Synergistic Effects Of Mangiferin And Sodium Houttuyfonate In Combination With Fluconazole Against Fluconazole-resistant Candida Albcians

Candida albicans is a kind of parasitic fungi and could lead to superficial skin and mucous membrane infection,severe pneumonia,meningitis and other deep infection at a hither concentration.Resistance of Candida albicans to fluconazole is continuously increasing and becomes one of the main reasons for the failure of clinical treatment.Therefore,the research of new antifungal agents is of great significance.Natural compounds have been the important resources for drug research because of novel structures,strong biological activities,low toxicities and abundancees.This article is based on the previous findings,antifungal effects of mangiferin and Sodium Houttuyfonate at higher concentrations,and focused on the synergistic effects and mechanisms of mangiferin and Sodium Houttuyfonate with fluconazole against fluconazole-reisitant Candida albicans.First part of our study is to explore the effect and mechanism of mangiferin in combination with fluconazole against fluconazole-reisitant Candida albicans.By CHROMagar medium,we identified 24 laboratory clinical Candida albicans strains to ensure the reliability of the experiment.With checkerboard microdilution assay,growth inhibition test,time-growth experiment and time-kill experiment time repeatedly verified the synergistic effect of mangiferin and fluconazole against fluconazole-reisitant Candida albicans.By establishing systematic fungal infection model in mice,we explored the antifungal effect in vivo.Results indicated that the combination of two drugs could not improve the survival rate but could significantly prolonged the average survival days of mice.We further investigated synergistic mechanism of mangiferin and fluconazole against Candida albicans by real-time RT-PCR methods on molecular levels.By real-time RT-PCR,we noticed that the combination of mangiferin and fluconazole could inhibit expression levels of CDR1,CDR2,MDR1,suggesting possible molecular mechanisms of synergistic effect may be related to the inhibitions of drug resistant proteins(Cdr1p?Cdr2p?Mdr1p)and sensitivity recovery of strains to fluconazole.The second part of this study is to explore the synergistic effect and mechanism of Sodium Houttuyfonate with fluconazole against fluconazole-resistant Candida albicans.In this study,we used checkerboard microdilution assay to prove that Sodium Houttuyfonate has a synergistic antifungal effct with azoles.Further study with agar disk diffusion experiment,checkerboard microdilution assay,time-growth curves,time-kill curves to explore synergistic effects on planktonic Candida albicans.Results showed that the combination of two drugs could have obvious inhibitory effects and fungidical effects on Candida albican.Further experiments by hypha inhibition test,XTT reduction of biofilm assay,biofilm observation by fluorescence microscopy and relative CSH assay were carried out to explore the synergistic anti-biofilm effects of Sodium Houttuyfonate in combination with fluconazole.Our results showed that the combination of two drugs could significantly inhibit hypha and biofilm formation and reduce relative CSH.And the inhibition effents were positive related to drug concentrations.Further research by establishing systematic fungal infection model in mice and biofilm model in rats to explore the synergistic antifungal effet of Sodium Houttuyfonate with fluconazole in vivo.Results showed that the combination of two drugs could significantly improve the survival rate of systematic fungal infection model and inhibited biofilm formation in vivo.Experiments were carried out to further explore the mechanism of synergistic effect of Sodium Houttuyfonate with fluconazole.Reactive oxygen species assay and the determination of the intracellular ATP levels indicated that the mechanism may be related to the reduction of intracellular ATP and inhibiton of cell metabolism.Immunofluorescence assay was used to observe effects on cell wall components.Results showed that cells treated with drugs became bigger and "beaded" and the exposure of ?-1,3-glucan fluorescence was decreased.By flow cytometry,with the effct of the combination of 8?g /m L Sodium Houttuyfonate and 4?g /mL fluconazole,84.46% Candida albicans losed selective permeability ability.Finally.we used quantitative real-time RT-PCR to explore the synergistic effect on expression levels of drug resistance related genes.Results showed that the combination of two drugs could selectively inhibit expressions of drug resistance genes CDR1,CDR2,MDR1,FLU1.In conclusion,the synergistic antifungal effect of Sodium Houttuyfonate and fluconazole against fluconazole-resistant Candida albicans may be related to the inhibition of cell metabolism levels,reducing the expressions of drug-resistance genes and the loss of membrane permability.The third part of this study is to explore the synergistic mechanism of Sodium Houttuyfonate with fluconazole against Candida albicans by iTRAQ analysis.2333 proteins were identified by iTRAQ,150 credible proteins were detected in two-drug-treated cells excluding FLC-treated and SH-treated cells alone.Among them,34 proteins were verified from Uniprot database.By biological function analysis of differential expressed proteins,energy metabolism proteins were up-expressed(ACO1p?HXK2p?KGD1p?ACS2p),?-1,3 glucan synthesis protein(FKS1p)was down-expressed and ?-1,3 glucan decomposition proteins(XOG1p,ENG1p)were up-expressed.The results suggested the mechanism of synergistic antifungal effect of SH and FLC was related to the regulation of ?-1,3-glucant synthesis and catabolism,blocking of ?-1,3-glucant synthesis or enhancement of its catabolism,and the damage of cell wall integrity.