左肺组织,采用苏木素-伊红(HE)染色和过碘酸-雪夫(PAS)染色观察支气管肺组织形态学变化;分别收集三组小鼠右肺支气管肺泡灌洗液(BALF),采用瑞氏吉姆萨染色法计数BALF中的炎症细胞数量,采用ELISA检测BALF中白介素(IL)-4、IL-10、IL-17。采用Western blotting法及免疫组化法检测支气管肺组织中CXCL10、CXCR3相对表达量。结果哮喘模型组、雷公藤甲素组气道上皮杯状细胞占比高于对照组(P<0.05),雷公藤甲素组气道上皮杯状细胞占比低于哮喘模型组(P<0.05)。哮喘模型组、雷公藤甲素组BALF中细胞总数、嗜酸粒细胞计数、中性粒细胞计数、淋巴细胞计数、IL-4、IL-17高于对照组,IL-10低于对照组(P<0.05);雷公藤甲素组BALF中细胞总数、嗜酸粒细胞计数、中性粒细胞计数、淋巴细胞计数、IL-4、IL-17低于哮喘模型组,IL-10高于哮喘模型组(P<0.05)。Western blotting法及免疫组化法结果显示,哮喘模型组、雷公藤甲素组支气管肺组织中CXCL10、CXCR3相对表达量高于对照组,雷公藤甲素组支气管肺组织中CXCL10、CXCR3相对表达量低于哮喘模型组(P<0.05)。结论雷公藤甲素能够减少哮喘小鼠气道上皮杯状细胞及BALF中细胞总数、嗜酸粒细胞、中性粒细胞、淋巴细胞,下调促炎因子IL-4、IL-17,上调抑炎因子IL-10,同时抑制CXCL10、CXCR3表达;雷公藤甲素减轻哮喘小鼠气道炎症的机制可能与其可调控CXCL10/CXCR3轴有关。

Objective To explore effect of triptolide on airway inflammation in asthmatic mice via CXCL10/CXCR3 axis,in order to provide theoretical basis for clinical application of triptolide in treating asthma.Methods From July to December 2020,36 female SPF BALB/c mice were selected,and randomly divided into control group,asthma model group and triptolide group,with 12 mice in each group.Asthma model group and triptolide group were established asthmatic mouse model by intraperitoneal injection sensitization and atomization inhalation provocation with ovalbumin.Control group was intraperitoneally injected with 0.9%sodium chloride solution.Triptolide group was intraperitoneally injected with 0.2 ml triptolide(40μg/kg)1 hour before aerosol inhalation provocation of ovalbumin at every time,and control group and asthma model group were intraperitoneally injected with 0.2 ml 0.9%sodium chloride solution,for consecutive 14 days.After intervention,the behavior of the three groups of mice was observed;within 24 hours after the last atomization,the mice were killed by cervical dislocation method.The left lung tissues of the three groups of mice were taken,the hematoxylin-eosin(HE)staining and periodic acid-schiffstain(PAS)staining were used to observe the morphology of bronchopulmonay tissue.The right lung bronchoalveolar lavage fluid(BALF)of the three groups of mice was collected respectively,and the inflammatory cell count in BALF was counted by Reich Giemsa staining,and IL-4,IL-10,IL-17 in BALF were detected by ELISA.The relative expression of CXCL10 and CXCR3 in bronchopulmonay tissue were detected by Western blotting and immunohistochemistry.Results The proportion of goblet cells in airway epithelium of asthma model group and triptolide group was higher than that of control group(P<0.05),and the proportion of goblet cells in airway epithelium of triptolide group was lower than that of asthma model group(P<0.05).The cell count,eosinophil count,neutrophil count,lymphocyte count,IL-4,IL-17 in BALF of asthma model group and triptolide group were higher than those of control group,IL-10 in BALF of asthma model group and triptolide group was lower than that of control group(P<0.05);and the cell count,eosinophil count,neutrophil count,lymphocyte count,IL-4,IL-17 in BALF of triptolide group were lower than those of asthma model group,IL-10 in BALF of triptolide group was higher than that of asthma model group(P<0.05).Western blotting and immunohistochemistry showed that,the relative expression of CXCL10 and CXCR3 in bronchopulmonay tissue of asthma model group and triptolide group were higher than those of control group,and the relative expression of CXCL10 and CXCR3 in bronchopulmonay tissue of triptolide group were lower than those of asthma model group(P<0.05).Conclusion Triptolide can reduce the goblet cells in airway epithelium and the cell count,eosinophil,neutrophil,lymphocyte in BALF,down regulate the pro-inflammatory factors IL-4,IL-17,up regulate the anti-inflammatory factor IL-10,and inhibit the expression of CXCL10 and CXCR3.Triptolide may improve airway inflammation of asthma mice by inhibiting CXCL10/CXCR3 axis.