食管上皮连接屏障受损是难治性胃食管反流病（RGERD）难以缓解的原因。胰蛋白酶是RGERD的主要致病因子，可激活食管上皮细胞蛋白酶活化受体-2（PAR-2），但其损伤食管上皮连接屏障的机制尚未明了。褪黑素可保护多种细胞连接屏障，但目前尚无褪黑素对RGERD食管上皮连接屏障作用的相关研究。申请者前期研究发现：褪黑素通过调节ERK/MLCK途径，保护食管上皮连接屏障。本项目基于前期工作和前人研究，拟通过动物实验和细胞实验，探讨：1）胰蛋白酶是否激活食管上皮细胞PAR-2/PKC/ERK/MLCK途径；2）胰蛋白酶是否促进食管上皮细胞产生IL-8，使中性粒细胞活化、释放弹性蛋白酶调节食管上皮细胞PAR-2/Rho/ROCK/MLCP转导；3）褪黑素能否阻断上述信号途径；最终阐明褪黑素对PAR-2介导胰蛋白酶损伤食管上皮连接屏障的保护机制。本项目可以为RGERD发病机制及治疗药物研究提供实验依据。

Sustained damage of esophageal epithelial junction barrier plays a key role in the pathogenesis of RGERD, however, the exact mechanism is still not yet clear. Trypsin is the main pathogenic factor of refractory gastroesophageal reflux disease (RGERD). It can activate protease activated receptor-2 (PAR-2) of the esophageal epithelial cells. Melatonin can protect the cellular junction barrier of different types of cells, but there isn't any research about melatonin effects on the esophageal epithelial junction barrier of RGERD. It was found in our previous research: the activating of ERK/MLCK signaling pathway mediated the disruption of esophageal epithelial junction barrier, and melatonin protected the esophageal epithelial junction barrier through inhibiting this signal transduction. The present project bases on the previous work and the predecessors' research, aims to explore: 1) whether trypsin activates PAR-2/PKC/ERK/MLCK signal transduction in the esophageal epithelial cells, 2) whether trypsin stimulates esophageal epithelial cells to produce IL - 8, makes the neutrophils chemotaxis and releases elastase, then elastase adjusts PAR-2/Rho/ROCK/MLCP signal transduction in the esophageal epithelial cells, 3) whether melatonin protects the esophageal epithelial junction barrier through blocking the above signal transduction. Finally, we wish to clarify that melatonin protects esophageal epithelial junction barrier against trypsin via PAR-2. The present study lays a theory foundation for further understand the mechanism of esophageal epithelial junction barrier damage in RGERD, and provides the theoretical and experimental basis for RGERD drug therapy.