基于TLR/自噬/NF-κB信号系统探讨复方肿节风雾化剂对大鼠细菌性慢性咽炎的干预作用

Bacterial chronic pharyngitis is pharyngeal diffuse inflammation caused by Group A Streptococcus Pyogens (GAS) based Gram-positive bacteria, the existence of drug resistance, delayed healing and other issues. Our group found that the therapeutic effect to esophageal reflux pharyngitis by using Fufangzhongjiefeng aerosol significantly better than that of western medicine, and this traditional Chinese medicine had significant antibacterial activity for GAS in vitro. This medicament is optimization of the proved recipe made by Prof. Li Shengyue who is famous Chinese traditional doctor. The TLR / autophagy / NF-κB signaling system is a key to regulate immune and inflammatory response. Autophagy can regulated inflammation by clearing endogenous inflammatory agonists and influencing the secretion of immune mediators. In this project, we will investigate Fufangzhongjiefeng aerosol the therapeutic effect and intervention mechanism to the rat model of chronic pharyngitis which is be infected with GAS. In view of the references about immune regulation of chronic pharyngitis and the preliminary studies of our group, we plan to detect the expression of serum SIgA and TLR / autophagy / NF-κB signal system in the model rat by the experiment methods of histopathology, ELISA, immunohistochemistry, real-time fluorescence quantitative PCR, Western blot, and transmission electron microscope. This research will provide a scientific laboratory basis for the treatment of bacterial chronic pharyngitis by using traditional Chinese medicine.

细菌性慢性咽炎是由乙型溶血性链球菌（GAS）为主的革兰阳性细菌引起的咽部弥漫性炎症，存在耐药性、迁延不愈等问题。课题组前期临床发现以全国名老中医李声岳教授验方改进的复方肿节风雾化剂对食管返流性咽喉炎的治疗效果显著优于西医疗法，且具有显著的体外GAS抗菌作用。已知TLR/自噬/NF-κB信号系统是调节免疫和炎症反应的关键信号系统，自噬可以通过去除内源性炎症小体激动剂和对免疫介质分泌的影响来控制炎症。因此，基于上述慢性咽炎免疫调控理论背景及课题组前期研究基础，拟通过GAS慢性咽炎大鼠体内、外动物实验，采用组织病理学、ELISA、免疫组织化学、实时荧光定量、Western blot、电子透射显微镜等方法对血清SIgA、TLR/自噬/NF-κB信号系统相关指标进行检测，了解复方肿节风雾化剂对大鼠乙型溶血性链球菌慢性咽炎的影响及可能的干预机制，为中医药治疗细菌性慢性咽炎提供科学的实验室依据。

细菌性慢性咽炎是由乙型溶血性链球菌（GAS）为主的革兰阳性细菌引起的咽部弥漫性炎症，存在耐药性、迁延不愈等问题。课题组前期临床发现以全国名老中医李声岳教授验方改进的复方肿节风雾化剂对食管返流性咽喉炎的治疗效果显著优于西医疗法，且具有显著的体外GAS抗菌作用。已知TLR/自噬/NF-Κb信号系统是调节免疫和炎症反应的关键信号系统，自噬可以通过去除内源性炎症小体激动剂和对免疫介质分泌的影响来控制炎症。因此，基于上述慢性咽炎免疫调控理论背景及课题组前期研究基础，本项目通过GAS慢性咽炎SD大鼠体内外实验，采用组织病理学、ELISA、免疫组织化学、实时荧光定量、Western blot、电子透射显微镜等方法对血清与黏膜炎症因子及SIgA、TLR/自噬/NF-κB信号系统相关指标进行检测，并通过网络药理学方法，预测复方肿节风雾化剂治疗慢性咽炎的活性成分、靶点及通路，对筛选出的关键靶点及信号通路进行体外实验验证，研究复方肿节风雾化剂基于TLR/自噬/NF-κB信号系统对细菌性慢性咽炎的可能作用机制。结果显示，复方肿节风雾化剂可显著改善咽部黏膜上皮脱落、坏死、炎性细胞浸润情况，显著下调TNF-α、IL-1β、IL-6及IFN-β等炎症因子的表达，上调SIgA的表达，下调TLR2、TLR4、TRAF6、MyD88和NF-κB P65 mRNA及蛋白的表达，上调咽部黏膜组织中Beclin1、P62和LC3表达水平以恢复咽部黏膜的细胞自噬。再通过网络药理学方法，挖掘出复方肿节风活性成分51个、潜在作用靶点11个及12个经典通路，对筛选出的关键靶点及信号通路进行体外实验验证。结果表明“复方肿节风雾化剂”治疗慢性咽炎的作用是多成分、多靶点、多通路的，复方肿节风雾化剂对细菌性慢性咽炎的作用机制之一可能是促进咽部黏膜细胞自噬、干预TLR/NF-κB信号通路表达以下调TNF-α、IL-1β、IL-6及IFN-β等炎症因子、上调SIgA的表达，从而对慢性咽炎的炎症反应发挥抗炎作用，这为中医药治疗细菌性慢性咽炎提供科学的实验室依据，也为后续复方肿节风雾化剂治疗慢性咽炎的具体靶点作用机制提供研究新方向。