基于EGFR-MEK/ERK/Sp1通路探讨泽漆化痰方通调水道抑制COPD气道粘液高分泌的作用机制

Airway mucus hypersecretion has been closely related with the occurrence, progression and clinical outcome of COPD, invention and control on airway mucus hypersecretion has a great significance to the prevention and therapy of COPD. According to preliminary clinical and experimental research, Zeqihuatan decoction is the proven prescription of national famous Professor HuangJiGeng, it has the effect of regulation of water passages and eliminating phlegm, it also can down-regulate TNF-a and MUC5AC in rats of airway mucus hypersecretion. The protein content and transcriptional level of MUC5AC has positive correlation of airway mucus hypersecretion, while recent researches have discovered the cascade of “TNF-α→EGFR-MEK/ERK/Sp1→MUC5AC”. Based on these observations, we propose that Zeqihuatan decoction can inhibit airway mucus hypersecretion by regulating the pathway of EGFR-MEK/ERK/Sp1. This study will be done to establish the rat model with double elements of smoking and LPS infused into trachea, and the cell model induced by TNF-α, then we will observe the following items: 1)the interventional effects of Zeqihuatan decoction on airway mucus hypersecretion in different pathological stages of COPD; 2)The effects of Zeqihuatan decoction on associated proteins activity and gene transcription in EGFR-MEK/ERK/Sp1 pathway. Thus we will aim to illustrate the mechanism of Zeqihuatan decoction inhibiting airway mucus hypersecretion in COPD, and set the scientific basis for the TCM pathology on regulation of water passages.

气道粘液高分泌与COPD的发生、进展和临床结局相关，干预并控制粘液高分泌在COPD防治研究中具重大意义。泽漆化痰方是黄吉赓教授的经验方，具通调水道，化痰消饮的作用，前期研究证实该方能下调气道TNF-α、MUC5AC表达，抑制COPD气道粘液高分泌。MUC5AC的含量和转录水平与气道粘液分泌呈正相关，结合 “TNF-α→EGFR-MEK/ERK/Sp1→MUC5AC”这一认识，提出“泽漆化痰方可能通过调节EGFR-MEK/ERK/Sp1通路抑制COPD气道粘液高分泌”的假说。本研究拟烟熏合LPS气道内滴入、TNF-α诱导分别建立COPD气道粘液高分泌大鼠模型和细胞模型，观察该方对COPD不同病理阶段气道粘液高分泌的干预作用及对EGFR-MEK/ERK/Sp1信号通路相关蛋白活性和基因转录的影响。阐明泽漆化痰方抑制COPD气道粘液高分泌的作用机制，并为“通调水道”的中医病机理论提供科学依据。

气道粘液高分泌与COPD的发生、发展及临床结局密切相关，干预并控制粘液高分泌在COPD的防治研究中意义重大。泽漆化痰方是黄吉赓教授的经验方，具通调水道，化痰消饮的作用，前期研究证实该方能下调气道TNF-α、MUC5AC表达，抑制COPD气道粘液高分泌。其中MUC5AC的含量和转录水平与气道粘液分泌呈正相关，本研究结合 “TNF-α→EGFR-MEK/ERK/Sp1→MUC5AC”这一通路开展相关研究。.该研究体内以烟熏合LPS气道内滴入建立COPD气道粘液高分泌大鼠模型，体外以TNF-α刺激人类支气管上皮细胞株16HBE建立COPD气道粘液高分泌细胞模型，观察泽漆化痰方对模型大鼠气道及肺组织细胞因子，病理形态学改变，EGFR通路上下游蛋白活性的影响；对模型细胞活力及凋亡，EGFR、ERK、SP1、MUC5AC相关基因和通路蛋白表达的影响。.体内实验结果显示，泽漆化痰方可以减少模型大鼠气道上皮杯状细胞增生，降低BALF中炎症因子TNF-α、IL-8释放，下调肺组织MUC5AC基因表达，降低EGFR、SP1、MUC5AC蛋白水平；但与EGFR抑制剂AG1478组组间比较，后者干预EGFR蛋白表达水平更为显著；说明泽漆化痰方抑制气道粘液高分泌的过程还可能存在EGFR之外的未知通路，值得我们进一步的研究和探索。.体外实验结果显示，TNF-α诱导可以建立COPD高分泌细胞模型；并证实EGFR mRNA及MUC5AC mRNA、EGFR与MUC5AC的蛋白水平在模型细胞中均有高表达；进一步qPCR与WB探究证实，在模型中EGFR抑制MUC5AC的表达，而抑制EGFR则增加了模型细胞的活性及凋亡水平，从而证实气道粘液高分泌“TNF-α→EGFR→MUC5AC”细胞通路的存在。进一步重复模型，应用泽漆化痰方浸膏粉不同剂量干预，发现该方有降低模型细胞MUC5AC基因表达的趋势，且机制可能与抑制EGFR下游蛋白ERK1/2的磷酸化有关。说明在细胞实验层面，该方抑制粘液高分泌的通路机制仍需进一步探究。.本研究从“调控气道粘蛋白表达”角度出发，围绕EGFR相关通路，探讨中药复方防治COPD气道黏液高分泌的作用机制，为“化痰消饮”的中医治法提供科学依据。