Mechanism of Jingpixian Tincture against Trichophyton rubrum

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.