捏脊法改善SPMs炎症调控防治脾虚哮喘的机制研究

Dysregulation of endogenous specialized proresolving mediators(SPMs) leads to the unresolved inflammation in asthma. In TCM, spleen deficiency is a key mechanism of asthma among children, which is also complicated with inflammation dysregulation. Spine-pinching manipulation is effective in regulating zangfu-organs and effectively strengthening the spleen , which has good clinical effect in preventing and treating asthma among children. Based on the study experience in America as a visiting scholar and the previous research, improving SPMs dysregulation in spleen deficiency may be an important mechanism of spine-pinching manipulation on preventing and treating asthma among children. In this project, a rat asthma model will be adopted in which ovalbumin (OVA) sensitization and challenge is based on a spleen-deficiency model. Spine-pinching manipulation will be applied as an intervention method. At several time points before and after OVA sensitization and challenge, the following will be observed. The pathology and microstructure of the jejuna and lung will be assessed by HE staining and electron microscope. The ratio of subgroups of spleen CD4+T cells will be detected by flow cytometry. Serum OVA-sIgE, inflammation-related cytokines in serum and bronchoalveolar lavage fluid will be detected by ELISA. The levels of SPMs-precursor fatty acids in the jejuna and lung as well as SPMs and pro-inflammatory lipid mediators (LMs) in the lungs will be detected by HPLC-MS/MS. The protein distribution and mRNA expression of key enzymes related to inflammation-related LMs in the lung will be detected by immunohistochemistry and RT-PCR respectively. The protein expression of IκBα/p-NF-κB p65 and the major receptors of inflammation-related LMs will be detected by western blotting. This project will help with clarification of the spleen deficiency influence on inflammation regulation of SPMs in asthma and then the mechanism of spine-pinching manipulation via improving inflammation regulation of SPMs on prevention and treatment of spleen-deficiency asthma.The project may not only enrich the connotation of the spleen deficiency but also provide clinical application of spine-pinching manipulation with scientific support.

内源性特异性促炎症消退介质（SPMs）调控障碍是哮喘慢性炎症的重要原因。脾虚为儿童哮喘重要病机，脾虚存在炎症调控障碍。捏脊法可调脏腑，善健脾，可较好防治儿童哮喘；结合前期研究提示，改善脾虚哮喘SPMs炎症调控可能是其重要机制。本项目在脾虚慢性炎症大鼠模型基础上再造哮喘模型，运用捏脊法干预脾虚及哮喘造模过程，在不同时间点观察，HE染色和电镜检测空肠和肺组织的病理及超微结构变化，流氏细胞术检测脾CD4+T细胞亚类比例；ELISA检测血清OVA-sIgE及血清和支气管肺泡灌洗液中炎症细胞因子表达，高效液相色谱-质谱法检测肺和空肠SPMs前体脂肪酸、肺组织主要SPMs及促炎脂质介质（LMs）水平，免疫组化和RT-PCR分别测LMs合成酶表达分布及基因表达，免疫印迹测IκBα/p-NF-κB p65及SPMs主要受体表达，明确捏脊法改善SPMs炎症调控防治脾虚哮喘的机制，为防治儿童哮喘提供科学依据。

中医学认为，脾虚是儿童哮喘的重要病理基础。研究提示，脾虚可能负面影响体内炎症的自我调控。内源性特异性促炎症消退介质（SPMs）参与限制炎症并促进炎症的及时消退。哮喘的发生发展与SPMs炎症调控障碍密切相关。推拿防治儿童哮喘效果良好，擅长健脾的捏脊法是常用的主要手法。为了探讨脾虚对幼鼠哮喘SPMs炎症调控的影响以及捏脊法的效用机制，本项目首先建立脾虚模型，并运用捏脊法干预，观察脾虚是否诱导幼鼠免疫失衡及炎症状态、是否影响SPMs前体脂肪酸水平，以及捏脊法的干预效应；然后，建立脾虚哮喘模型，运用捏脊法干预，观察脾虚对幼鼠哮喘过敏性气道炎症（AAI）程度及炎症消退的影响，观察脾虚对肺组织炎症介质和SPMs、相关合成酶和受体等SPMs炎症调控相关因素的影响以及捏脊法的效用机制。研究结果显示，脾虚诱导幼鼠出现Th2免疫优势、肺和空肠的前体脂肪酸水平不同程度下降，尤以空肠AA和肺DHA水平下降显著；捏脊法干预有效抑制了脾虚诱导的Th2免疫优势和空肠AA水平的下降。脾虚诱导幼鼠哮喘AAI炎症程度增加且消退延迟，并伴有肺组织PGE2、CysLTs和LTB4等炎症介质含量进一步升高、PGE2合成酶COX-2蛋白及基因表达以及炎症介质主要受体表达进一步升高；脾虚抑制了LXA4、RvE1和RvD1等SPMs含量的适时升高，抑制了致敏后SPMs主要受体蛋白表达的增加，诱导合成酶蛋白表达改变，促进了p-NF-κB和p-IκBα蛋白表达的进一步增加，诱导了肺上皮屏障紧密连接ZO-1和Occludin蛋白表达进一步下降。捏脊法干预有效抑制了脾虚对幼鼠哮喘AAI炎症以上负面的影响，促进了AAI消退。因此，本研究提示，脾虚诱导幼鼠哮喘SPMs炎症调控障碍和肺上皮屏障的破坏，这可能是脾虚加重幼鼠哮喘AAI炎症程度并延缓消退的重要机制之一。捏脊法干预可有效促进SPMs炎症调控、保护肺上皮屏障，阻止脾虚对幼鼠哮喘AAI的负面影响，促进炎症的消退。本研究为脾虚是儿童哮喘重要病理基础提供科学依据，也为捏脊法等小儿推拿防治儿童哮喘等过敏性疾病的推广运用提供科学依据。