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甲氧基黄酮类化合物对囊性纤维化跨膜电导调节因子的激活作用

曹欢欢, 房芳, 于波, 栾剑, 姜雨, 杨红^*

辽宁师范大学生命科学学院，大连 116081

摘要

囊性纤维化跨膜电导调节因子(CFTR)是一种cAMP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 = 2 μmol/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cAMP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。

关键词： 囊性纤维化跨膜电导调节因子; 氯离子转运; 3’,4’,5,5’,6,7-六甲氧基黄酮; 5-羟基-6,7,3’,4’-四甲氧基黄酮; 激活剂

分类号：R965.2

[Polymethoxylated flavonoids activate cystic fibrosis transmembrane conductance regulator chloride channel.] [Article in Chinese]

CAO Huan-Huan, FANG Fang, YU Bo, LUAN Jian, JIANG Yu, YANG Hong^*

College of Life Sciences , Liaoning Normal University, Dalian 116081,China

Abstract

Cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-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 cAMP 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.

Key words: CFTR; Cl−; transport; 3’,4’,5,5’,6,7-hexamethoxyflavone; 5-hydroxy-6,7,3’,4’-tetramethoxyflavone

收稿日期：2014-09-23　　录用日期：2014-12-31

通讯作者：杨红　　E-mail: hyanglnnu@gmail.com

引用本文：

曹欢欢, 房芳, 于波, 栾剑, 姜雨, 杨红. 甲氧基黄酮类化合物对囊性纤维化跨膜电导调节因子的激活作用[J]. 生理学报 2015; 67 (2): 225-234.

CAO Huan-Huan, FANG Fang, YU Bo, LUAN Jian, JIANG Yu, YANG Hong. [Polymethoxylated flavonoids activate cystic fibrosis transmembrane conductance regulator chloride channel.] [Article in Chinese]. Acta Physiol Sin 2015; 67 (2): 225-234 (in Chinese with English abstract).