Synergistic Activity And Molecular Mechanism Of Tetrandrine On Fluconazole To Candida Albicans In Vitro

1 ObjectiveTo investigate the synergistic effect of tetrandrine on fluconazole to Candida albicans, and to explore the molecular mechanism in the transcriptional level.2 MethodsThe minimum concentrations of tetrandrine which are not cytotoxic to the yeast form of 16 C.albicans were determined according to microdilution test.and the minimum inhibitory concentration(MICs) of fluconazole alone and combination with tetrandrine were determined in 16 C.albicans strains by the CLSI proposed M27-A microdilution method.The synergistic effect of tetrandrine on fluconazole was further confirmed by time-kill curves method.The expressions of MDR1,FLU1,CDR1,CDR2,EGR11 were measured by reverse transcription-polymerase chain reaction(RT-PCR).3 ResultsThe minimum concentrations at which tetrandrine had no cytotoxic effect to the yeast form C.albicans was 30μg/mL,the survivability of the yeast form of 16 C.albicans in tetrandrine at the concentration of 30μg/mL was greater than 95%.The MICs of fluconazole alone to the yeast form C.albicans were 0.250～64μg/mL and 0.125～16μg/mL when combined with tetrandrine.The time-kill curve indicated that after 24 or 48 hours' exposure to differently treated groups,fluconazole or fluconazole in combination with tetrandrine were fungistasis,the CFU of every strain of C.albicans exposed to fluconazole in combination with tetrandrine decreased a more 21og₁₀ CFU/mL when compared with that of the strains exposed to fluconazole alone,and conspicuous synergistic activity was observed between fluconazole and tetrandrine,and which had statistical significance(P = 0.000).Before exposed to tetrandrine,the expressions of MDR1,FLU1,CDR1,CDR2 were different among the fluconazole-susceptible,dose-dependent fluconazole-susceptible and fluconazole-resistant C. albicans(P＜0.05),but the expression of ERG11 gene was not significant(P＞0.05).After exposed to tetrandrine for 24h,the expressions of MDR1 in fluconazole-resistant C.albicans, FLU1 in dose-dependent fluconazole-susceptible & fluconazole-resistant C.albicans,CDR1 & CDR2 in all C.albicans and the expressions of ERG11 in fluconazole-susceptile isolates were lower than in tetrandrine-free groups(P＜0.05).4 Conclusion4.1 Tetrandrine's synergistic activity on fluconazole to C.albicans showed time dependence. After 24 h and 48 h,significant synergistic activity was observed between fluconazole and tetrandrine.4.2 At the transcriptional level,tetrandrine can inhibit the mRNAs' expressions of the effux pumps genes------MDR1,FLU1,CDR1,CDR2 of C.albicans.4.3 At the transcriptional level,tetrandrine can inhibit the mRNA expression of the target enzyme gene ERG11 of C.albicans.