Ras-cAMP Pathway-based Investigation Of Inhibitory Effects And Mechanism Of Baicalin Alone Or In Combination With Fluconazole Against Candida Albicans Biofilms

Objective:This study aimed to investigate the antifungal activity of baicalin alone or in combination with fluconazole against Candida albicans biofilms via apoptotic pathway and to explore the related mechanism.Methods:1.The growth curve of Candida species was used to evaluate after with baicalin treatment; Microbroth dilution method was performed to determine the minimal inhibitory concentration(MIC) and sessile minimal inhibitory concentration(SMIC) of baicalin; Transmission electron microscope(TEM) was performed to observe the ultrastructural of C. albicans; Propidium iodide(PI) was used to determine the membrane permeabilization of C. albicans; Flow cytometry was used to determine the cell cycle following the treatment of baicalin; CLSM and flow cytometry were used to observe the phosphatidyl serine(PS) and calculate the apoptosis rate; Flow cytometry was used to determine the reactive oxygen species(ROS) and mitochondrial membrane potential(MMP) with the treatment of baicalin; FITC-VAD-FMK staining assay was performed to evaluate the metacaspase activity; Dihydrorhodamine(DAPI) and terminal deoxynucleotidyl transferase-mediated d UTP nick end labeling(TUNEL)staining were performed to determine the nuclear damages; Spectrophotometry was performed to measure the cytochrome C(Cyt C) release; q RT-PCR was used to measure the expression of redox and apoptosis-related genes; Western-blot assay was performed to determine the expression of Ras1 p and Tpk1 p.2.Checkerboard method was used to study the interrelation of baicalin and fluconazole on C. albicans; Time-kill curve was used to evaluate the effect of baicalin alone and incombination with fluconazole on C. albicans; XTT reduction assay and measurement of biofilm biomass assay were performed to investigate the antibiofilm activity of baicalin alone and in combination with fluconazole; Scanning electron microscopy(SEM) and confocal laser scanning microscopy(CLSM) were used to observe the three-dimensional structure of C. albicans biofilms; The liquid and solid medium were to observe the effect of baicalin alone and in combination with fluconazole on yeast-hypha transformation in C. albicans; Cellular surface hydrophobicity(CSH)assay was performed to investigate the effect of baicalin alone and in combination with fluconazole on adherence in C. albicans; High-performance liquid chromatography(HPLC) method was used to test the ergosterol bio-synthesis; Quantitative real time PCR(q RT-PCR) was performed to measure the expression of adhesion and hypha related genes.Results:1. The MIC value of baicalin to C. albicans were between 500-1000 μg/m L, which has moderately antifungal effect; Mechanism of apoptosis-induction of baicalin to C.albicans biofilm included by influencing the expression of redox and apoptosis-related genes and proteins; blocked the cell cycle and influenced the ultrastructural of C.albicans biofilms; caused PS externalization, nuclear fragmentation, chromatin condensation, metacaspase activation and Cyt C release.2. Synergistic effect was showed in anti-biofilm by baicalin in combination with fluconazole, and FICI was between 0.28-0.5; SEM and CLSM showed that baicalin alone and in combination with fluconazole could destruct and resulted in scant or nonexistent biofilms; Mechanism of synergistic effect of baicalin in combination with fluconazole to C. albicans biofilm included by influencing the expression of adhesion,hypha related genes, CSH and ergosterol bio-synthesis controlling genes; inhibited thegerm tube, yeast-hypha transformation and adhesion; decreased the CSH and inhibited ergosterol biosynthesis.Conclusion:Collectively, we found baicalin has moderately antifungal effect, and could baicalin could induce C. albicans biofilms apoptosis. Not only that, baicalin could as a synergist in combination with fluconazole against C. albicans biofilms by more than one ways and targets. Our findings are beneficial for understanding the mode of action of baicalin,and provide a novel thought for researching and development of new antifungal agents.