月桂酸单甘油酯(GML)协同苦参碱对奶牛乳房炎白色念珠菌抗菌机理研究

Clinically, direct antibiotics injection to udder is preferred to dairy cows bacterial mastitis therapy. However, these antibiotics injections easily enhance the proliferation of Candida albicans. In this way, Candida albicans biofilm can be easily generated in udder and usually brings dairy cows severe fungal mastitis.. Here, we main focus on the pathogenic mechanism and virulence development process of Candida albicans in dairy cows. We suppose Matrine, as local features Chinese traditional medicine, can disturb the biofilm generation though special biological process and pathway. Firstly, we evaluate the inhibition effect on biofilm formation with Matrine by MTT and FACS, and make out the functional changes on structure stability, electrical potential and permeability of cell membrane. By the observation under electron microscope and laser confocal scanning microscope, we will explore the disruption mechanism on cell wall structure and metabolism pathway. Besides, the ROS production, activity of respiretory enzymes, synthesis of virulence factors and nucleic acid will be analyzes by fluorescent dyed makers and real-time qPCR. On this ground, we will further explore the specific anti-Candida albicans mechanism in different stages during biofilm formation. Hence, its details on disruption ways in interference quorum sensing system and inhibition activity on conversion between spore and mycelial phase will be also investigate thoroughly.Secondly, there is synergistic effect between GML and Matrine. And GML can induce cell apoptosis though ROS pathway. On this ground, we will expore that whether GML can improve the anti- biofilm activity of Matrine by induced cell apoptosis through ROS/Caspase pathway. Furtherly, we will investigate whether the drug combination with GML and Matrine can induce Candida albicans death through regulation of Ca2+pump function. This study will provide a detailed mechanism of synergistic effect of GML and matrine on Candida albicans, and will support the Chinese drugs pharmaceutics to benefit the treatment on dairy cows mastitis.

临床上将抗生素注入奶牛乳区来治疗细菌性乳房炎，但抗生素能促进白色念珠菌在乳房的繁殖，同时易形成生物被膜的白色念珠菌通过污染的注入器进入乳区，导致了奶牛真菌性乳房炎发生。本研究从白色念珠菌致病机制及毒力形成机理出发，采用MTT法和流式细胞术、电镜观察和激光共聚焦扫描显微镜分析法、荧光染料标记及荧光实时定量PCR等方法，研究特色中药苦参碱对白色念珠菌膜结构、膜通透性和膜电位的作用；对菌株细胞壁的破坏和代谢通路影响；对菌株代谢过程中的呼吸活性氧、有关的酶、毒力因子和核酸合成的影响；对生物被膜形成不同时期结构的作用、干扰群体密度感应系统以及孢子相与菌丝相转换的作用方式；同时利用月桂酸单甘油酯（GML）与苦参碱协同抗菌作用，研究协同药物通过活性氧ROS/Caspase信号途径诱导细胞凋亡；通过调节Ca2+泵功能途径诱导白色念珠菌死亡机制。研究结果为临床开发治疗奶牛真菌性乳房炎的中药制剂提供理论依据。

引起奶牛乳房炎的病原菌有100多种，机体免疫力低下或者抗生素等的滥用导致奶牛乳头正常菌群失衡，其中真菌大量繁殖破坏了奶牛乳腺组织进而造成真菌性乳房炎症的发生。GML作为溶剂应用于医药、保健产品中，作为治疗性药物正在受到关注。本课题从真菌分离鉴定、生物学特性分析；GML和苦参碱对分离的真菌作用特征及机制；GML逆转真菌对唑类抗真菌药物的耐药性三方面研究。结果表明，2016年之前宁夏地区引起真菌性奶牛乳房炎病原菌大多为白色念珠菌，而2016年之后发现主要是克柔念珠菌等为主非白色念珠菌。白色念珠菌溶血性、磷脂酶活性和成膜能力都较强，并具有不同毒力，对中药和常用抗真菌药物表现不同程度敏感。克柔念珠菌分离株和近平滑念珠菌分离株携带所检测的全部毒力因子。克柔念珠菌对氟康唑、酮康唑和伊曲康唑耐药，多药外排泵和唑类靶标基因高表达。在奶牛乳腺生理环境变化范围内，各念珠菌生长情况、耐药性及毒力因子表达情况均受到不同程度的影响。铜离子能够显著抑制念珠菌的生长，同时能够提高念珠菌对抗真菌药物的敏感性和降低毒力因子的表达。抗生素按照治疗奶牛乳房炎浓度作用于念珠菌能够促进念珠菌生长且易使念珠菌产生更强耐药性。GML通过破坏菌体结构的完整性，使细胞核受损、细胞体积缩小，影响细胞各种生理活动，刺激胞内产生大量ROS，造成细胞死亡。GML联合唑类药物、两性霉素B、5氟胞嘧啶、茶皂素、氟尼辛葡甲胺组合作用于念珠菌表现为协同作用。GML还具有降低念珠菌毒力因子、抑制生物被膜和降低菌株耐药性的作用。通过降低念珠菌细胞表面疏水性，抑制其黏附能力；抑制菌株形态转换；显著下调念珠菌相关耐药基因的表达。有效降低生物被膜的厚度和活力，降低和逆转耐药性。