Synergistic Effect Of Berberine Hydrochloride Combined With Fluconazole Against Resistant Candida Albicans

Objective:In recent years,the conditional pathogenic infection caused by Candida albicans?CA?has become more and more serious.With the wide use of Fluconazole?FLC?,the drug resistance and resistance rate are gradually increased.A broad-spectrum antibacterial Chinese medicine with remarkable effect and less resistance has the potential to solve the enhancement of drug resistance.In the previous study,we found that high concentration?128?g/mL?of Berberine hydrochloride?BBH?inhibited Candida albicans and biofilm,and the combination of berberine hydrochloride and fluconazole had the effect of synergistic inhibition against fluconazole-resistant Candida albicans.Therefore,this study aims to investigate the antibacterial effect of drug combination on Candida albicans resistant strains.To explore the molecular mechanism of synergistic effect by drug transporter activity,biofilm formation ability and intracellular calcium ion homeostasis.Multi-angle exploration of possible molecular targets for synergistic effect,providing an effective solution for clinical treatment of drug-resistant strains.Methods:1.The minimum inhibitory concentration?MIC?of berberine hydrochloride and fluconazole against Candida albicans was determined by broth microdilution method,and the fractional inhibitory concentration index?FICI?of the combination was determined by the checkerboard broth microdilution method,screening the lowest drug concentration combination with strong inhibition,XXT assay to determine the dynamic antifungal effect of each group of drugs.Study the inhibitory effect of drug combination on fluconazole resistant strain.2.The changes of drug transporter activity were detected by Rhodamine 6G?Rh6G?absorption and efflux experiments.The expression levels of key genes CDR1,CDR2 and MDR1 were detected by RT-qPCR.Study the effect of berberine hydrochloride combined with fluconazole on the drug transporter in Candida albicans.3.Microscopy observed the effect of drug combination on hypha growth and biofilm formation.The expression levels of key genes HWP1,ECE1 and ALS3 were detected by RT-qPCR.Study the drug combination on the inhibition of hyphae,biofilm and its key genes in Candida albicans.4.Inverted fluorescence microscopy and flow cytometry were used to observe the effect of drug combination on intracellular calcium ion homeostasis.RT-qPCR was used to detect the expression levels of gene CCH1,MID1,CNB1,YVC1,VCX1,PMC1 and PMR1.Study the effect of drug combination on the regulation of calcium flux and its genes,to explore the possible molecular mechanism of drug combination synergy of fluconazole-resistant Candida albicans.Results:1.The MIC of berberine hydrochloride was 64?g/mL,and the MIC of fluconazole was 512?g/mL or more.The FICI of the five fluconazole-resistant strian?CA 0253,CA 2119,CA 12038,CA 21065,CA 1460?were all less than 0.5,and the lowest combination concentration was 2?g/mL berberine hydrochloride plus 1?g/mL Fluconazole.The combination of drugs made MIC of FLC and BBH decrease from512?g/mL and 64?g/mL to 1?g/mL and 2?g/mL,respectively,which significantly increased the sensitivity of resistant Candida albicans to drugs.The dynamic antibacterial effect of the combination group was significantly better than the others within 0-48h?P<0.05?.The combination group had the synergistic effect of inhibiting resistant Candida albicans.2.The efflux effect of Rh6G in Candida albicans cells significantly decreased after single or combined treatment of BBH and FLC?P<0.05?.The fluorescence intensity of the supernatants of the combination group were lower than 1.43 and 1.28times of the single treatment of BBH and FLC respectively at 2 hours,and the antibacterial effect of the combination group was significantly better than the two drugs alone?P<0.05?.However,none of the four groups of treatment could promote the absorption of Rh6G in resistant strains.Compared with the control,the combination group decreased the expression of CDR1 and CDR2 genes by 3.45 and3.85 times,respectively,but the expression of MDR1 increased by 2.27 times?P<0.05?.Compared with treatment of FLC and BBH alone,the expression of CDR1gene in the combination group decreased by 5.24 and 2.66 times,respectively?P<0.05?,and MDR1 gene decreased by 1.68 and 1.70 times,respectively?P<0.05?.3.The combination group could significantly inhibit the growth of hyphae.The FLC group had a small number of longer germ tubes,the BBH group and the control group formed a large number of hyphae.The combination group could prevent the formation of biofilm.The FLC group could see a large number of cells sprouting to form pseudohyphae,the cells adhered to each other and aggregated into a mass.The BBH and the control group could form mature biofilm,a large number of hyphae were interwoven into a network,in which many yeasts accumulated.Compared with the FLC group,the combination group decreased the expression of HWP1,ECE1 and ALS3 gene by 7.26,12.20 and 3.73 times,respectively?P<0.05?.Compared with the BBH group,the combination group decreased the expression of HWP1,ECE1 and ALS3 gene by 54.11,34.20 and 13.62 times,respectively?P<0.05?.There was no significant difference in the expression of HWP1 and ALS3 genes between the FLC group and the control group,while the ECE1 gene expression level was 5.68 times higher than that of the control group?P<0.05?.The expression levels of HWP1,ECE1and ALS3 genes were increased in the BBH group compared with the control group?P<0.05?.4.The combination group and BBH group showed green fluorescence in Candida albicans cells,and Ca²⁺influx in cytoplasm.There was no visible intracellular fluorescence in the FLC group and control group.The results of flow cytometry showed that both the intracellular Ca²⁺concentration of the drug combination treatment?2?g/mL BBH,1?g/mL FLC?and BBH group was higher than that of FLC and control group?P<0.05?,and the intracellular Ca²⁺concentration of the combination group was higher than the BBH group?P<0.05?,but there was no difference between the FLC group and the control group.The expression levels of VCX1 and PMR1 genes in other groups were lower than control group?P<0.05?,and the VCX1 and PMR1 genes in the combination group were 8.89 and 1.69 times lower than the BBH group?P<0.05?.There was no significant difference in the expression of VCX1 and PMR1 between the combination group and the FLC group?P>0.05?.The YVC1 gene was significantly up-regulated in the combination group and BBH group,and the combination group was 1.62 times higher than the BBH group?P<0.05?,while the combination group was 6.47 times higher than FLC group.The PMC1 gene was down-regulated in the combination group and the BBH group?P<0.05?.The expression of PMC1 gene in the FLC group was increased,which was 5.23 and 5.15times of the combination group and BBH group?P<0.05?.Conclusion:1.The combination of 2?g/mL BBH and 1?g/mL FLC has synergistic antibacterial effect,and is superior to the drugs alone.The antifungal agent combined with traditional Chinese medicine can synergistically inhibit the bacterial.2.The molecular mechanism of synergistic antibacterial effect of drug combination includes:down-regulation drug transporter gene CDR1 and CDR2,inhibits efflux process of fluconazole.Down-regulation HWP1,ECE1,ALS3 gene,inhibits initial adhesion and aggregation of biofilm,inhibits the form of hyphae,leads to defects in biofilm formation.It can also up-regulate YVC1 gene to promote Ca²⁺transfer into cytoplasm,and down-regulate VCX1,PMC1,and PMR1 genes to inhibit intracellular Ca²⁺transfer into vacuoles and Golgi,causing cellular Ca²⁺influx,destroys the calcium homeostasis in Candida albicans.