紫草素对白色念珠菌的抑制作用机制

Inhibitory mechanism of alkannin on Candida albicans

摘要:

[目的]研究紫草素抑制白色念珠菌的作用机制。[方法]通过微量稀释法测定紫草素对白色念珠菌的最低抑菌浓度（MIC）和最低杀菌浓度（MFC）；紫外分光光度法测定紫草素对白色念珠菌细胞膜渗透性的影响；扫描电镜观察紫草素对菌体形态的影响；激光共聚焦显微镜测定紫草素对白色念珠菌细胞内钙离子浓度的影响；卵黄平板培养基法检测紫草素对白色念珠菌的细胞膜磷脂酶活性的影响；RT-PCR检测紫草素对白色念珠菌PLB1和PLB2基因表达量的影响。[结果]紫草素对白色念珠菌有较强的抑制作用，其对白色念珠菌的MIC和MFC分别为16μg/mL和32μg/mL。紫草素能破坏白色念珠菌细胞膜的完整性，使细胞膜的通透性增加，导致细胞内DNA和RNA等大分子物质的泄漏和细胞内钙离子的流失。其中MIC的紫草素作用菌体16 h后，上清液中的DNA和RNA等大分子含量与对照组相比增加了117.32%（P<0.01）；细胞内的[Ca²⁺]降低了72.02%（P<0.01）。扫描电镜结果也证明了紫草素对白色念珠菌细胞膜的破坏作用。紫草素也能抑制白色念珠菌分泌磷脂酶，且呈浓度剂量依赖。其中，与对照组相比，MIC的紫草素能使白色念珠菌分泌磷脂酶的量下降56.3%（P<0.01）。RT-PCR结果显示，紫草素能抑制编码磷脂酶B的基因PLB1和PLB2的表达量，其中1/2 MIC的紫草素作用白色念珠菌16 h后，与对照组相比，PLB1和PLB2基因的相对表达量分别降低了56.4%和61.4%（P<0.01）。[结论]紫草素对白色念珠菌有较强的抑杀作用，其作用机制是通过破坏白色念珠菌细胞膜的完整性，增加菌体细胞膜的通透性，导致细胞内DNA和RNA等大分子的泄漏和细胞内[Ca²⁺]的流失，最终引起菌体的死亡。而紫草素对白色念珠菌磷脂酶分泌的抑制作用，致使其不能及时维护和修复由紫草素造成的细胞膜的破坏和损伤，也是导致菌体死亡的原因。

Abstract:

[Objective] This study aims to explore the inhibitory effect of alkannin on Candida albicans and the mechanism. [Methods] The minimal inhibitory concentration and minimal fungicidal concentration of alkannin on C. albicans were measured by microdilution method. The effect of alkannin on the membrane permeability of C. albicans was determined by ultraviolet spectrophotometry. The effect of alkannin on the morphology of C. albicans was observed by scanning electron microscope and the effect of alkannin on intracellular calcium ion concentration of C. albicans was determined by laser scanning confocal microscope. The effect of alkannin on membrane phospholipid enzyme activity of C. albicans was determined by egg-yolk agar plate medium method. The effect of alkannin on amount of gene expression of PLB1 and PLB2 was determined by RT-PCR. [Results] Alkannin had potent inhibitory effect on C. albicans, the minimal inhibitory concentration and minimal fungicidal concentration of alkannin on C. albicans were 16 mg/mL and 32 mg/mL respectively. Alkannin could damage the cytomembrane integrity of C. albicans and increased the permeability of cytomembrane, leading to the leakage of macromolecular substances such as DNA and RNA in cells and the loss of intracellular calcium ions. The concentration of macromolecular substances such as DNA and RNA in the supernatant increased by 117.32% (P<0.01) after alkannin acted on C. albicans for 16 h and the intracellular[Ca²⁺] decreased by 72.02% (P<0.01). The scanning result of electron microscopy also proved the destructive effect of alkannin on C. albicans cytomembrane. Alkannin could inhibit secretion of phospholipase by C. albicans and showed the dose-dependence. Alkannin in minimal inhibitory concentration decreased phospholipase secretion of C. albicans by 56.3% (P<0.01) compared with the control group. RT-PCR results demonstrated that alkannin could inhibit the expression of PLB1 and PLB2, which were a pair of gene encoding phospholipase B. After alkannin in 1/2 minimal inhibitory concentration acted on C. albicans for 16 h, the expression of PLB1 and PLB2 decreased by 56.4% and 61.4% respectively (P<0.01) compared with the control group. [Conclusion] Alkannin had a strong inhibitory effect on C. albicans by damaging cytomembrane integrity of C. albicans and increasing the permeability, leading to leak of intracellular macromolecule such as DNA and RNA and loss of[Ca²⁺], eventually causing cell death. The inhibition of alkannin on phospholipase secretion of C. albicans prevented cells from maintaining and repairing the damage of cytomembrane caused by alkannin, was also one of reasons of cell death.