MiR-124-3p经调控claudin-1在A型肉毒毒素抑制下颌下腺分泌的机制研究

紧密连接是唾液经旁细胞途径转运的结构基础和限制因素，其中claudin-1是影响旁细胞屏障的最重要的分子。申请人前期研究发现A型肉毒毒素（botulinum toxin type A，BTXA）通过激活ERK1/2通路上调大鼠腺泡细胞内claudin-1表达，高通量测序和生物信息学分析发现miR-124-3p可调控ERK1/2通路，但对于BTXA是否可以通过miR-124-3p调控claudin-1的表达进而抑制唾液分泌尚未阐明。本项目拟采用western blot、qRT-PCR、和免疫荧光等方法在动物水平和细胞水平上检测BTXA对紧密连接结构和功能的影响；通过构建过表达miR-124-3p的动物模型以及工具药和基因转染等方法在分子水平上明确miR-124-3p调控claudin-1的信号通路。该研究将为更加全面地认识BTXA抑制唾液分泌的机制提供新思路，为扩展临床应用范围提供理论基础。

Tight junction (TJ) is a limiting factor for salivary secretion through paracellular pathway of salivary gland. The claudin family is a significant component of TJ, of which claudin-1 plays an important role in barrier function. Botulinum toxin type A (BTXA) can effectively inhibit salivary secretion, but most of the research on the mechanism focus on the inhibition of the nervous system, and the direct effect of BTXA on salivary gland cells is rarely reported. Our previous work indicated that BTXA could promote the expression of claudin-1through the ERK1/2 pathway on SMG-C6 cells. It was also found that miR-124-3p could regulate the ERK1/2 pathway through high-throughput sequencing and bioinformatics analysis. However, the exact changes of TJ and regulation of microRNA when BTXA inhibits salivary secretion have not been elucidated. We will investigate the effects of TJ structure and function by using western blot, qRT-PCR, FITC-dextran test and immunofluorescent methods in vivo and in vitro experiments. Meanwhile, the signaling pathway of regulation of claudin-1 by miR-124-3p will be determined by establishing an animal model of miR-124-3p overexpression and using pharmacological inhibitors and gene transfection techniques. This study may provide new clues for further research on the mechanism of BTXA inhibiting salivary secretion which is pivotal for the development of new clinical applications for BTXA.