| BackgroundIn recent years,the number of invasive fungal infections has increased dramatically,with higher morbidity and mortality among patients with immune system deficiencies.Candida albicans(C.albicans,CA)is a relatively common invasive fungus.People with severe candidiasis caused by Candida albicans can threaten the lives of patients.At present,there are limited options for antifungal preparations.Because of their low toxicity and high efficiency,azole drugs are often used in long-term large-dose clinical applications to prevent or treat fungal infections,resulting in the emergence of drug-resistant strains.The development of new anti-fungal drugs is time-consuming and expensive,and the current combination of anti-fungal drugs and non-anti-fungal drugs is a research hotspot for anti-fungal drug resistance.Ginkgo lide B(GB)is a platelet-activating factor(PAF)receptor antagonist with anti-platelet effect.There are reports in the literature that GB can increase the calcium concentration in mammals,and many studies have shown that disturbing the intracellular calcium homeostasis can resist fungal resistance.At present,there is no research report on the combination of GB and Fluconazole(FLC)against resistant Candida albicans.ObjectivesExplore the combined effects of GB and FLC on resistant Candida albicans both in vivo and in vitro,and explore their potential synergistic antifungal mechanisms.MethodFirst of all,the in vitro effect evaluation was carried out,and the effect of GB and FLC on the planktonic cells of resistant Candida albicans was determined by the micro broth dilution method;XTT colorimetric method was used to determine the effect of GB and FLC on the formation of biofilm in Candida albicans at different time periods of 4,8,12 and 24h.Secondly,the in vivo effect was conducted to establish a fungal infection model of the Galleria mellonella infection model,and the in vivo effect of drug combination was carried out by measuring and comparing the survival rate,fungal burden analysis and histological study of different drug groups.Finally,the potential synergistic antifungal mechanisms of drug combination were explored.Culture with RPMI-1640 medium with hyphae induction ability and observe the morphological changes of Candida albicans after drug intervention under the microscope;using fluorescent probe Fluo-3/AM to explore the effect of drug combination on intracellular calcium homeostasis;using fluorescent probe Rh 6G to study the effect of GB on drug transporters of drug-resistant Candida albicans,thereby further clarifying the mechanism of GB and FLC synergistic effect against drug-resistant fungi.Results(1)GB alone exhibited weak antifungal activity,while combined with FLC showed a synergistic effect against drug-resistant Candida albicansThe in vitro study of drug combination using micro broth dilution method found that GB only had a weak antifungal effect on the two resistant strains with MIC of 64?g/mL and 128?g/mL,respectively.However,when GB was combined with FLC,the MIC of FLC could be reduced from more than 512?g/mL to 0.25-1?g/mL,and the FICI value was 0.06-0.25,both of which was less than 0.5,showing strong synergy.(2)Combined use of GB and FLC exhibited synergistic anti-biofilm effectBiofilm is an important virulence factor for Candida albicans and an important factor leading to its resistance.GB alone showed no effect on the biofilms of Candida albicans at different time periods.When used in combination with FLC,it exhibited a synergistic effect against the biofilms formed by Candida albicans at 4,8,and 12 hours.The FICI value was between 0.06-0.25.After combined use,the MIC of FLC could be reduced from>1024?g/mL to 0.25-2?g/mL,and for different strains,the shorter the biofilm formation time,the lower the FLC MIC would be.However,with the increase of time,the combination of drugs showed an irrelevant effect on the bio film formed by each strain at 24h,and the FICI values were all greater than 0.5.(3)The combined use of GB and FLC could improve the survival rate of Galleria mellonella larvae,reduce its fungal burden and reduce its tissue damageIn this chapter,the infection model of the Galleria mellonella larvae was used for in vivo studies.The experimental results showed that the combination of GB and FLC could improve the survival rate and reduce the death of the Galleria mellonella larvae;after combined use of drugs,it could significantly reduce the number of Candida albicans in the larvae of Galleria mellonella;histopathological sections show that the black colonies in the growth control group and the drug alone group were dense and large,and the larval tissues have been damaged.In the combined GB and FLC group,the damage was significantly reduced,and the number and area of the colonies were greatly reduced.(4)The combination of GB and FLC could inhibit the morphological transformation of Candida albicansIn the RPMI-1640 medium with hypha induction ability,Candida albicans in the control group and the drug-alone group showed dense and narrow hyphae,while the number of cells in the combined GB and FLC group not only decreased significantly,the length of the hyphae were also significantly shortened,and some yeast cells could also be observed in the field of view,indicating that the combination of GB and FLC could inhibit the morphological transformation of Candida albicans.(5)The combination of GB and FLC could interfere with the intracellular calcium homeostasisIon homeostasis is an important factor for maintaining the normal physiological activity of cells.The experimental results showed that the trends of intracellular calcium concentration in the drug single-use group and the control group were almost the same,while the fluorescence intensity of the GB+FLC group increased after 20 minutes,which was significantly different from the other three groups(P<0.001).This indicated that the synergistic antifungal combination of drugs may be related to the disturbance of intracellular calcium homeostasis.(6)GB inhibits the activity of drug transportersBoth FLC and Rh 6G were substrates of drug transporters on the cell membrane of Candida albicans.In this experiment,the fluorescent tracer Rh 6G was used as a substitute for FLC.The experimental results showed that GB could increase the uptake of FLC,and at the same time inhibit the high expression of efflux pumps associated with drug resistance on the cell membrane,so that FLC entering the cell reduces efflux and the concentration of intracellular antifungal drugs increased,thereby improved the azole-resistant Candida albicans sensitivity to FLC.ConclusionIn summary,this study reports for the first time that GB could increase the antifungal effect of FLC on azole-resistant Candida albicans.The combination of two drugs produced a synergistic effect both in vitro and in vivo.Mechanism studies have shown that the synergistic effect was related to the inhibition of morphological transformation,increase of intracellular calcium concentration and suppression of efflux pump activity.More mechanisms of synergism need further investigation in the future,this study will provide ideas for finding new antifungal drug targets and developing new antifungal agents. |
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