The Activation Effect Of Polymethoxylated Flavones On Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel

Cystic fibrosis transmembrane conductance regulator(CFTR) is a cAMP-activated chloride channel expressed in the apical membrane of serous epithelial cells. CFTR plays a crucial role in transepithelial fluid homeostasis because it is the primary driver of fluid and water secretion. CFTR activators may have potential use for the treatment of CFTR-related diseases. So, looking for the CFTR activator is current research hotspot.Polymethoxylated Flavone is an important member of flavonoid, which has a variety of pharmacological activities, such as anti-tumor, anti-inflammatory, anti-oxidant, anti –cardiovascular, anti-atherosclerosis and so on. This research found a new activity they have potentiation effect on CFTR Cl~- channel. In the present study, three Polymethoxylated Flavones(5-Hydroxy-6,7,3′,4′-tetramethoxyflavone, HTF; 3’,4’,5,5’,6,7-hexamethoxyflavone, HXF; 3,5,6,7,8,3’,4’-heptamethoxyflavone, HPF) are identified as CFTR activators using high-throughput screening based on FRT cell-based fluorescence assay, but the precise molecular mechanism whereby Polymethoxylated Flavones-induced chloride secretion remained largely elusive.The present study is aimed to investigate the potentiation effects of HTP, HXF and HPF on CFTR(Wt-CFTR, ?F508-CFTR)Cl~- channel activities using a cell-based fluorescence assay and the short circuit Ussing chamber assay. MTT test is done to estimate whether the FRT cell produce toxicity. The results of cell-based fluorescence assay showed that HTP, HXF, HPF 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. The activation of CFTR by HTF, HXF and HPF are forskolin(FSK) dependent. Thress 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). These results suggest that Polymethoxylated Flavones may potentiate CFTR Cl~- channel activities through both elevation of cAMP level and binding to CFTR protein pathways. In ex vivo studies, they could stimulate transepithelial Cl~- secretion in rat colonic mucosa and enhance fluid secretion in mouse trachea submucosal glands. Cytotoxicity test results show that HTP and HXF fail to produce toxicity in 24 h. Notably, HXF show the highest affinity to CFTR protein among the flavonoid CFTR activators identified so far. Polymethoxylated Flavones might be developed as a new drug in the therapy of CFTR-related diseases such as CF, bronchiectasis and habitual constipation.