| BackgroundIn the past few decades,fungus has emerged as an important cause of severe and fatal infections.Candida albicans is one of the most common opportunistic pathogens in humans,and the infections caused by it are associated with a high mortality often in excess of 30%due to the limited types of available antifungal agents and the emergence of drug resistance.Only a few types of antifungal agents are available now,such as azoles,echinocandins,polyenes,miazines and allyamines.Among them,fluconazole(FLC),one of the azoles,is the most comonly used agent to treat infections caused by Candida albicans due to its high bioavailability and low toxicity.However,the extensive and frequent use of FLC has led to the development of drug resistance in Candida albicans,and brought great challenges for anti-fungal infections.In addition,Candida albicans is easy to form biofilms on medical devices,which enhances the drug resistance of Candida albicans to antifungal agents.Therefore,it is becoming increasingly difficult to ignore the phenomenon of drug resistance in Candida albicans.Approaches to treat these infections caused by drug resistant Candida albicans are lacking,and patients must receive pharmacotherapy of toxic antifungals.Thus,the development of improved antifungal treatments to combat the drug resistance is urgently needed.Drug combination of FLC with non-antifiingal agents,now has been one of the most successful approaches to cope with the resistance of fungi.Utilizing synergistic drug combinations could potentially reduce toxicity that may occur when patients are exposed to high doses of drugs.Ambroxol hydrochloride(ABH)is widely used to treat chronic obstructive pulmonary disease,chronic bronchitis,bronchial asthma,spastic bronchitis,as well as bronchopulmonary complications after chest surgery,due to its potent expectoranting action,respiratory function improvement and low toxicity.Furthermore,it has also been reported to exert antioxidant activity and relieve the inflammation by reducing the release of inflammatory cytokines.So far,the efficacy,tolerability and safety of ABH have been verified in clinic.Interestingly,ABH used alone at high concentrations of 1-60mg/mL has been shown to exhibit antimicrobic effects against Candida albicans,Candida parapsilosis and Pseudomonas aeruginosa.However,no study has investigated the combined antifungal effects of ABH and FLC so far.ObjectiveThe present study was designed firstly to determine the potential antifungal effects of FLC and ABH against Candida albicans in viro and in vivo,and further detecte the synergistic mechanisms associated with anti-drug resistance of Candida albicans.MethodsThe in vitro antifungal effects of FLC and ABH alone or in combination against Candida spp.isolates with different susceptibilities were determined by a broth microdilution method,according to Clinical and Laboratory Standards Institute method M27-A3 document.Fractional inhibitory concentration index(FICI)model and percentage of growth difference(?E)model were used to interpret the in vitro antifungal effects of FLC and ABH on Candida spp.isolates.Using Galleria mellonella as the infection model,survival rates,fungal burden and histopathology of infected larvae were used to determine the in vivo antifungal effects of FLC and ABH alone or in combination.For the synergistic mechanism explorations,the effect of ABH on the uptake and efflux of FLC was measured using rhodamine 6G as fluorescence dye,with the help of a flow cytometer.In addition,two important virulence factors,hyphal growth and phospholipase activity,were determined,and then hhypha related gene HWPI and phospholipase encoding genes(PLBI and PLC1)were respectively determined by the RT-PCR.Results1 ABH combined with FLC reversed the resistance of Candida albicans cells in vitroAddition of ABH made the MIC of FLC for CA10 and CA16 decrease from>512?g/mL to 2?g/mL,with both of these FICIs<0.5,indicating that ABH combined with FLC has synergistic effects against resistant Candida albicans.Synergy was not observed when this combination therapy was used against susceptible Candida albicans,with the FICIs>1.2 ABH combined with FLC showed an effect against resistant Candida albicans in vivoDrugs.FLC and ABH,used alone had no significant effect on the survival rates of infected larvae but the combination of them significantly improved the survival rates,thus,confirming the efficacy of this drug combination in vivo.Encouragingly,the combination of FLC and ABH resulted in a greater reduction in fungal burden compared to the control and indivicdual drug treatments.By observing the slices of infected larvae in four groups,it can be found that there is a significant decrease in the number of' melanized nodules in larvae treated with this drug combination,comparing to no drug or individual drug treated larvae.Our results verified the synergistic effects of FLC and ABH in combination against resistant Candida albicans in vivo.3 ABH inhibited the activity of drug transportersBoth rhodamine 6G and FLC are substrates of drug transporters in Candida albicans.Rhodamine 6G was used as the alternate of FLC in the present study.Intracellular rhodamine 6G uptake in ABH-treated group increased over time,and ABH group increased faster comparing with control group.Cells treated with ABIH pumped out lower concentrations of rhodamine 6G than cells without drug treatment.Thus,it can be seen that the synergistic effect of ABH combined with FLC against resistant Candida albicans may relate to the inhibition of drug transporters,which resulted in the increase of intracellular FLC.4 ABH combined with FLC inhibited the virulence factors of resistant Candida albicans4.1 ABH combined with FLC inhibited hyphal growthControl group,FLC group and ABH group presented large amount of biofilms consisting of abundant filamentous and yeast forms,but no hypha was observed in drug combination group,as shown by the microscope images.It was further revealed that the expression level of hypha-related genes HWP1 in resistant Candida albicans was significantly down-regulated with the combination treatment of FLC and ABH.It is certain to say that this drug combination exhibited inhibitory effect on resistant Candida albicans hyphae.4.2 ABH combined with FLC inhibited the extracellular phospholipase activityThe egg yolk agar medium allows the rapid detection of extracellular phospholipase activity in Candida albicans.No difference in the Pz values was detected between drug alone groups and control group.Wondrously,the Pz value in drug combination group was significantly higher than that in other three groups,indicating ABH combinated with FLC has a strong inhibitory effect on the extracellular phospholipase activity of resistant Candida albicans.Phospholipase-related genes PLB1 and PLC1 in Candida albicans were significantly down-regulated by the combined treatment of FLC and ABH,which further confirmed the inhibitory effect of this drug combination on resistant Candida albicans phospholipase activity and suggested PLB1 and PLC1 may be the potential targets for this drug combination against resistant Candida albicans.ConclusionThis study revealed that combined FLC and ABH treatment results in synergistic effects against resistant Candida albicans in vitro and in vivo.Furthermore,it was found that the changes of drug uptake and efflux,hyphal growth inhibition and extracellular phospholipase inhibition were the exact mechanisms that participate in the synergistic effects conferred by FLC and ABH.Our study reveals that ABH can be combined with FLC to reverse the resistance of Candida calbicans for the first time.In addition,our study may also lead to the identification of new potential antifungal targets. |
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