OVA特异性免疫治疗对哮喘小鼠IL-23/Th17轴的影响及机制研究

Study on influence of OVA specific immunotherapy on IL-23/Th17 axis of a mouse asthma model and its mechanism

目的腹部皮下注射大剂量卵白蛋白(OVA)建立小鼠哮喘免疫治疗模型,探讨IL-23/Th17轴在小鼠哮喘模型经皮免疫治疗过程中的作用。方法选择18只BALB/c小鼠为建模研究对象,按随机数字表法分为3组:空白对照组、哮喘对照组和哮喘免疫治疗组,每组6只小鼠。哮喘免疫治疗组予10μg OVA于0、7 d腹腔注射致敏,21~27 d持续1周予1 mg OVA腹部皮下注射诱导免疫耐受,35~41 d予1%OVA雾化激发;哮喘对照组在21~27 d OVA皮下注射等量生理盐水,其余处置同哮喘免疫治疗组;空白对照组采用等量生理盐水致敏及激发。50 d予10%OVA加强激发1次。末次激发24 h内检测气道反应性;收集支气管肺泡灌洗液(BALF)计数细胞总数及细胞分类计数;ELISA法检测血清OVA特异性Ig E、BALF中IL-5、IFN-γ、IL-23、IL-10的表达;HE染色观察肺组织病理改变;流式细胞仪检测各组脾、肺组织Treg、Th17细胞比例;q-PCR检测肺组织转录因子。结果哮喘免疫治疗组气道反应性、BALF中嗜酸性粒细胞计数、IL-23水平及血清OVA特异性Ig E水平明显低于哮喘对照组,差异有统计学意义(P<0.05);而BALF中IFN-γ水平与哮喘对照组比较差异无统计学意义(P>0.05);同时HE染色发现哮喘免疫治疗组肺组织炎症较哮喘对照组明显减轻;流式细胞技术检测发现外周血Treg细胞百分比明显高于哮喘对照组,差异有统计学意义(P<0.05);q-PCR检测肺组织转录因子表明免疫治疗组Foxp3明显高于哮喘对照组,而RORγt明显低于哮喘对照组,差异均具有统计学意义(P<0.05)。结论大剂量OVA特异性免疫治疗能够减轻哮喘小鼠气道慢性炎症反应,同时引起Th17细胞下降伴随IL-23表达降低;其机制可能与纠正肺部IL-23/Th17轴有关。

Objective To establish a mouse model of immune treatment of asthma through subcutaneous injection with high dose of ovalbumin（ OVA） in the abdomen and investigate the role of IL-23/Th17 axis response in its mechanism. Methods With a random number table method,18 female BALB/c mice were divided into 3 groups（ normal control group,asthma group and asthmatic immune tolerance model group）,with 6 mice in each group. The mice in the asthmatic immune tolerance model group were sensitized with 10 μg OVA by intraperitoneal injection in the abdomen on day 0 and day 7. The mice in the model group induced immune tolerance with 1 mg OVA by subcutaneous injection in the abdomen every day for a week（ day 21 to day 27）. Both the model group and asthma control group were challenged with 1%OVA on day 35 to day 41. The mice in the normal control group were challenged with the equal amount of saline. On the 50 th day,each group were sforzando challenged once with 10% OVA. The airway reactivity was detected at 24 after the last challenge. The enhanced pause（ Penh）was measured to evaluate the airway responsiveness with a lung functional instrument. Bronchoalveolar lavage fluid（ BALF） was collected to count the total cells and eosinophils,and the cytological studies were conducted. The OVA-specific Ig E in peripheral blood,and the IL-5,IFN-γIL-23,IL-10 in BALF were detected by enzyme-linked immunosorbent assay（ ELISA）. The lung tissue was obtained to perform histological analysis by HE staining. The percentagea of Treg and Th17 cells in spleen and lung tissue were calculated by the flow cytometry（ FCM）. Then the expression of transcription factors was detected by q-PCR. Results For the asthmatic immune tolerance model group,the airway responsiveness,the cell count of eosinophilic granulocytes in BALF,the levels of IL-23 and OVA-specific Ig E in the serum were significantly lower than the asthma group and the difference is of statistical significance（ P〈0. 05）,while the difference in the IFN-γlevel in BALF compared with the asthma group is of no statistical significance（ P〈 0. 05）. The transcription factor of lung tissue detected with q-PCR showed Foxp3 in the asthmatic immune tolerance model group was significantly higher than the asthma group,while RORγt was significantly lower than the asthma group（ P〉 0. 05） and the differences were of statistical significance（ P〈 0. 05）. Conclusion Large dose of OVA specific immunotherapy can alleviate the chronic inflammatory response of asthmatic mice,and the decrease of Th17 cells associated with the expression of IL-23 was decreased. The mechanism may be related to the correction of the lung Il-23/Th17 axis.