中文摘要：

囊性纤维化跨膜电导调节因子（CFTR）是一种c AMP依赖的Cl-通道蛋白,其在上皮液体分泌过程中具有重要作用。本研究组在前期工作中观察到两种甲氧基黄酮类化合物3’,4’,5,5’,6,7-六甲氧基黄酮（HMF）和5-羟基-6,7,3’,4’-四甲氧基黄酮（HTF）能够有效地激活CFTR Cl-通道,但是作用机制尚不清楚。本研究旨在利用细胞荧光淬灭模型和短路电流技术系统研究HMF和HTF对CFTR Cl-通道的激活作用。荧光淬灭实验结果显示两种化合物均能以剂量依赖的方式激活CFTR Cl-通道,该激活作用具有快速、可逆的特点,可被CFTR特异性抑制剂CFTRinh-172完全抑制;引人注目的是,HMF（EC50=2μmol/L）是迄今发现的亲和力最高的黄酮类CFTR Cl-通道激活剂。HMF和HTF对CFTR Cl-通道的激活作用具毛喉素（forskolin,FSK）依赖特性,与FSK和3-异丁基-1-甲基黄嘌呤（3-Isobutyl-1-methylx,IBMX）的作用存在相加效应,但是与三羟基异黄酮（genistein,GEN）的作用之间不存在协同效应。离体组织研究结果显示,HMF和HTF能够显著促进大鼠结肠粘膜Cl-电流及小鼠气管粘膜下腺液体分泌。以上结果提示,HMF和HTF能够通过提高c AMP水平和直接与CFTR蛋白作用两条途径发挥CFTR Cl-通道激活作用。本研究为深入揭示黄酮类CFTR Cl-通道激活剂结构与功能之间的关系奠定了基础。

英文摘要：

Cystic fibrosis transmembrane conductance regulator（CFTR）, a c AMP-dependent chloride channel, plays key roles in fluid secretion in serous epithelial cells. Previously, we identified two polymethoxylated flavonoids, 3＇,4＇,5,5＇,6,7-hexamethoxyflavone（HMF） and 5-hydroxy-6,7,3＇,4＇-tetramethoxyflavone（HTF） which could potentiate CFTR chloride channel activities. The present study was aimed to investigate the potentiation effects of HMF and HTF on CFTR Cl- channel activities by using a cell-based fluorescence assay and the short circuit Ussing chamber assay. The results of cell-based fluorescence assay showed that both HMF and HTF could dose-dependently potentiate CFTR Cl- channel activities in rapid and reversible ways, and the activations could be reversed by the CFTR blocker CFTRinh-172. Notably, HMF showed the highest affinity（EC50 = 2 μmol/L） to CFTR protein among the flavonoid CFTR activators identified so far. The activation of CFTR by HMF or HTF was forskolin（FSK） dependent. Both compounds showed additive effect with FSK and 3-Isobutyl-1-methylx（IBMX） in the activation of CFTR, while had no additive effect with genistein（GEN）. In ex vivo studies, HMF and HTF could stimulate transepithelial Cl- secretion in rat colonic mucosa and enhance fluid secretion in mouse trachea submucosal glands. These results suggest that HMF and HTF may potentiate CFTR Cl- channel activities through both elevation of c AMP level and binding to CFTR protein pathways. The results provide new clues in elucidating structure and activity relationship of flavonoid CFTR activators. HMF might be developed as a new drug in the therapy of CFTR-related diseases such as bronchiectasis and habitual constipation.