Effect And Mechanism Of Butyl Alcohol Extract Of Baitouweng Decoction On Cell Membrane,Cell Wall And Vacuole Of Candida Albicans

Objective:To investigate the effect and mechanism of Butyl alcohol extract of BaiTouWeng decoction?BAEB?on cell membrane,cell wall and vacuole of Candida albicans.Method:Firstly,the MIC₈₀0 and SMIC₈₀0 of Butyl alcohol extract of BaiTouWeng decoction?BAEB?against C.albicans were determined by micro-solution dilution method.The effects of BAEB on the activity of C.albicans were observed by Spot assay.The changes of intracellular osmotic pressure of C.albicans after BAEB intervention were detected by microtiter plate reader.The effect of BAEB on cell membrane permeability of C.albicans were observed by fluorescence microscopy.The content of ergosterol in C.albicanscellmembranewasdetectedbyhighperformanceliquid chromatography,and the expression of ergosterol biosynthesis related genes in cell membrane was detected by qRT-PCR.The effect of BAEB on C.albicans cell activity was detected by spot assay.The changes of?-1,3-glucan and chitin was measured by flow cytometry and microplate reader after BAEB treatment.The expression of?-1,3-glucan related genes and chitin related genes in cell wall was detected by qRT-PCR.Transmission Electron Microscopic was used to observe the effect of BAEB on cell wall structure of C.albicans.The effect of BAEB on vacuolar membrane of C.albicans was observed by fluorescence microscope.The expression of vacuole synthesis-associated genes of C.albicans was detected by qRT-PCR.Results:The results showed that the activity of C.albicans was significantly decreased in 256,512 and 1024?g/mL BAEB group.The intracellular glycerol content of C.albicans was significantly increased in 512 and 1024?g/mL BAEB group?P<0.05?.The gene HOG1 associated with intracellular osmotic pressure of C.albicans was down-regulated by 9.1,9.3 and 5.5 times,respectively.C.albicans with red fluorescent were increased significantly in 512 and 1024?g/mL BAEB group.The peak area of ergosterol in the 1024?g/mLBAEB group was 35.88495,with a significant difference?P<0.05?;ERG1,ERG2,ERG3,ERG4,ERG5,ERG6,ERG10,ERG11,ERG13,ERG24,ERG25,ERG251,ERG26 and UPC2 were down-regulated by 6.57,4.88,4.14,9.23,3.40,9.83,3.07,7.49,5.52,5.89,2.44,3.24,1.97,10.06 times respectively in 1024?g/mL BAEB group.The C.albicans activity decreased with the BAEB intervention.Exposure of chitin and?-1,3-glucan increased gradually in the 256,512,and 1024?g/mL BAEB groups?P<0.05?.FKS1,CHS1,CHS2,CHS3 and CHS8 were down-regulated by 5.57,1.35,3.29,4.47,3.00 times in the group of 1024?g/mL BAEB treatment respectively.TEM showed the cell wall structure of C.albicans was damaged.Through the yeast vacuolar membrane dye MDY-64 staining,as the concentration of BAEB increases,the green fluorescence generated by the vacuolar membrane of C.albicans decreases and the vacuole membrane breaks down.In the 1024?g/mL BAEB intervention group,APM1,CLA4,PEP7,PEP12,PKH2,VPS15,VPS34,VPS41,and YPT72 were down-regulated by 4.410,4.534,4.070,2.184,4.506,11.435,11.316,3.192,2.372 times,respectively.Conclusion:BAEB can inhibit the cell membrane integrity of C.albicans by destroying the cell membrane structure and reducing the content of cell membrane components,thereby inhibiting the activity of C.albicans cells,and at the same time inhibiting the expression of ergosterol synthesis-related genes in the cell membrane of C.albicans and reducing the synthesis of ergosterol.BAEB can expose C.albicans cell wall?-1,3-glucan and chitin to destroy the structure of the cell wall.At the same time,by inhibiting the expression of genes related to?-1,3-glucan and chitin biosynthesis,the structure of cell wall is destroyed and the cell wall integrity of C.albicans is affected.BAEB can damage vacuolar membrane of C.albicans and down-regulates genes related to vacuole synthesis of C.albicans.