辣椒素受体经由occludin和ZO-1调控颌下腺分泌的机制研究

We previously showed for the first time that activation of transient receptor potential vanilloid 1 (TRPV1) promotes saliva secretion through modulating tight junction-mediated paracellular route. However, the exact molecule and mechanism involved in the TRPV1 modulation is still unknown. By using immunobloting, immunofluorescence, siRNA and plasmid transfection, we will detect the effects of TRPV1 on expression, distribution, and function of tight junction components in rat isolated submandibular glands (SMGs) as well as SMG-C6 cells, aiming to determine that occludin and ZO-1 are potential targets for TRPV1 to regulate saliva secretion in rat SMG from organic, cellular and molecular levels. We will further investigate the signal pathway associating TRPV1 with occludin and ZO-1, especially focusing on ERK1/2 and RhoA/ROCK/MLC2 pathways. Moreover, we plan to establish hypofunctional SMG models in vivo and SMG-C6 cells in vitro induced by TNF-α, an important inflammation factor, and explore the alteration of occludin and ZO-1 together with their pathophysiological significance. We will also investigate the possibility of using TRPV1 to promote saliva secretion on the inflammatory, hypofunctional SMGs. This study will allow us to further understand the physiological and pathophysiological significance of TRPV1-regulated occludin and ZO-1 in SMG in vivo and in vitro, providing a novel insight into a future therapeutic target for hyposecretion of SMG.

我们首次发现细胞间紧密连接是辣椒素受体调控颌下腺分泌的新途径，但辣椒素受体调控的具体分子及作用机制尚待明确。本课题拟采用免疫印迹、免疫荧光、siRNA和质粒转染等方法检测激活辣椒素受体对大鼠离体颌下腺和SMG-C6细胞上紧密连接蛋白表达、分布和功能的影响，在器官、细胞和分子水平证实occludin和ZO-1是辣椒素受体特异性调控的紧密连接靶分子；围绕ERK1/2和RhoA/ROCK/MLC2通路探讨辣椒素受体调控occludin和ZO-1的信号转导途径；特别是在炎症因子诱导的整体和细胞模型上检测occludin和ZO-1的变化及病理生理意义，探讨激活辣椒素受体经调节紧密连接分子改善颌下腺分泌功能低下的可行性。该研究将揭示辣椒素受体调控occludin和ZO-1的分子机制，丰富和完善紧密连接介导颌下腺分泌的理论，并为探讨以紧密连接为靶点改善颌下腺分泌提供新的思路。

紧密连接是位于上皮细胞之间的蛋白复合体，是介导水和溶质经旁细胞转运的重要途径。本项目以颌下腺紧密连接膜蛋白occludin和胞浆蛋白ZO-1/-2为重点，明确它们的表达和分布特点，分别敲低occludin和ZO-1/-2的表达可降低跨上皮细胞电阻和增加FITC-右旋糖酐透过率，而过表达紧密连接蛋白则恢复旁细胞途径通透性，证实occludin和ZO-1/-2是构成颌下腺紧密连接的关键蛋白。激活辣椒素受体可改变occludin和ZO-1/-2的细胞内分布，增加ERK1/2磷酸化。使用ERK1/2激酶抑制剂PD98059或转染siRNA敲低ERK1/2表达后，激活辣椒素受体引起的跨上皮细胞电阻降低及occludin重分布的作用均被抑制。辣椒素可增加颌下腺组织及SMG-C6细胞GTP-RhoA和磷酸化MLC2的表达；RhoA抑制剂C3转移酶或Rho激酶抑制剂Y27632预处理可抑制辣椒素引起的旁细胞途径通透性增加，阻断ZO-/-2重分布，说明激活辣椒素受体以ERK1/2依赖的方式调控occludin的功能，通过RhoA-ROCK信号通路调控ZO-1和-2分布以及紧密连接的开放。在家兔模型上，移植颌下腺紧密连接的宽度明显减小，ZO-1等紧密连接蛋白的表达量降低。辣椒素治疗可减轻移植颌下腺紧密连接的结构损伤，恢复紧密连接蛋白的表达。针对颌下腺移植患者早期分泌不足的临床问题，我们联合使用辣椒素和卡巴胆碱促进腺体分泌，明显降低移植颌下腺的导管阻塞率。该研究揭示了激活辣椒素受体调控occludin和ZO-1/-2的分子机制，为改善颌下腺分泌功能低下提供了新策略。