Gomisin M1协同氟康唑对耐药白念珠菌毒力因子的作用研究

Effect of gomisin M1 combined with fluconazole on virulence factors of drug-resistant Candida albicans

目的探讨gomisin M1(从五味子科植物中提取的木脂素类成分)联合氟康唑抗耐药白念珠菌的作用及其机制。方法采用倍比微量稀释法检测gomisin M1联合氟康唑对白念珠菌的最小抑菌浓度;通过时间-生长曲线动态观察gomisin M1联合氟康唑对白念珠菌的抑制作用;采用倒置显微镜观察gomisin M1联合氟康唑在Spider和SD+10%胎牛血清(fetal bovine serum,FBS)液体培养基中对白念珠菌菌丝生长的影响;采用卵黄琼脂平板法测定gomisin M1联合氟康唑对白念珠菌胞外磷脂酶活性的影响;采用qRT-PCR法检测gomisin M1联合氟康唑对白念珠菌菌丝相关基因及水解酶相关基因表达的影响。结果Gomisin M1联合氟康唑对耐药白念珠菌具有明显的抑制作用,并能明显抑制耐药白念珠菌菌丝的生长;Gomisin M1联合氟康唑能够降低白念珠菌胞外磷脂酶活性,显著下调菌丝相关基因RAS1、CDC35、增强丝状生长蛋白1(enhanced filamentous growth protein 1,EFG1)、细胞伸长蛋白1(cell elongation protein 1,ECE1)、凝集素样蛋白3(agglutinin-like protein3,ALS3)、菌丝壁蛋白(hyphal wall protein,HWP1)和水解酶相关基因分泌型天冬氨酸蛋白酶1(secreted aspartyl protease1,SAP1)、SAP2、SAP3以及磷脂酶B1(phospholipase B1,PLB1)的表达(P<0.05、0.01)。结论Gomisin M1协同氟康唑能够抑制耐药白念珠菌菌丝形成相关蛋白和胞外磷脂酶等毒力因子的表达,从而发挥抗菌作用。

Objective To explore the effect and mechanism of gomisin M1(lignans extracted from Schisandraceae plants)combined with fluconazole against drug-resistant Candida albicans.Methods Multiple microdilution method was used to detect the minimum inhibitory concentration of gomisin M1 combined with fluconazole against C.albicans.Time-growth curve was used to observe the inhibitory effect of gomisin M1 combined with fluconazole on C.albicans.Inverted microscope was used to detect the effect of gomisin M1 combined with fluconazole on hyphae formation in Spider and SD+10%fetal bovine serum(FBS)liquid medium.Yolk agar plate method was used to determine the effect of gomisin M1 combined with fluconazole on extracellular phospholipase activity of C.albicans.qRT-PCR method was used to detect the effect of gomisin M1 combined with fluconazole on gene expressions of myceliumrelated proteins and hydrolase.Results Gomisin M1 combined with fluconazole had a significant inhibitory effect on drug-resistant C.albicans and significantly inhibited the hyphae growth of drug-resistant C.albicans.Gomisin M1 combined with fluconazole reduced C.albicans extracellular phospholipase activity,significantly inhibited hypha-related genes RAS1,CDC35,enhanced filamentous growth protein 1(EFG1),cell elongation protein 1(ECE1),agglutinin-like protein 3(ALS3),hyphal wall protein(HWP1)and hydrolase-related genes secreted aspartyl protease 1(SAP1),SAP2,SAP3 and phospholipase B1(PLB1)expressions(P<0.05,0.01).Conclusion Gomisin M1 combined with fluconazole can play an antifungal role through inhibiting the expression of virulence factors including hyphae formation related proteins and extracellular phospholipase of drug-resistant C.albicans.