The Synergistic Antifungal Effect Of Licofelone Combined With Fluconazole Against Candida Albicans

AimsCandida albican is a kind of common opportunistic fungal pathogen,which often existed in the oral,upper respiratory,intestinal and vagina of health individual,and not causing disease.But it will lead to disseminated or chronic infections in critically ill patients and immunocompromised individuals.This study aimed to evaluate the synergistic antifungal effects of dual mPGES-1/LOX inhibitor licofelone combined with fluconazole against C.albicans.MethodsThis paper was mainly consists of three parts,in vitro study,in vivo study and potential mechanisms study.The in vitro antifungal studies were included minimum inhibitory concentrations and sessile minimum inhibitory concentrations determination.FICI and ΔE model were used to assess the combined antifungal effect.The in vivo studies were used Galleria mellonella model to assay the antifungal activity of the drug combination including survival curve assay,histopathology observation and fungal burden determination.In addition,drug resistance and fungal virulence are two major factors that disturbing the treatment of C.albicans,here we determined the secreted aspartyl proteinase gene family,RAS/cAMP/PKA pathway related genes and biofilm-specific gene expression levels by RT-PCR,and extracellular phospholipase activities by egg yolk agar method to investigate the antifungal mechanism of licofelone combined with fluconazole.ResultsIt was found that in vitro,licofelone(128 μg/mL)alone can inhibit resistant C.albicans,and synergistically effect against resistant C.albicans was observed when licofelone(16 μg/mL)combined with fluconazole(1 μg/mL).The drug combination can inhibit the resistant C.albicans biofilm formation over 4,8,12 h,this inhibitory effect was scarcely observed over 24 h.The interactions of the combination were in accordance with the in vivo study.It was found that for the combination group,not only did survival rate higher than drug alone and control group,but the fungal burden determination was also shown less than other three groups.Furthermore,compared to the drug alone groups,licofelone(16 μg/mL)combined with fluconazole(1 μg/mL)can down-regulate virulence related genes SAP1 to SAP4,biofilm-specific related gene BCR1,ALS1,ALS3,HWP1 and RAS/cAMP/PKA pathway related genes RAS1,CYR1,TPK2 expression level,and the precipitation zone of combination was 0.91,which means the combination can decrease the extracellular phospholipase activities.ConclusionAbove all,licofelone as the novel enzyme inhibitor,its antifungal ability has not been discovered yet.These results first shown that licofelone combined with fluconazole can work against resistant C.albicans,and this combination also had certain inhibit effect on biofilm formation over 12 h.The mechanism study support the hypothesis that the synergistic effects of combination of fluconazole and licofelone against resistant C.albicans might be related to attenuating virulence factor such as secreted aspartyl proteinase,extracellular phospholipase activities,and inhibiting the transition between yeast and hyphal growth forms,but more in-depth study are needed.