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该研究探讨龙胆泻肝汤氯仿提取物(CELX)对白念珠菌VVC临床株外分泌水解酶的影响。分别采用牛奶培养基、蛋黄培养基、聚氧乙烯脱水山梨醇单油酸酯(吐温-80)培养基对15株白念珠菌VVC临床株进行分泌型天冬氨酸蛋白酶(Sap)、磷脂酶(PL)、脂肪酶(Lip)阳性菌株的筛选;牛奶平板法、蛋黄平板法、吐温-80平板法分别检测CELX干预后,Sap,PL与Lip的活性变化;qRT-PCR检测基因SAP1~7,10,PLB1~2,LIP3~6的表达量。结果可见,15株VVC临床株的Sap,PL均为阳性,11株Lip阳性菌株。随着CELX剂量的递增,与空白对照组相比,各含药培养基(牛奶培养基、蛋黄培养基、吐温-80培养基)上菌落的沉淀圈逐渐减小,当药物达256 mg·L-1时,菌落沉淀圈几乎消失,酶活力显著减弱。qRT-PCR结果显示,256mg·L-1CELX时,除SAP5,SAP6外,SAP1,SAP2,SAP3,SAP4,SAP7,SAP9,SAP10分别下调了62%,55%,62%,84%,61%,51%,68%;PLB1下调了67%;LIP3,LIP4,LIP6分别下调了51%,54%,55%。研究表明,CELX能抑制白念珠菌重要毒力因子天冬氨酸蛋白酶、磷脂酶与脂肪酶的活性。
This study was to investigate the effect of Longdan Xiegan decoction (CELX) on the clinical extracellular secretion hydrolase of Candida albicans VVC. Fifteen Candida albicans VVC clinical strains were treated with secreted aspartic proteinase (Sap), phospholipid (PL) and Lip (Lip) positive strains. The changes of Sap, PL and Lip activity were detected by milk plate method, yolk plate method and Tween-80 plate method. The expression of SAP1 ~ 7,10, PLB1 ~ 2, LIP3 ~ 6 expression levels. The results showed that 15 strains of VVC clinical Sap, PL were positive, 11 Lip positive strains. With the increasing dose of CELX, the sedimentation circle of colonies on each drug-containing medium (milk medium, egg yolk medium, Tween-80 medium) was gradually reduced compared with the blank control group. When the drug reached 256 mg · L-1, the colonies precipitation ring almost disappeared, enzyme activity was significantly weakened. The results of qRT-PCR showed that the levels of SAP1, SAP2, SAP3, SAP4, SAP7, SAP9 and SAP10 decreased by 62%, 55%, 62%, 84% and 61% 51%, 68%; PLB1 down 67%; LIP3, LIP4, LIP6 were down 51%, 54%, 55%. Studies have shown that CELX can inhibit Candida albicans important virulence factors aspartic protease, phospholipase and lipase activity.