解表方调控JAK/STAT蛋白激酶信号通路增强鼻黏膜屏障的作用机制研究

The upper respiratory mucosal barrier (URMB) is the first line defense against the pathogenic microorganism from mouth and nose. The dysfunction of this barrier is one of the major causes of acute upper respiratory infection(AURI). The application of “Exterior-releasing” is the basic method for the treatment of “Exterior syndrome” of traditional chinese medicine. Mahuang decoction and Yinqiao powder are used as the common medicine of “Exterior-releasing prescriptions”. The symptom of “Exterior syndrome” is similar with AURI very much. So, “Exterior-releasing prescriptions” are usually used in the treatment of AURI. However, the pharmacodynamic studies of the “Exterior-releasing prescriptions” have not shown strong activity in anti-inflammatory and anti-viral. Which cannot explain the action mechanism of the “Exterior-releasing prescriptions” in the treatment of AURI. Our previous studies showed that the “Exterior-releasing prescriptions” can reduce mortality induced by bacteria or viruses infected through the nasal cavity of mouse with URMB dysfunction. Part of the mechanism is related to the increasing the sIgA, lysozyme and interferon in saliva. Then, we think “Exterior-releasing prescriptions” maybe have effect on URMB dysfunction. In this project, we hope to reveal the scientific connotation of the “Exterior-releasing prescriptions” in the treatment of AURI. We will study the mechanism of “Exterior-releasing prescriptions” in promoting the upper respiratory mucosal mechanical and immune barrier from regulating the JAK/STAT protein kinase signal pathway. The techniques such as gene silencing will be used. We are going to explore the mechanisms of “Exterior-releasing prescriptions” in preventing deferred and downward development of AURI by promoting synthesis of sIgA and regulation of DCs/Treg cell imbalance.

上呼吸道黏膜屏障（URMB）是机体防御病原微生物由口鼻入侵的第一道防线，该屏障功能低下是急性上呼吸道感染（上感）发生的重要原因。解表方是中医用于表证治疗的方药，表证的症状与上感极为相似，临床报道以麻黄汤、银翘散为代表的解表方用于“上感”治疗均取得较好的疗效，但迄今为止从抗炎、抗病毒等方面对解表方的研究却未表现出较强的活性，不能阐释解表方防治“上感”的机制。课题组前期研究发现，解表方可降低URMB功能低下模型动物滴鼻感染细菌或病毒的死亡率，提高唾液中sIgA、溶菌酶、干扰素含量，提示解表方可提高URMB功能，但作用机制不清。本课题拟从调节JAK/STAT信号通路角度，采用基因沉默等技术，研究解表方增强上呼吸道黏膜机械屏障及免疫屏障的作用机制，从促进效应分子sIgA合成及调节DCs/Treg细胞平衡的角度，探讨解表方提高URMB以防止上感迁延及向下发展的机制，以期揭示解表方治疗上感的科学内涵。

本课题从调控JAK/STAT蛋白激酶信号通路的角度，对解表方增强鼻黏膜屏障的作用机制进行了探讨。采用冷-热循环刺激成功复制了大鼠及小鼠鼻黏膜屏障功能低下（RNMID及MNMBD）模型。转录组的差异基因和蛋白组的差异蛋白联合分析发现，SD大鼠经冷-热循环刺激后，可导致鼻黏膜局部免疫功能抑制。麻黄汤可通过促进RNMID大鼠鼻黏膜组织中BAFF、IgA、pIgR的蛋白表达，进而促进鼻黏膜IgA的蛋白表达和sIgA的分泌，并通过激活TLR4/NF-κB/BD信号通路促进RNMBD大鼠鼻黏膜β-防御素（BD2）的表达，该作用麻黄汤优于银翘散。银翘散减轻炎症作用更明显，可降低血清中炎症因子IL-1β、TNF-α的含量。转录组、蛋白组测序，以及qPCR、Western blot验证分析发现，两个解表方均可激发鼻腔局部免疫功能，修复鼻黏膜上皮屏障，作用机制研究发现，解表方可通过激活IL-10/IL-22/STAT3信号传导，诱导下游抗炎因子、抗菌肽、粘液蛋白、趋化因子、基质金属蛋白等发挥促进上皮屏障修复和激活鼻腔局部免疫功能。麻黄汤和银翘散均可通过激活JAK1/ STAT1/2信号通路促进MNMBD模型小鼠鼻黏膜组织中IFN-λ及相关信号转导分子的蛋白表达，其中银翘散促进IL-10Rβ和抗病毒蛋白Mx1的蛋白表达的作用较麻黄汤更为明显。采用聚肌胞苷酸（Poly I:C）模拟病毒经鼻刺激MNMBD模型后，两个解表方仍然可升高模型小鼠鼻黏膜组织中IgA和IFN-λ的蛋白表达。采用脂多糖（LPS）模拟炎症刺激，造成鼻黏膜机械屏障破坏，给予两种解表方后可显著增加Occludin蛋白表达，说明两方具有较好的修复鼻黏膜机械屏障的作用。在炎症环境下，银翘散可通过抑制TLR4/NF-κB/BD信号通路减轻脂多糖刺激诱导的上呼吸道炎症反应。以银翘散中主要药味牛蒡子的活性成分牛蒡子苷及苷元作为研究对象，对促进鼻黏膜上皮屏障的作用进行探讨，发现整体和离体实验中，它们均可促进损伤的鼻黏膜上皮屏障修复，减少炎症因子的释放，其作用机制与激活IL-22信号通路有关。研究还发现，辛凉解表方银翘散和麻杏石甘汤均可通过抑制LPS诱导小鼠急性肺损伤TLR4-JNK信号通路，阻止小鼠肺部中性粒细胞胞外诱捕网（NETs）的形成，这可能是辛凉解表方阻止上感向下呼吸道迁延的重要途径之一。