Kobusin对趾间毛癣菌的抗真菌作用及其机制

Antifungal activity and its mechanism of Kobusin against Trichophyton interdigitale

摘要:

目的 探究Kobusin对趾间毛癣菌(Trichophyton interdigitale)的抗真菌作用及其机制。方法 采用微量稀释法测定Kobusin的最低抑菌浓度(minimal inhibitory concentration, MIC)；通过显微镜观察Kobusin对孢子萌发的抑制作用；利用含药平板法分析菌丝径向生长情况；采用扫描电子显微镜(scanning electron microscope, SEM)表征菌丝形态结构变化；结合荧光显微镜观察与核酸和蛋白质泄漏实验评估细胞膜完整性；使用马尔文激光粒度分析仪检测Zeta电位变化；采用酶标仪测定跨膜电势、碱性磷酸酶(alkaline phosphatase, AKP)活性、丙二醛(malondialdehyde, MDA)含量、活性氧(reactive oxygen species, ROS)积累量、超氧化物歧化酶(superoxide dismutase, SOD)活性、过氧化氢酶(catalase, CAT)活性、过氧化物酶(peroxidase, POD)活性、线粒体膜电位(mitochondrial membrane potential, MMP)、ATP含量、琥珀酸脱氢酶(succinate dehydrogenase, SDH)及苹果酸脱氢酶(malate dehydrogenase, MDH)活性。结果 Kobusin对趾间毛癣菌的MIC为39 μg/mL，可显著抑制孢子萌发与菌丝径向生长。SEM显示菌丝形态结构严重受损；荧光显微镜观察证实细胞膜完整性遭到破坏，核酸与蛋白质泄漏量显著增加，Zeta电位及跨膜电势明显紊乱。同时，Kobusin导致MDA含量和ROS累积量显著上升，AKP、SOD、CAT、POD活性受到抑制，MMP显著下降，ATP合成减少，SDH与MDH活性降低。结论 Kobusin通过抑制孢子萌发与菌丝生长、破坏细胞膜及细胞壁完整性、干扰膜电位稳定性、诱导氧化应激损伤、破坏线粒体能量代谢等机制发挥抗真菌作用。

Abstract:

Objective To investigate the antifungal activity of Kobusin against Trichophyton interdigitale and its underlying mechanisms.Methods The minimal inhibitory concentration (MIC) of Kobusin was determined by the broth microdilution assay. The inhibitory effect of Kobusin on spore germination was observed microscopically, while that on hyphal radial growth was assessed on the agar plates containing Kobusin. Scanning electron microscopy (SEM) was employed to examine the morphological alterations in hyphae. Fluorescence microscopy and nucleic acid and protein leakage assays were employed to evaluated cell membrane integrity. Malvern Zetasizer was used to measure the changes in Zeta potential. A microplate reader was used to measure transmembrane potential, alkaline phosphatase (AKP) activity, malondialdehyde (MDA) content, reactive oxygen species (ROS) accumulation, superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) activities, mitochondrial membrane potential (MMP), ATP levels, as well as succinate dehydrogenase (SDH) and malate dehydrogenase (MDH) activities.Results Kobusin exhibited a MIC of 39 μg/mL against T. interdigitale, significantly inhibiting spore germination and hyphal growth. SEM revealed severe ultrastructural damage to hyphae. Fluorescence microscopy confirmed compromised membrane integrity, evidenced by increased nucleic acid and protein leakage and disrupted Zeta/transmembrane potentials. Meanwhile, Kobusin significantly increased the MDA content and ROS accumulation, inhibited the activities of AKP, SOD, CAT, and POD, markedly reduced MMP, decreased ATP synthesis, and weakened the activities of SDH and MDH.Conclusion Kobusin exerts antifungal effects by inhibiting spore germination and hyphal growth, disrupting cell membrane and cell wall integrity, interfering with membrane potential stability, inducing oxidative stress damage, and impairing mitochondrial energy metabolism.