Study On The Effect And Mechanism Of Sodium Houttuyniae Alone Or Combined With Fluconazole On Candida Albicans Biofilm

ObjectiveThe aims of this study were to explore the mechanism of apoptosis induced by Sodium Houttuyfonate(SH)against Candida albicans biofilm cells and investigate the effect and mechanism of SH and fluconazole(FLC)against C.albicans.Methods1.The minimum inhibitory concentration(MIC)of SH was detected by Microdilution method;4',6-diamidino-2-phenylindole(DAPI)staining was used to observe the morphological changes of the cell nucleus of the resistant C.albicans induced by SH;FITC-VAD-FMK staining was used to observe the changes of Caspase activity in resistant C.albicans biofilm cells induced by SH;JC-1 staining was used to detect the changes of mitochondrial membrane potential(MMP)in the resistant C.albicans biofilm cells by flow cytometry;the level of Reactive oxygen species(ROS)in C.albicans biofilm cells induced by SH were detected by2,7-Dichlorodi-hydrofluorescein diacetate(DCFH-DA)staining;AnnexinV-FITC / PI staining was used to detect the apoptosis rate of C.albicans biofilm cells induced by SH.2.The MIC90 and SMIC90 of SH,combined with FLC in C.albicans were determined by M27-A3(CLSI)dilution method;the synergistic effect of SH,combined with FLC was evaluated by the conventional checkerboard method(fractional inhibitory concentration index(FICI));scanning electron microscopy was used to observe the morphological structure of C.albicans biofilm cells induced by SH combined with FLC;real-time fluorescence quantitative PCR(qRT-PCR)was used to detect the relative changes in the expression of beta-1,3-glucan associated genes related in biofilm maturation of C.albicans treated with SH combined with FLC.Results1.The MIC of clinical fluconazole resistant C.albicans 385 induced by SH was32 ?g/mL but the value induced by FLC was MIC>1024 ?g/m L.A certain concentration of SH could increase the ROS level of clinical drug resistance in C.albicans biofilm cells but the MMP decreased,and the apoptosis rate of clinical drug resistance in C.albicans biofilm cells were significantly increased;the metacaspase activity were increased as well as nuclear fragmentation and collapse,the difference were statistically significant.2.SH combined with FLC shows synergy effect(FICI< 0.13~0.5);scanning electron microscopy observed that the number of C.albicans induced by SH combined with FLC were significantly less than the number of control group;When16 ?g/mL SH used for strain C.albicans 1601,all of the above gene expression were significantly increased in the range of 3.71 times to 12.63 times(P<0.05);The treatment with 8 ?g/mL FLC,compared with the expression of ACT1 gene,the relative expression of gene ZAP1 and gene ADH5 was unchanged;The relative expression of genes IFD6 and PHR1 were significantly decreased;The relative expression of BGL2 gene,XOG1 gene and FKS1 gene were increased.In the combination(8 ?g/mL FLC + 16 ?g/mL SH):(1)The relative expression of BGL2 gene was not affected;(2)The relative expression of gene IFD6 and gene PHR1decreased;(3)The relative expression of ZAP1 gene,ADH5 gene,XOG1 gene and FKS1 gene increased significantly in the range of 1.98 times to 4.10 times(P<0.05).Experiments showed that the SH and FLC combination were effective on the related biofilm cells gene expression of ?-1,3-glucan synthase in antifungal action and the mature biofilm cells.ConclusionsSH against C.albicans shows promising effect.SH can induce a series of apoptosis symptom including pyknosis,fragmentation,PS valgus nucleus,MMP damage and.SH in combination with fluconazole shows a synergistic effect mainly through the influence of the related genes expression of ?-1,3-glucan synthase,thusinhibiting the formation of C.albicans biofilms to form the antifungal effect.Therefore our presented results preliminarily explore the mechanism of antifungal effect of SH,and provide the possible basis for the development of new antifungal drugs against C.albicans.