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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。
Cystic fibrosis transmembrane conductance regulator (CFTR) is a c AMP-dependent Cl-channel protein that plays an important role in epithelial fluid secretion. In our previous work, we observed that the two methoxy flavonoids, 3 ’, 4’, 5,5 ’, 6,7-hexamethoxyflavone (HMF) and 5-hydroxy-6,7,3’ , 4’-tetramethoxyflavone (HTF) can effectively activate CFTR Cl-channel, but the mechanism of action is unclear. The purpose of this study was to investigate the activation of CFTR Cl-channel by HMF and HTF by using fluorescence quenching model and short-circuit current technique. Fluorescence quenching results showed that both compounds can activate CFTR Cl-channel in a dose-dependent manner. The activation is fast and reversible and can be completely inhibited by CFTRinh-172, a CFTR-specific inhibitor. , HMF (EC50 = 2μmol / L) is by far the most affinity flavone CFTR Cl-channel activator. The activation of CFTR Cl-channels by HMF and HTF is characterized by a forskolin (FSK) -dependent property that is associated with the binding of FSK and 3-Isobutyl-1-methylx (IBMX) There is additive effect, but there is no synergistic effect with genistein (GEN). The results of in vitro study showed that HMF and HTF can significantly promote the Cl-current in rat colonic mucosa and the secretion of submucosal gland fluid in mice. The above results suggest that HMF and HTF exert CFTR Cl-channel activation by increasing the level of c AMP and directly interacting with the CFTR protein. This study laid the foundation for further revealing the relationship between the structure and function of flavonoid CFTR Cl-channel activators.