In Vitro And In Vivo Activity Of N-butylphthalide Against Candida Albicans And Underlying Mechanisms

Background:Candida albicans(C.albicans)is a kind of common opportunistic pathogen that may cause invasive fungal bloodstream infections.It brings great challenge to the treatment of candidiasis due to the increasing resistance of C.albicans to traditional antifungal drugs,such as azoles,on account of the long-term applications of them.N-butylphthalide(NBP)is a main component of Apium graveolens oil and is currently used for the treatment of acute and chronic ischemic stroke in clinic.It has been reported that the compounds that are the structural analogues of NBP and also extracted from Apium graveolens seeds have antifungal activity against C.albicans,however,there are no reports investigating the antifungal activity of NBP against C.albicans.Objectives:To investigate the antifungal effects of NBP against C.albicans both in vitro and in vivo and preliminarily explore the potential antifungal mechanisms.Method:A broth microdilution method was used to determine antifungal activity of NBP used alone and combined with fluconazole(FLC)against planktonic C.albicans and FICI model was used to interpret the in vitro interaction of the drug combination.XTT colorimetric assay was used to evaluate the anti-biofilm activity of NBP against C.albicans biofilms at different stage of maturation.Time-killing curves were performed to evaluate dynamical antifungal activity of NBP.Galleria mellonella infection model was established and survival rate,fungal burden and histological study were used to evaluate antifungal activity of NBP in vivo.To evaluate the effects of NBP on the virulence factors of C.albicans,hyphal morphologic switch and phospholipase activity were determined.The effects of NBP on the levels of intracellular reactive oxygen species and mitochondrial membrane potential were detected by fluorescent probe DCFH-DA and rhodaminel23.To explore potential synergistic mechanism of NBP combined with FLC,the effects of NBP on drug uptake and efflux were measured using rhodamine 6G.Results:(1)NBP exhibited antifungal activity against C.albicans and also exhibited synergistic effects combined with FLC against resistant C.albicans.NBP exhibited antifungal activity against six tested C.albicans strains with MICs of 128 ?g/mL,and also exhibited synergistic effects combined with FLC against resistant C.albicans with FICIs<0.25,resulting in a decrease in the MICs of FLC from>512 to 0.25-1 ?g/mL.Time-killing curves verified the antifungal activity in dynamic.(2)NBP exhibited anti-biofilm activity against C.albicans biofilms pre-formed<12 h and also exhibited synergistic effects combined with FLC against resistant C.albicans biofilms pre-formed<12 h.NBP exhibited anti-biofilm activity against C.albicans biofilms pre-formed<12h with sMICs of 128-256 ?g/mL,and also exhibited synergistic effects combined with FLC against resistant C.albicans biofilms pre-formed<12 h with FICIs<0.5,resulting in a decrease in the sMICs of NBP from 128-256 to 32-64 ?g/mL and the sMICs of FLC from>1024 to 0.5-8 ?g/mL.Besides,no synergism was observed when NBP combined with FLC against susceptible C.albicans biofilms pre-formed<12h.In addition,NBP alone or combined with FLC hardly inhibited biofilms pre-formed over more than 24h.(3)NBP combined with FLC exerted synergistic antifungal effects to C.albicans infected Galleria mellonella larvae.NBP monotherapy could enhance survival rate of larvae infected by C.albicans,reduce the fungal burden in larvae and cause less damage to larval tissues.However,the therapeutic effects were significantly improved when NBP combined with FLC,indicating an evident synergistic antifungal activity of the combination of NBP and FLC in vivo.(4)NBP attenuated the virulence factors of C.albicans.NBP significantly reduced the activity of extracellular phospholipase of C.albicans from very high activity to low activity.Besides,NBP prevented C.albicans hyphal morphologic switch in a dose dependent manner.These results suggested NBP exhibited antifungal activity via attenuating virulence factors.(5)NBP induced intracellular reactive oxygen species accumulation and a decrease in mitochondrial membrane potential.As the increase of NBP concentration,the levels of intracellular reactive oxygen species in C.albicans increased,at the same time,the mitochondrial membrane potential decreased and the percentage of C.albicans cells with the reduced mitochondrial membrane potential increased significantly,suggesting that NBP exhibited antifungal activity via inducing intracellular reactive oxygen species accumulation and a loss mitochondrial membrane potential.(6)NBP inhibited the activity of drug transporters in C.albicans.NBP significantly promoted drug uptake and suppressed drug efflux pumps,leading to a high levels of FLC concentrations in fungal cells,which was the potential mechanism of the synergistic effects between NBP and FLC.Conclusions:NBP exerted antifungal activity alone and synergized with FLC against planktonic C.albicans and preformed biofilms in vitro.Besides,NBP combined with FLC exerted in vivo antifungal effects,prolonging the survival rate of Galleria mellonella larvae infected with C.albicans,reducing the fungal burdern and causing less damage to tissue.The underlying mechanism was correlated with attenuating C.albicans virulence factors,including activity of extracellular phospholipase and hyphal morphologic switch,inducing accumulation of intracellular reactive oxygen species and a loss in mitochondrial membrane potential and depressing activity of drug transporters.