Effect Of Longdan Xiegan Decoction On The Invasion Of Candida Albicans VVC Clinical Strain And The Related Mechanism

Objective : To investigate the intervention effect and mechanism of Chloroform extracts of Longdan xiegan decoction(CELX)against Candida albicans invasion.Methods : 1.The extraction methods of different polar parts of Longdan xiegan decoction are that 80% ethanol water solution is used to extrac total composition of Longdan Xiegan decoction by condensation reflux,successively with petroleum ether,chloroform,ethyl acetate,n-butanol and the corresponding extractions are evaporated to solid powder which are the extractions of Longdan xiegan decoction of different polarity parts.2.By comparing the minimal inhibitory concentration of extractions against 15 strains of clinical isolates of Candida albicans,confirm the best extraction site of Longdan xiegan decoction.3.In vivo experiment,after the mouse models of vaginal candidiasis were successfully constructed 20mg/kg,32 mg/kg,64 mg/kg CELX are given by gavage respectively.The positive drug treatment group is treated by 20 mg/kg Fluconazole,The normal group and model group are given physiological saline.Continuous medication for 7days,the clinical manifestations,microscopic examination,fungal load and histopathological examination are observed.4.Experiment in vitro is divided into two parts.(1)After the intervention of CELX,the colony morphology and the ability of hyphal filament invasion were observed by solid medium and semi-solid medium respectively.XTT and Fluorescence microscope assay were applied to determine the metabolic activity of hyphae of C.albicans treated by CELX for 6h.hyphae morphology were observed by scanning electron microscope.The CFU variation of Candida albicans in 6 hours was counted by Plate assay.The expression of ALS1,ALS3,SSA1,SUN41,HWP1,Ca PDE2,UME6 and ECE1 were detected by real-time quantitative PCR(q RT-PCR).(2)Secreted aspartyl proteinase(Sap),phospholipase(PL)and lipase(Lip)positive strains were sieved from 15 strains of Candida albicans by milk culture medium,egg yolk culture medium and Tween-80 medium respectively.And the PZ value was used to express the enzyme activity.After CELX intervention the activities of Sap,PL and Lip were measured by milk plate method,egg yolk plate method and Tween-80 plate method respectively q RT-PCR was used to detect the expression of aspartic protease SAP1 ~ 7,10,phospholipase B(PLB1 ~ 2)and lipase(LIP3 ~ 6).Results:1.Comparing with other extractions of different polar parts of Longdan xiegan decoction,Chloroform extracts of Longdan xiegan decoction has the strongest antibacterial capacity to 15 strains of clinical isolates of VVC and the MICs of CELX against C.albicans strains are between 32 and 64 ?g/m L;2.In vivo experiments showed that the chloroform extract of Longdan Xiegan decoction has a good therapeutic effect on vulvovaginal candidiasis.High dose treatment group(64mg/kg)can significantly reduce the number of vaginal fungal load,improve vaginal mucosal damage in mouse;3.CELX with concentration of 256 ?g/m L inhibit hyphae formation and weaken the ability of mycelial invasion significantly,which the results from fluorescence microscopy,scanning electron microscopy and solid culture medium and semi-solid medium.And as well as the results of q RT-PCR showed that the expression of hyphae formation related genes ALS1? ALS3?SSA1?SUN41?HWP1?UME6 and ECE1 were down-regulated by 47%?81.6%?60.2%?69.8%?73.7%?49.5%?52.8%.The negative control gene Ca PDE2 up 5.5 times.By the way,the change of the negative control gene Ca PDE2 was not dose-dependent.4.The Secreted aspartyl proteinase and phospholipase of 15 VVC clinical strains were positive,11 strains of lipase positive strains and the enzymes activities were strong.The results of drug CELX-containing medium(milk medium,egg yolk culture medium,Tween-80 medium)experiment showed that compared with the blank control group,the sedimentation of the colonies decreased gradually in each culture medium with theincrease of CELX dose.when the concentration of CELX was 256 ?g/m L,the colony was almost disappeared which indicated enzyme activity was weakened and the results of q RT-PCR showed that SAP1,SAP2,SAP3,SAP4,SAP7,SAP9 and SAP10 were down-regulated by 62%?55%?62%?84%?61%?51%?68% respectively except for SAP5 and SAP6;PLB1,LIP3,LIP4,LIP6 were down-regulated by 67%?51%?54%?55% respectively.Conclusion : The Chloroform extracts of Longdan xiegan decoction(CELX)may reduce Candida albicans mucosal penetration by inhibiting the formation of hyphae and interfere with the secretion of various hydrolytic enzymes of Candida albicans to reduce its damage to vaginal mucosa.Finally,the effect of reducing the invasion of Candida albicans was achieved.