酿酒酵母NAU-LYH-SGS株对猪OTA中毒的干预作用及其机制研究

Ochratoxin A (OTA) is a world-wide potent mycotoxin that naturally occurs in food and feeds， and there are many reports of pig OTA poisoning. Saccharomyces cerevisiae NAU-LYH-SGS is isolated by our lab, and can produce GSH and selenomethionine. Preliminary study showed that Saccharomyces cerevisiae NAU-LYH-SGS can degrade OTA, and alleviate the OTA-induced enterotoxigenesis, nephrotoxicity and immunotoxicity. The contents of the project are as follows: ① verifying the protective effects of Saccharomyces cerevisiae NAU-LYH-SGS on OTA-induced toxicity and exploring the mechanism from intestinal microbial community diversity, OTA degradation, the intestinal barrier function, SIRT1 pathways and autophagy by using piglets as the model; ② investigating the alleviating effects of Saccharomyces cerevisiae NAU-LYH-SGS on OTA-induced enterotoxigenesis and their signaling pathway mechanism by using IPEC-J2 cells to co-culture with Saccharomyces cerevisiae NAU-LYH-SGS; ③ investigating the alleviating effects of zymosan, GSH or/and selenomethionine on OTA-induced nephrotoxicity and immunotoxicity and their autophagy mechanism by using the porcine kidney 15 cells and porcine primary T lymphocyte cells as the models. Someadvanced technologies such as transmission electron microscopy, flow cytometry, western blotting, GFP-LC3 transfection, confocal laser, and RNA interference will be used in the project. The objective is to clarify the mechanism of the protective effects of Saccharomyces cerevisiae NAU-LYH-SGS on OTA poisoning, and provide a scientific basis for Saccharomyces cerevisiae NAU-LYH-SGS widely use in livestock production, and for the control of OTA poisoning and human health.

赭曲霉毒素A(OTA)是广泛存在于饲料中的一种具有很强毒性的霉菌毒素，有大量猪OTA中毒的报道。酿酒酵母NAU-LYH-SGS株由本研究室自主分离，能产GSH和硒蛋氨酸。初步研究表明，该菌株能降解OTA，减弱OTA的肠、肾和免疫毒性。本项目首先是通过仔猪试验，进一步确认该菌株防控猪OTA中毒的效果，并从肠内微生物群落多样性、OTA降解、肠道屏障功能、SIRT1通路和自噬等方面探讨机制。其次用该菌株与猪上皮细胞IPEC-J2共培养，应用电镜、流式细胞术、免疫印迹和RNA干扰等技术研究其减弱OTA肠毒性的效果与信号通路机制。最后以猪肾细胞和原代T淋巴细胞为模型，进一步应用GFP-LC3转染和激光共聚焦等技术研究酵母多糖、硒蛋氨酸或/和GSH减弱OTA的肾毒性和免疫毒性的自噬机制。目的是阐明该菌株防控猪OTA中毒的机制，为其广泛应用提供科学依据，项目无疑对猪OTA中毒的防控和人类健康有重要意义。

赭曲霉毒素A(ochratoxin A，OTA）是继黄曲霉毒素后被确定的又一类具有很强毒性的霉菌毒素，广泛存在于各种谷物及动物源性饲料中。猪对OTA敏感性最强，在美国、加拿大、欧洲各国及我国等均有猪OTA中毒的报道，给养猪业和人类健康带来了严重危害。酿酒酵母NAU-LYH-SGS株由本研究室自主分离，是农业部规定可以在饲料中使用的益生菌，发酵生产后胞内硒蛋氨酸以硒计为90.0～110.0μg/g，胞内GSH含量为32.0～35.0 mg/g。本项目从以下9个方面进行了系统研究：①体外研究了OTA的肠毒性及其机制；②体外研究了非毒性浓度OTA对DON诱导的肠上皮细胞屏障功能障碍的影响及其机制研究；③体内外研究了OTA暴露对肾小球的毒性与机理；④体外研究了OTA的免疫毒性作用及其诱导的免疫抑制的机制；⑤体外研究了甘露寡糖（一类从酵母中提取的免疫抗原活性物质）对OTA诱导免疫抑制的保护作用及机理；⑥体外研究了有机硒缓解OTA引起PK15毒性和DNA损伤的效果与机制；⑦体外研究了硒蛋氨酸和N-乙酰半胱氨酸（GSH前体物质）缓解AB1和OTA诱导的氧化应激和免疫毒性作用及其机制；⑧体外研究了硒、锌联合应用缓解OTA诱导的肾细胞纤维化；⑨猪体内研究了以酿酒酵母NAU-LYH-SGS株为核心的富硒益生菌缓解OTA诱导的猪肾毒性及其可能机制。研究涉及到诸多技术，包括细胞凋亡、自噬、流式细胞术、免疫印迹、基因敲除、GFP-LC3转染和激光共聚焦等。研究结果进一步阐明了OTA的肾毒性、肠毒性和免疫毒性的机制，再次确认了酿酒酵母NAU-LYH-SGS株防控猪OTA中毒的效果，并从OTA降解、肠道屏障功能、硒蛋白表达、表观遗传学以及细胞信号通路和自噬等方面阐明了酿酒酵母NAU-LYH-SGS株防控猪OTA中毒的机理，为酿酒酵母NAU-LYH-SGS株在养猪生产上的广泛应用提供了科学依据。