Effect And Mechanism Of Butyl Alcohol Extract Of Baitouweng Decoction On Adhesion And Biofilm Formation Of Candida Albicans Based On PH Pathway

Objective: This study aimed to investigate the effect and mechanism of Butyl alcohol extract of Bai Tou Weng decoction on adhesion and biofilm formation of Candida albicans based on p H signal pathway.Method: Under acidic conditions: Spot assay method was used to detect the sensitivity of p H mutants to BAEB.XTT assay was used to detect the effect of BAEB on metabolic activity of p H mutants.The effect of BAEB on the adhesion activity of p H mutant cells were observed by fluorescence microscopy.The effect of BAEB on hydrophobicity of p H mutant was determined by n-octane inclusion method.The effect of BAEB on the expression of adhesion genes related to p H mutants was detected by q RT-PCR.Under Alkaline conditions: Spot assay was used to detect the sensitivity of p H mutants to BAEB.Scanning electron microscopy was used to observe the effect of BAEB on the biofilm morphology of p H mutants.CLSM was used to measure the effect of BAEB on the biofilm thickness of p H mutants.XTT assay was used to detect the effect of BAEB on the metabolic activity of p H mutants biofilm.The damage of BAEB to p H mutants biofilm was detected by flow cytometry.The effect of BAEB on the expression of biofilm related genes to p H mutants was detected by q RT-PCR.Results: Under acidic conditions: Spot assay observation showed that p H mutants were less sensitive to BAEB,512 g/m L BAEB interfered with p H mutants for 24 h and48h,there was no significantly decrease in bacterial colony.XTT assay showed that the metabolic activity of WT,PHR2 complementation,rim101/rim101 and RIM101 complementation was significantly inhibited in 512μg/m L BAEB,and there was no significantly difference in the inhibition of phr2/phr2 metabolic activity.Fluorescence microscopy showed that the cell adhesion activity of WT,PHR2 complementation,rim101/rim101,RIM101 complementation was significantly inhibited in 512μg/m L BAEB,the cell adhesion activity of phr2/phr2 had no obvious effect in 512μg/m L BAEB.The n-octane inclusion method showed that the cell surface hydrophobicity of WT,phr2/phr2,PHR2 complementation,rim101/rim101,RIM101 complementation was decreased in 512μg /m L BAEB.The q RT-PCR assay showed that the adhesion genes of p H mutants had inhibited in 1024μg /m L BAEB.Under alkaline conditions: Spot assay was used to observe the susceptibility of p H mutants to BAEB,and 512 g/m L BAEB significantly inhibited the bacterial colony of p H mutants.The SEM showed that the biofilm structure of rim101/rim101 and RIM101 complementation was damaged in 512 μg/m L BAEB,the biofilm structure of phr1/phr1 and PHR1 complementation was no obviously damaged.The CLSM showed that the biofilm thickness of rim101/rim101 and RIM101 complementation was reduced and phr1/phr1 and PHR1 complementation was not reduced in 512μg/m L BAEB.The XTT assay showed that the biofilm activity of WT,phr1/phr1,PHR1 complementation,rim101/rim101 and RIM101 complementation was significantly inhibited in 512 μg/m L BAEB.The Flow cytometry showed that the biofilm of PHR1 complementation,rim101/rim101 and RIM101 complementation was obviously damaged and phr1/phr1 was not significantly damaged in 512 μg/m L BAEB.The q RT-PCR method that except to the up-regulation of HSP90 gene expression,ALS3,SUN41,HWP1,UME6 and PGA10 genes of PHR1 deletion,PHR1 complementation,RIM101 deletion,and RIM101 complementation strains showed a downward expression trend.Conclusion:Under acidic conditions,the Candida albicans p H mutants had low sensitivity to BAEB by the results of the spot assay,and BAEB effectively inhibited the adhesion process of strains other than the phr2/phr2.Under alkaline conditions,the Candida albicans p H mutants was highly sensitive to BAEB by the results of the Spot assay,and BAEB effectively inhibited biofilm formation of of p H mutants.This experiment provides an experimental basis for the clarification of the antifungal mechanism based on p H pathway.