| With the wide spread of human immunodeficiency virus (HIV), misusing of tremedous antibiotic and immunosuppressive therapy to treat cancer or organ transplant, and also increasing number of biological material applying in human beings, the incidence of systemic fungal infections has been risen dramatically in recent years. Candida albicans infection is the most common isolates among them. It shows that C. albicans accounts about 50-60% in the major systemic fungal pathogen during 1996 to 2002. The triazole fluconazole is the most widely used antifungal drug to treat Candida infections. Unfortunately, wide spread uses of fluconazole have led to the rapid development of drug resistance which has severely hindered antifungal therapy.Baicalein (BE) is a major component originally isolated from the roots of Scutellaria baicalensis Georgi. Several different functions of BE have been reported. In addition to its inhibition effect on lipoxygenase, BE was found to have antioxidant, neuroprotective, antibacterial, antiviral activities. It was reported that BE also has antifungal activities, but could not be applied in clinical therapy because of its few effect. Attempts have been made to cope with treatment failures by using combination therapy. To seek for a novel combination therapy, we investigated the in vitro interaction of fluconazole and BE against fluconazole-resistant clinical isolates of C. albicans.In this study, we first investigated the effect of BE on C. albicans by microdilution assay and the synergistic activities between FLC and BE against FLC-resistant C. albicans by checkerboard microdilution assay. We found that the drug-sensititive C. albicans but not the fluconazole-resistant C. albicans was inhibited when baicalein was used alone,the effective concentration was 1μg/ml-2μg/ml. When used in drug-sensitive C. albicans with the combination of fluconazole and BE, each drug showed the little change comparing with drugs used alone. The fractional inhibitory concentration (FIC) index was all above 0.5,meaning no synergism in fluconazole-BE combination. However, the fluconazole-BE combination markedly reduced MIC80s, especially the MIC80 of either individual agent while used in fluconazole-resistant C. albicans. The corresponding median FIC index was 0.069 (range, 0.037 to 0.098). Synergism was observed in all isolates .Further time-kill studies were conducted with fluconazole and BE against one chosen clinical fluconazole-resistant C. albicans 0603433. BE did not affect the growth curve at 16mg/ml after 24 h, regardless of the initial inoculum is 103 or 105 CFU/ml,the fluconazole showed a little antifungal activity for initial inoculum is 103 CFU/ml . The fungistatic activity of fluconazole was dramatically enhanced by addition of BE when used in combination for 24h.Then we further investigated whether BE could play a same role with other antifungal agent or in other fungus. Respectively,the combination of baicalein and KCZ or MCZ reduced the MIC below to 0.5μg/ml both and showed significant synergistic activities against fluconazole-resistant C. albicans (FIC index <0.5).And the combination of BE and AmB reduced the MIC from 1-2μg/ml to 0.062μg/ml and also showed the synergistic activities .When used in Candida parapsilosis and Candida glabrata with the combination of fluconazole and BE, no synergism was observed (FIC>1.5).In order to examine the effect of baicalein (BE) on C. albicans biofilm formation,we dyed the BE-treated biofilm and untreated biofilm with the fluorescent stains FUN-1 and concanavalin A-Alexa Fluor 488 conjugate (ConA).Confocal scanning laser microscopy (CSLM) showed that normal C. albicans biofilm exhibited a typical three-dimensional nature, composed mainly of true hyphae. When cells were treated with 8μg/mL BE, biofilm development was inhibited and growth was predominantly composed of yeast cells and pseudohyphae.True hyphae were rarely observed, a factor that contributed to the poor biofilm architectures. To provide a more quantitative assessment of the extent of cell growth of biofilms in the presence or absence of BE, the ability of cells to reduce XTT was measured after the treatment for biofilms with different concentration of BE for 48h .The inhibitory effect on biofilms appeared to be dose-related. Over 70% inhibition was observed at BE concentrations between 4μg/ml and 32μg/ml A lower concentration (2μg/ml) produced 15% inhibition and 1μg/ml BE had almost no effect on biofilm formation.To examine the mechanism by which BE inhibits biofilm formation, the CSH of biofilm was measured by the biphasic separation method. Negative correlations were observed between BE concentration and the CSH of biofilm. High CSH was observed in normal mature biofilm. When the cells were treated with BE, CSH decreased. The relative CSH was 0.85 and 0.11 at BE concentrations of 1μg/ml and 32μg/ml, respectively.The mRNA expression levels of CSH1, which codes for the CSH-associated protein in C. albicans, were determined by real-time RT-PCR. The results showed that BE-treated cells expressed lower levels of CSH1 mRNA than the cells grown in the absence of BE, which correlated with the CSH exhibited by the cells.Here, we describe cellular changes that accompany death in C. albicans after exposure 12h to a range of different concentrations of BE , including treatment with the antifungal agent amphotericin B (AMB) as a positive control .Flow cytometry (FACS) assay showed that 10% or 20% apoptosis of C. albicans were induced respectively at baicalein concentrations of 4μg/ml or 8μg/ml.DAPI-staining reassured the occurrence of the apoptosis. Cells dying under 8μg/ml BE treated displayed several markers characteristic of apoptosis. These include the rapid exposure of phosphatidylserine (PS) at the outer cell membrane, cellular dyskaryosis,the generation of vacuole in cytoplasm, the margination of chromatin in nuclei, nuclear fragmentation, and the degradation of DNA.Compared to the normal cells, the apoptotic cells induced by BE showed increased endogenous reactive oxygen species (ROS) generation, and it shows the dose-dependent and time-dependent activity.The generation of ROS induced by 32μg/ml BE for 48h was 5 times higher than control . 16μg/ml BE induced cells shows less mitochondrial membrane potential and for 12h decreased 50% comparing with control. After treated with 32μg/ml BE for 3h , the mRNA expression of CAP1, SOD2 and TRR1 were apparently up-regulated.Considered the expression of CAP1 was up-regulated more than 24times than control, further experiment we did to investigate survival of wild type strains ,CAP1 knock-out strains and overexpression CAP1strains treated by BE. Reaults showed after induced by 32μg/ml BE for 3h,the survival of three strains were 35%,41.7%and54%.That means CAP1 played a role in baicalein-induced apoptosis. CAP1-deleted cells was very sensitive to baicalein.Gene chip analysis showed that the genes affected by baicalein were related to cell cycle, drug efflux, virulence, substrate transport and heat shock.
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