Regulatory Effects And Selectivity Of Natural And Synthetic Flavonoids On Volume-regulated Anion Channels

Natural flavonoids are ubiquitous in dietary plants and vegetables and have been proposed to have antiviral,antioxidant,cardiovascular protective and anticancer effects.Volume-regulated anion channels?VRACs?,which are essential for cell volume regulation,have been proposed to play a key role in cell proliferation and migration,apoptosis,transepithelial transport,and cancer development.Therefore,the aim of this study is to explore the highly specific blockers of VRACs channel and preliminarily study the mechanism of flavonoids regulating angiogenesis.A whole-cell patch technique was used to record VRAC currents in the human embryonic kidney?HEK?293 and human umbilical vein endothelial?HUVEC?cells.The 5'-bromo-2-deoxyuridine?BrdU?assay technique was used to investigate cell proliferation.This study demonstrates that natural and synthetic flavonoids are potent VRAC current inhibitors,and VRAC inhibition by flavonoids might be responsible for their anti-angiogenic effects.This study mainly includes the following two parts:?1?the regulatory effect of natural and synthetic flavonoids on volume-regulated anion channels;?2?studies on the regulatory effect and selectivity of synthetic flavonoid flavoxate and its metabolites 3-methylflavone-8-carboxylic acid?MFCA?on volume-regulated anion channels and calcium-activated chloride channels.Part 1 The effect of natural and synthetic flavonoids on VRAC channelsObjective:The aim of the study was to investigate the regulatory effects of natural and synthetic flavonoids on VRAC channels,and indicating that flavonoids were effective inhibitors of VRAC channels.At the same time,it is suggested that the blocking effect of flavonoids on VRAC channels may be an important reason for its inhibition of angiogenesis.Methods:Whole-cell patch clamp technique was used to record the effects of natural and synthetic flavonoids on VRAC currents in HEK293 and HUVEC cells,and BrdU technique was used to detect the effect of drugs on cell proliferation.Results:1.The effects of natural flavonoids on VRAC currents in HEK293 cells.At 100?M,34 of the 53 flavonoids inhibited VRACs current by more than 50%.Luteolin,baicalein,eupatorin,galangin,quercetin,fisetin,karanjin,Dh-morin,genistein,irisolidone and prunetin have the highest inhibition rate?the mean inhibition rate is greater than 80%?.2.The effects of natural flavonoids on dose-response curve of VRACs in HEK293 cellsWe recorded the effects of different doses of drugs on VRAC currents at-100 mV and established a dose-response relationship curve.The results show that the IC₅₀s range is 5-13?M and the E_maxs range is 87-99%.3.The effects of natural flavonoids on current-voltage?I-V?curves of VRACs in HEK293 cellsWe observed the effects of 3 and 30?M drugs and 10?M DCPIB on the I-V curve of VRAC channels.The results showed that these drugs could inhibit VRACs current in a concentration-dependent manner,but did not affect its reversal potential.4.The effects of natural flavonoids on VRAC currents in HUVEC cellsConsistent with the VRAC currents results observed for the drugs observed in HEK293 cells,100?M of luteolin,baicalein,galangin,quercetin,fisetin,genistein and 10?M DCPIB caused significant inhibition of VRAC currents in HUVEC cells.However,flavone,herbacetin,daidzein,genistin,and catechin have a weaker inhibitory effect on VRAC currents in HUVEC cells.5.The effects of natural flavonoids on proliferation of HUVEC cellsThe results showed that 11 natural flavonoids with strong inhibition of VRACs?except Dh-morin?significantly inhibited the proliferation of HUVEC cells.6.The effects of synthetic flavonoids on VRAC currents in HEK293 cellsAt 100?M,flavoxate,alovcidib and diosimin significantly inhibited VRACs current with inhibition rates of 87.84±0.86%,71.33±1.31%,56.98±5.12%.Flavoxate had the strongest inhibitory effect on VRACs.7.The effect of flavoxate on the dose-effect relationship and current-voltage?I-V?curve on VRAC currents in HEK293 cellsWe established a dose-response curve of flavoxate regulating VRAC currents in HEK293 cells.And the IC₅₀0 of flavoxate is 2.25±0.30?M and the E_maxax is 91.81±2.74%.We observed the effects of flavoxate of 3 and 30?M and DCPIB of 10?M on the I-V curve of VRAC currents.It was found that flavoxate could significantly inhibit the currents of VRAC,but did not affect its reversal potential.Part 2 The selective study of synthetic flavonoid flavoxate and its active metabolites on VRAC channels.Objective:To explore the selective effects of flavoxate and its active metabolite MFCA on VRAC channels.Methods:Whole cell patch clamp technique was used to record the effects of flavoxate and its active metabolite MFCA on VRACs and TMEM16A/CaCCs.Results:1.The current recorded in the experiment is TMEM16A/CaCC currentsCalcium enter the cell membrane through the electrode tip,and the increasing intracellular calcium concentration will gradually activate the current.The intracellular free calcium concentration was 384 nM.Therefore,it is determined that the recorded current is TMEM16A/CaCC currents,which can be administered when the current tends to stabilize.2.The regulatory effects of flavoxate and MFCA on VRACsAt 100?M,flavoxate significantly inhibited VRAC currents activated by hypotonic solution,and the inhibition rate was 89.43±0.92%.MFCA also significantly inhibited VRAC currents at 100?M with an inhibition rate of72.62±1.58%.3.The effects of flavoxate and MFCA on TMEM16A/CaCCsThe inhibition rate of flavoxate on TMEM16A/CaCC currents are about35%at 100?M.By observing the effects of 30?M flavoxate and MFCA on the I-V curve of TMEM16A/CaCC currents,it was found that both flavoxate and MFCA could inhibit TMEM16A/CaCC currents.The inhibition rate of flavoxate on TMEM16A/CaCC currents were 21.11±2.74%at 30?M,and the inhibition rate of MFCA on TMEM16A/CaCC currents were 19.25±2.30%at30?M.Conclusions:1.Natural and synthetic flavonoids can significantly inhibit VRAC channel currents,which may block angiogenesis by blocking VRAC channel currents.2.Flavoxate and its active metabolite MFCA have a high selectivity on VRAC channels.