异黄绵马酸PB对红色毛癣菌生物被膜的抑制作用观察

Inhibiting effect of isoflavaspidic acid PB on biofilm of Trichophyton rubrum

目的观察异黄绵马酸PB对红色毛癣菌生物被膜的抑制作用,并探讨其可能作用机制。方法构建红色毛癣菌生物被膜,用XTT法测定异黄绵马酸PB、两性霉素B、盐酸特比萘芬对红色毛癣菌生物被膜的最低抑菌浓度(s MIC),计算抑制50%及80%被膜生长的药物浓度即s MIC50、s MIC80。取红色毛癣菌生物被膜分为正常组、异黄绵马酸PB组、AMB组、TBF组,后三组分别用125. 00、2. 00、125. 00μg/m L异黄绵马酸PB、两性霉素B、盐酸特比萘芬处理,于扫描电镜下观察药物对生物被膜形态变化的影响,通过超高效液相色谱法测定红色毛癣菌生物被膜细胞膜成分麦角甾醇在药物作用前后的含量变化,采用qRT-PCR法测定红色毛癣菌生物被膜ERG1、MEP4 mRNA的表达。结果异黄绵马酸PB对红色毛癣菌生物被膜的s MIC50及s MIC80分别为31. 25、62. 5～125μg/m L。扫描电镜结果显示,异黄绵马酸PB组红色毛癣菌生物被膜生长明显受到抑制,菌丝呈扁平状且出现大量断裂,未见分生孢子,仅有少量干瘪状细胞外基质。正常组、异黄绵马酸PB组红色毛癣菌生物被膜中麦角甾醇含量分别为(3. 68±0. 05)、(2. 36±0. 01)μg/g,ERG1 mRNA相对表达量分别为1. 36±0. 03、1. 00±0. 06,MEP4 mRNA相对表达量分别为2. 86±0. 08、1. 00±0. 08,异黄绵马酸PB组与正常组相比P均<0. 01。结论异黄绵马酸PB对红色毛癣菌生物被膜具有较好的抑制作用,其作用机制可能与抑制红色毛癣菌麦角甾醇的生物合成,抑制麦角甾醇合成相关基因ERG1及毒性基因MEP4有关。

Objective To observe the inhibitory effect of isoflavaspidic acid PB on biofilm of Trichophyton rubrum(T.rubrum)and its possible mechanism.Methods After the mature biofilm of T.rubrum was cultured,the minimal inhibitory concentrations of isoflavaspidic acid PB,amphotericin B(AMB)and terbinafine hydrochloride(TBF)against the biofilm were determined by the XTT method,then the concentration of drugs inhibiting 50%and 80%biofilm growth(sMIC 50 and sMIC 80)were calculated.The biofilm of T.rubrum were divided into groups of normal,isoflavaspidic acid PB,AMB and TBF,and the latter 3 groups were treated with isoflavaspidic acid PB,AMB and TBF in the concentration of 125.00,2.00,125.00μg/mL,respectively.The morphologic change of biofilm was detected by scanning electron microscope(SEM)after treatment of these drugs.The change in content of ergosterol in cytomembrane of biofilm of T.rubrum was determined by ultra-performance liquid chromatography(UPLC).The expression of ERG1 and MEP4 mRNA in biofilm was determined by real-time fluorescence quantitative PCR(qRT-PCR).Results The sMIC 50 and sMIC 80 of isoflavaspidic acid PB against biofilm of T.rubrum were 31.25 and 62.5-125μg/mL,respectively.The SEM results indicated that,in the isoflavaspidic acid PB group,the growth of biofilm of T.rubrum was significantly restrained,the mycelium was flat and largely broken,and there was no conidiophores,and a small amount of withered extracellular matrix.The content of ergosterol in the normal group and the group of isoflavaspidic acid PB was(3.68±0.05)and(2.36±0.01)μg/g,the expression of ERG1 mRNA was 1.36±0.03 and 1.00±0.06,and the expression of MEP4 mRNA WAS 2.86±0.08 and 1.00±0.08,respectively,with significant difference(all P<0.01).Conclusion Isoflavaspidic acid PB has better inhibitory activity against biofilm of T.rubrum by reducing the biosynthesis of ergosterol and inhibiting the ergosterol synthesis-related gene ERG1 and toxic gene MEP4.