荆皮癣湿酊对红色毛癣菌的抗菌作用机制

Mechanism of Jingpixian Tincture against Trichophyton rubrum

摘要:

【目的】探讨荆皮癣湿酊（Jingpixian Tincture，JPXT）对红色毛癣菌（Trichophyton rubrum）的抗菌作用和机制。【方法】微量稀释法测定荆皮癣湿酊对红色毛癣菌的最低抑菌浓度（minimal inhibitory concentration，MIC）；荧光显微镜观察荆皮癣湿酊对红色毛癣菌孢子萌发和菌丝生长的影响；山梨糖醇保护试验测定荆皮癣湿酊对红色毛癣菌细胞壁的影响；流式细胞仪检测红色毛癣菌细胞内活性氧（reactive oxygen species，ROS）水平；酶标仪测定细胞内核酸释放量；高效液相色谱法（high performance liquid chromatography，HPLC）检测细胞膜麦角甾醇含量；酶标仪检测β-1，3-葡聚糖合酶（β-1，3-glucan synthase，β-1，3-GS）、几丁质合成酶（chitin synthetase，CS）、角鲨烯环氧酶（squalene epoxidase，SQLE）、14a-去甲基化酶（14-alpha demethylase，CYP51）活性；实时荧光定量PCR （real-time quantitative PCR，qRT-PCR）测定β-1，3-GS、CS、SQLE、CYP51 mRNA表达水平。【结果】荆皮癣湿酊能抑制红色毛癣菌生长，MIC为原液浓度1/8；对孢子萌发和菌丝生长具有良好的抑制效果；荆皮癣湿酊处理红色毛癣菌24、48 h后胞内核酸释放量明显增加；ROS水平显著升高；β-1，3-GS、CS酶活性和mRNA表达水平均无明显变化，但SQLE、CYP51酶活性和mRNA表达水平显著降低。【结论】荆皮癣湿酊能够抑制红色毛癣菌的生长，其作用机制可能与破坏红色毛癣菌细胞膜、增加细胞氧化应激水平、影响胞内物质合成有关。

Abstract:

[Objective] To investigate the mechanism of Jingpixian Tincture (JPXT) on Trichophyton rubrum. [Methods] Microdilution method was employed to determine the minimal inhibitory concentration (MIC), and fluorescence microscopy to observe the effect of JPXT on spore germination and mycelial growth of T. rubidium. Sorbitol was used to detect the effect of JPXT on fungal cell wall. Flow cytometry was employed to determine the intracellular reactive oxygen species (ROS) level of T. rubidium treated by JPXT. A microplate reader was used to determine the leakage of intracellular nucleic acid. High performance liquid chromatography (HPLC) was adopted to determine ergosterol content in the cell membrane. A microplate reader was used to measure the activities of β-1,3-glucan synthase (β-1,3-GS), chitin synthetase (CS), squalene epoxidase (SQLE), and 14-alpha demethylase (CYP51). The mRNA levels of β-1, 3-GS, CS, SQLE, and CYP51 were measured by real-time quantitative PCR (qRT-PCR). [Results] The MIC of JPXT against T. rubidium was determined as 1/8 of the stock solution concentration. JPXT inhibited the spore germination and mycelial growth of T. rubidium. It increased the leakage of intracellular nucleic acid significantly after treatment for 24 and 48 h, elevated the ROS level, and decreased the ergosterol content. It did not affect the activities or mRNA levels of β-1,3-GS and CS, while it inhibited the activities and down-regulated the mRNA levels of SQLE and CYP51. [Conclusion] JPXT may inhibit the growth of T. rubidium by destroying the cell membrane, increasing the oxidative stress, and affecting the synthesis of intracellular substances.