Mechanism Of Jingpixian Tincture Against Trichophyton Rubrum

1 Objective:This study aimed to evaluate the effect of Jingpixian Tincture(JPXT)on Trichophyton rubrum(T.rubrum)and explore the mechanism of action.The effect of JPXT on the apoptosis of T.rubrum was further investigated.These findings preliminarily elucidate the mechanism of JPXT against T.rubrum infection and provide novel thoughts and methods for clinical treatment of dermatomycosis.2 Methods:2.1 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.rubrum.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.rubrum treated by JPXT.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.Microplate reader was used to measure the activities ofβ-1,3-glucan synthase(β-1,3-GS),chitin synthetase(CS),squalene epoxidase(SQLE),and 14-alpha demethylase(CYP51).The m RNA levels of β-1,3-GS,CS,SQLE,and CYP51 were measured by real-time quantitative PCR(q RT-PCR).2.2 Cell Counting Kit-8(CCK-8)assay was used to detect the effect of JPXT on the growth activity of T.rubrum.The levels of Reactive oxygen species(ROS)and Mitochondrial membrane potential(MMP)were measured by flow cytometry.Annexin V-FITC/PI staining was used to observe the phosphatidyl serine(PS)externalization,and flow cytometry was used to detect the apoptosis rate.FITC-VAD-FMK staining assay was performed to evaluate the metacaspase activity.Ultraviolet spectrophotometer was performed to evaluate the activity of Cytochrome C oxidase.3 Results:3.1 The MIC of JPXT against T.rubrum was determined as 1/8 of the stock solution concentration.JPXT inhibited the spore germination and mycelial growth of T.rubrum.The leakage of intracellular nucleic acid was significantly increased after treatment for24 and 48 h.ROS level was elevated while the ergosterol content was decreased.The activities or m RNA levels of β-1,3-GS and CS were not been affected,while the activities and the m RNA levels of SQLE and CYP51 were down-regulated.3.2 The cell viability and MMP level of T.rubrum were down-regulated after JPXT treatment;ROS levels were significantly up-regulated;PS externalization and apoptosis rates were significantly increased;Metacaspase enzyme activity was significantly increased and cytochrome c oxidase activity was decreased.4 Conclusion:We found JPXT can significantly inhibited the growth of T.rubrum.Mechanism of antifungal effect may be related to damaging the structure and integrity of the cell membrane of T.rubrum,causing intracellular nucleic acid extravasation,affecting the biosynthesis of ergosterol,up-regulating the level of cellular oxidative stress,and inducing the apoptosis of T.rubrum.