| BackgroundIn the last few decades,opportunistic fungal infections have been increasingly recognized as major causes of human diseases,especially among immunocompromised patients,such as transplant recipients,HIV/AIDS patients,and cancer patients.Systemic infection caused by Candida species is the fourth leading cause of nosocomial bloodstream infection in modern hospitals.The increasing rate of non-Candida albicans isolation and the rapidly growing resistance of Candida species present a challenging clinical problem.C.albicans is the most common etiological agent of candidiasis,causing not only superficial mucosal candidiasis but also life-threatening systemic infection in immunocompromised patients.Only a few classes of antifungal agents,such as polyenes,azoles,allylamines,echinocandins,and miazines,are available,and their mechanisms are confined to targeting the cell envelope(wall and plasma membrane)and inhibiting DNA synthesis.In addition,most of these drugs exert serious unwanted effects on the host,such as nephrotoxicity caused by amphotericin B,visual disturbances caused by voriconazole,and congestive heart failure caused by itraconazole.In addition,some of these drugs,such as the echinocandins,are in limited clinical use due to high costs.Of particular importance today is the emergence of several Candida species resistant to many commonly used antifungal drags,especially fluconazole.Thus,there is an urgent and unmet need for the development of new antifungal drugs based on new antifungal targets.In view of the phenomenon that few antifungal drugs can be applied clinically,more and more anti-infection researchers hope to solve the problem of fungal drug resistance by combining drugs,but not focusing on the development of new drugs with high cost and long term.For combination drugs,it usually includes the combination of multiple antifungal agents and the combination of existing antifungal agents and non-antifungal agents.It is important to note that the efficacy of multiple antifungal agents may be not good,cost a lot and easy to increase patients' drug adverse reactions.Therefore,more and more researchers tend to the drug combinations of non-antifungal drugs and existing antifungal drugs.Drug combinations,such as ambroxol hydrochloride and fluconazole,budesonide and fluconazole,phenylbutyric acid and fluconazole,are found to show synergistic antifungal effects on the growth of resistant Candida albicans.Therefore,the combined application of antifungal drugs and non-antifungal drugs has become an important research method to overcome fungal resistance in recent years,and is also the research direction of our research group for many years.Calcium,as the second messenger in eukaryotic cells,plays a direct role in controlling the expression patterns of its signaling systems and is important in cell survival.Fungal calcium-calcineurin signaling network consists of various components,including calcium channels,sensors,transcriptional factors,exchanger,Ca2+ pumps as well as many calcium related proteins and enzymes.Most components as above have been found closely associated with numerous physiological processes in fungi,and there is a wealth of studies that appear to demonstrate the calcium-mediated and calcineurin mediated azole-resistance in fungi,suggesting the important role of this signaling network in the emergence of fungal resistance.Calcium channel blockers,which are commonly used drugs in clinic,are very safe,including nifedipien,amlodipine,benidipine,flunarizine,verapamil,diltiazem and so on.Considering the importance of calcium in fungal resistance and the fact that calcium channel blockers combined with fluconazole have in vitro synergistic effects against resistant Candida albicans in our previous studies,the present study was aimed at investigating the in vivo antifungal effects of calcium channel blockers(nifedipien,amlodipine,benidipine and flunarizine)and fluconazole on resistant Candida albicans,as well as exploring the mechanism of in vivo antifungal effects.This study will not only expand the clinical application of calcium channel blockers,but also generate important theoretical basis and ideas for the study of resistance mechanism against Candida albicans.ObjectiveIn the previous studies of our team,the drug combinations of fluconazole and calcium channel blockers showed in vitro synergistic effects against resistant Candida albicans.The present study aimed to identify whether the synergetic effects of fluconazole and calcium channel blockers could be replicated in vivo and further explore the potential mechanism.MethodsOne azoles-resistant C.albicans isolate,CA10,was used in this study.Calcium channel blockers was consist of amlodipine(AML),benidipine(BEN),flunarizine(FNZ)and nifedipine(NIF).The Galleria mellonella infection model was employed to study the in vivo antifungal effects of fluconazole and calcium channel blockers by survival assay,fungal burden determination,and histological study.Survival assay:Galleria mellonella was divided into four groups.The number of surviving larvae in each group was recorded every 24h for up to four days.Survival curves were drawn.Fungal burden determination:Infected Galleria mellonella was ground and diluted before culturing on solid YPD medium.CFUs were counted for each Galleria mellonella.Histological study:PAS staining was used to stain the histological sections of Galleria mellonella.The formation of clumps in the sections was observed to determine the damage of the tissue of Galleria mellonella,to further determine the combined antifungal effects of FLC and CCBs in vivo.In this study,further experiments were carried out in terms of virulence factors.Egg yolk agar method was used to determine the extracellular phospholipase activity of resistant Candida albicans.ResultsSurvival assay:Survival rates were significantly higher in the combination group than in the control group or the monotherapy group.There was no significant change between the control group and the single drug group.The survival rate of the combined group was about double that of the control group and the single group.At the same time,by comparing the results of fluconazole combined with four calcium channel blockers respectively,it can be seen that the survival rate of Galleria mellonella with FLC combined with amlodipine was significantly higher than that of the other three groups,and the improvement of survival rate was the best.Fungal burden determination:There was no significant difference between the control group and the calcium channel blocker group.Compared with the control group,the fungal burden of fluconazole alone group was relatively reduced.However,the fungal burden of the drug combination group was significantly reduced,and the combination group had the best effect.Histological study:Compared with the control group or the single drug group,the infected plaques in the combined drug group were significantly reduced.Egg yolk agar method:Compared with the precipitation circle value(0.77 0.03)in the control group,there was no significant difference in the single use group(FLC:0.79±0.01;BEN:0.76±0.06;AML:0.74± 0.08;NIF:0.75± 0.09;FNZ:0.76±0.04).The extracellular phospholipase activity in the drug combination group was significantly decreased.The absence of precipitation ring in FLC and AML showed that this combination could completely inhibit the activity of extracellular phospholipase.ConclusionThe results suggested that the combinations of fluconazole and calcium channel blockers have synergistic effects against azoles-resistant Candida albicans in vivo and the synergistic mechanism involves in inhibiting extracellular phospholipase activities.Among them,FLC combined with AML had the most obvious anti-C.albicans effect in vivo.These results were in agreement with our previous in vitro studies,showing that FLC combined with calcium channel blockers could resist drug-resistant C.albicans,and the mechanism was related to the activity of extracellular phospholipase in C.albicans. |
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