基于microRNA155调控Th17/Treg细胞探讨肝脾同治法对小儿支气管哮喘的作用及干预机制

Bronchial asthma (asthma) is the most common pediatric chronic respiratory disease, and seriously affects the physical and mental health of children. TCM holds that liver and spleen discord is “perennial root "formation of the important pathogenesis of asthma, liver and spleen Tongzhi is the basic solution that getting rid of the law of the" perennial root ", clinical curative effect is safe and effective. Asthma is characterized by chronic airway inflammation. Data display miR-155 regulate Th17/Treg cells in pathogenesis of chronic airway inflammation of immunological mechanism.The previous study demonstrated that Tianbei Decoction could inhibit chronic airway inflammation and adjust immune. So we assume that based on the treatment of liver and spleen Tongzhi with Tianbei Decoction can by intervening in miR-155 control Th17/Treg cells so as to effectively improve immune disorder caused chronic airway inflammation led to children with asthma. To confirm the hypothesis, we prepare for asthma model in mice and in vitro induced Th17/Treg cells,the application of molecular biology techniques, as a whole-organ- cell-molecular levels to confirm our hypothesis. Certainly, this study makes sense to reveal the pathogenesis of asthma from the perspective of microRNA, and illustrates the effect of liver and spleen Tongzhi preventing and controlling asthma with TCM targets, to reveal the liver and spleen discord is the essence of "perennial root formation, provide new way for the rich theory of children with asthma treatment.

支气管哮喘是小儿最常见的慢性呼吸系统疾病，严重影响小儿的身心健康。中医学认为肝脾不和是哮喘“夙根”形成的重要病机，肝脾同治法是剔除“夙根”的根本大法，临床疗效安全有效。哮喘以慢性气道炎症为特征。资料显示miR-155调控Th17/Treg细胞是哮喘慢性气道炎症发病的免疫学机制，前期研究显示天贝汤具有抑制慢性气道炎症和调节免疫的作用。因此我们假设基于肝脾同治法的天贝汤可通过干预miR-155调控Th17/Treg细胞从而有效改善免疫失常导致的慢性气道炎症所致的小儿哮喘。为证实该假说，我们拟制备小鼠哮喘模型和体外诱导Th17/Treg细胞分化，应用现代分子生物学技术，从整体-器官-细胞-分子水平证实我们的假说。本研究将从microRNA角度进一步揭示哮喘发病机制，阐明肝脾同治法防治哮喘的效应靶点，为揭示肝脾不和是小儿哮喘“夙根”形成的本质，为丰富小儿哮喘治法理论提供新思路。

目的：肝脾同治干预哮喘小鼠miR-155与Th17/Treg细胞免疫相关性实验性研究。SPF级雌性 BALB/c 小鼠，随机分为 空白(NC)组、模型(MC)组、肝脾同治低剂量组（GPTZ, 3.95/g·kg-1）、肝脾同治中剂量组（GPTZ, 7.90/g·kg-1）、肝脾同治高剂量组（GPTZ, 15.80/g·kg-1）、地塞米松组(1.56×10-3/g·kg-1)，OVA建立小鼠支气管哮喘气道炎症模型，建模后取肺组织行石蜡切片，常规染色，观察各组小鼠肺组织病理变化。采用ELISA法取小鼠血清及支气管肺泡灌洗液行白介素-4（IL-4）和干扰素（IFN-γ）检测。采用Real-time PCR技术检测脾脏miR-155、RORγt和Foxp3的表达情况。与空白组比较，模型组和各药物组支气管肺泡灌洗液IL-17A、TGF-β1值显著升高（P<0.05），IL-10值显著降低（P<0.05）；与模型组比较，各药物组IL-17A、TGF-β1值显著降低（P< 0.05），IL-10值显著升高（P<0.05）。与空白组比较，模型组Foxp3表达显著降低（P<0.05）；与模型组比较，各药物组Foxp3表达量显著升高（P<0.05），其中高剂量组Foxp3表达量最高。与空白组比较，模型组RORγt表达显著升高（P<0.05）；与模型组比较，各药物组RORγt表达量显著降低（P<0.05）；地塞米松组RORγt表达量最低，高剂量组RORγt表达量低于其余各剂量组。肝脾同治可能通过干预哮喘小鼠miR-155调节Th17/Treg及Foxp3/ RORγt表达，调控Treg/Thl7平衡，起到调节哮喘小鼠慢性气道炎症的作用。.支气管哮喘是小儿常见的呼吸系统疾病，基于小儿“脾常不足”、“肝常有余”的生理特点及五脏相关理论，治疗哮喘不能单纯针对体内宿有伏痰留饮，而单纯从肺、脾、肾三脏入手，本文从理论基础及临床实践两方面论证肝脾同治论治小儿支气管哮喘的有效性。通过阅读大量古代文献，分别从小儿肝脾生理、病理的特点，肝脾对小儿支气管哮喘宿根的形成的影响及肝脾与气机升降的关系进行论述，提出肝脾同治的理论依据及可行性；通过整理归纳相关文献，分析中医药治疗小儿支气管哮喘的疗效，发现肝脾同治在减轻患儿的临床症状，减少复发次数等方面具有独特的优势及应用前景。