The In Vitro And In Vivo Effects And Mechanisms Of Ribavirin Against Candida Albicans

BackgroundIn recent years,the incidence of fungal infection is increasing continuously with the rapid development of interventional therapy,organ transplantation and the widespread use of broad-spectrum antibiotics and immunosuppressants in clinical practice.Candida is the most common opportunistic fungi,and Candida albicans is the most common isolated strain.Although the widespread use of azoles,especially fluconazole（FLC）,has achieved satisfactory clinical results,the emergence of resistant strains has brought new challenges to antifungal therapy.In addition,the existing antifungal drugs are limited in variety and most of them are expensive,which imposes certain limitations on the combination of antifungal agents.In order to face the challenge and settle the problem,the present research,on one hand,is to discover new antifungal drugs;on the other hand,is to study the new effects of existing agents.This study evaluated the antifungal activity of existing clinical agents used alone or in combination with FLC from the standpoint of non-antifungal agents used alone or in combination with traditional antifungals.Inosine 5’-monophosphate dehydrogenase（IMPDH）inhibitors have good application prospects in immunosuppression,anti-tumor,antiviral,antimicrobial and antiprotozoal treatment.Studies have shown that inhibition of IMPDH will effectively attenuate the virulence of Cryptococcus or even kill it completely.Therefore,we speculated that IMPDH inhibitors might exert similar antifungal effects against C.albicans.Ribavirin（RBV）is a widely used broad-spectrum and potent antiviral drug,but there are no reports on pharm-acological effects other than antiviral activity.ObjectiveThe objective of this study was to determine the antifungal activity of RBV used alone or in combination with FLC against planktonic and biofilm of C.albicansin vitro,evaluate the antifungal effects of agents by establishing Galleria mellonella infection model in vivo,and further study the underlying mechanisms of its antifungal effects.Contents and MethodsIn this study,the minimum inhibitory concentration（MIC）of RBV used alone or in combination with FLC against C.albicans was determined by microdilution method,and the interaction between the two drugs was evaluated by the fractional inhibition concentration index（FICI）model.We also determined the antifungal effect of RBV in combination with FLC against C.albicans biofilm by XTT assay.In addition,we evaluated the antifungal effects of the agents in vivo from the survival assay and histological study using G.mellonella infection model.Furthermore,we explored the potential antifungal mechanisms of RBV used alone or in combination with FLC from the growth ability of hyphae,the activity of phospholipase,and the uptake or efflux of Rh6G.Results:1.The antifungal activity of RBV used alone or in combination with FLC against Candida.The results showed that the MIC₈₀of RBV was 2-4μg/m L for FLC-sensitive C.albicans and 8μg/m L for FLC-resistant C.albicans.RBV also exhibited significant synergistic effects when combined with FLC against resistant C.albicans,the MIC₈₀of FLC was decreased from>512μg/m L to 0.25-1μg/m L when the concentration of RBV was 1μg/m L.RBV used alone or in combination with fluconazole has no antifungal effect against non-C.albicans.2.The antibiofilm activity of RBV in combination with FLC against C.albicansAfter incubated the 96-well plate for 24 hours,RBV combined with FLC exhibited synergistic antifungal effects against sensitive C.albicans biofilms pre-formed over 4,8,12,and 24h（FICI<0.5）;While the synergism against resistant C.albicans biofilm was only observed over 4,8,and 12h,and had no effect on the biofilm pre-formed over 24h.3.RBV combined with FLC increased the survival rate of infected G.mellonella against resistant C.albicans.Survival assay showed that the survival rate of G.mellonella larvae infected with C.albicans in the control group was 25%,and the survival rates of the larvae in FLC and RBV alone groups were 45%and 35%,respectively.The survival rate of the infected larvae was as high as 70%.These results indicated that the survival rate of larvae was significantly improved when RBV in combination with FLC compared with the FLC-treated group over a 4-day period（P<0.05）.4.RBV combined with FLC reduced the tissue damage of infected G.mellonella against resistant C.albicans.The results of histological study showed that in the growth control group and drug monotherapy group,there were large areas of dark brown nodules scattered all over the tissue;in the drug combination group,only a few of dark brown nodules appeared in the tissue.5.RBV used alone or in combination with FLC decreased hyphal formation of C.albicans.In this experiment,the effect of agents on the hyphal formation of C.albicans was observed by fluorescence microscopy.Results showed that RBV had an inhibitory effect on the hyphal growth of C.albicans,and in the combination group,the hyphal length was significantly shorter and there was almost no hyphal aggregation compared to the drug monotherapy group and the control group.It is indicated that RBV used alone or in combination with FLC could decrease the hyphal formation of C.albicans.6.RBV combined with FLC inhibited the phospholipase activity of resistant C.albicans.The Pz values of the control group,FLC and RBV groups were 0.64±0.01,0.65±0.02and 0.66±0.01 respectively,showing very high phospholipase activity;the Pz value of drug combination group was 0.89±0.03,which was significantly different from that of the other groups（P<0.05）.It is indicated that the combination of RBV and FLC could significantly inhibit the phospholipase activity of resistant C.albicans.7.RBV had no significant effects on the uptake or efflux of Rh6GWe used Rh6G,which emits fluorescence,as an alternative to FLC for experimental studies.The results showed that there was no significant difference in fluorescence intensity between the experimental group and the control group（P>0.05）,indicating that RBV has no significant effect on the uptake or efflux of Rh6G.Conclusion and significanceRBV has antifungal activity when used alone and exhibits synergism against resistant C.albicans when used in combination with FLC in vitro and in vivo.Further mechanisms studies showed that the antifungal effect of agents might be related to inhibition of C.albicans biofilm,attenuation of phospholipase activity and inhibition of hyphal formation,and has no significant effect on drug uptake and efflux.This study is the first to report the antifungal effects of RBV used alone or in combination with FLC,which will provide valuable experimental data and research ideas for overcoming fungal resistance and the developing new antifungals.