硒化黄芪多糖减弱OTA对猪细胞毒性的作用与自噬机制研究

Ochratoxin A is a world-wide potent nephrotoxin and immunotoxin that naturally occurs in food and feeds. Selenylation modification of astragalus polysaccharide is an organic selenylation modification of polysaccharide, and has the better antioxidant function than selenium or/and astragalus polysaccharide. Our previous studies indicated that selenium alleviated ochratoxin A-induced nephrotoxicity by improving antioxidant function. However, the autophagy mechanism of ochratoxin A -induced toxicity and the effects of selenylation modification of astragalus polysaccharide on ochratoxin A -induced cytotoxicity and their mechanism have not been reported until now. The contents of the project are as follows by using the porcine kidney 15 cells and porcine alveolar macrophage cells as the models: investigation autophagy mechanism of ochratoxin-induced toxicity; the effects of selenylation modification of astragalus polysaccharide on ochratoxin A-induced nephrotoxicity and immunotoxicity; the autophagy mechanism of the protective effects of selenylation modification of astragalus polysaccharide on ochratoxin A-induced nephrotoxicity and immunotoxicity by the autophagy control agent (such as autophagy activator rapamycin, autophagy inhibitor chloroquine, 3 - MA) and interferencing autophagy-related gene (Atg5, Atg7, Beclin-1 and so on). Some advanced 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 project provides the prevention and control of ochratoxin A-poisoning in pig, and a scientific basis for the selenylation modification of astragalus polysaccharide use in livestock production.

赭曲霉毒素A(OTA)是广泛存在于饲料中的一种具有很强肾毒性和免疫毒性的霉菌毒素。硒化黄芪多糖是一种有机硒多糖，具有比硒、黄芪多糖或硒加黄芪多糖更好的抗氧化功效。我们前期研究表明硒可通过抗氧化作用减弱OTA对猪肾细胞的毒性。但迄今有关OTA毒性的自噬机制，硒化黄芪多糖减弱OTA毒性的作用与机制未见研究。本项目拟以猪肾细胞和猪肺泡巨噬细胞为模型，应用透射电镜、流式细胞术、蛋白免疫印迹等技术研究硒化黄芪多糖减弱OTA对猪肾毒性和免疫毒性的作用；进一步应用GFP-LC3转染、激光共聚焦、RNA干扰等技术，通过应用自噬调控剂（如自噬激活剂雷帕霉素，自噬抑制剂氯喹、3-MA）和干扰自噬相关基因（Atg5、Atg7、Beclin-1等）研究OTA对猪肾毒性和免疫毒性的自噬机制，硒化黄芪多糖减弱OTA肾毒性和免疫毒性的自噬机制。本项目可为猪OTA中毒的防控及硒化黄芪多糖在畜牧生产上的应用提供科学依据。

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，可引起猪肾毒性和免疫毒性，但其机制不清。猪圆环病毒病是猪最重要的传染病之一，可引起肾脏损伤，但其对OTA引起肾毒性的作用与机制不清楚。我们前期研究表明硒可通过抗氧化作用减弱OTA对猪肾细胞的毒性，但硒化黄芪多糖或富硒益生菌对猪圆环病毒2型复制和OTA毒性的作用与机制未见研究。本项目以猪，猪肾细胞和猪肺泡巨噬细胞为模型，应用流式细胞术、激光共聚焦、蛋白免疫印迹，LC-MS/MS、RNA干扰等技术，研究了饲料中OTA对断奶仔猪的毒性和机制，证明含有0.4和0.8 mg/kg的OTA饲料，可导致血清和组织内OTA浓度升高（从高到低依次为血液、肾脏和脾脏），生长性能降低，肾功能损伤，使肾小管上皮细胞发生退行性变化和细胞凋亡，脾脏中可以观察到细胞核固缩和CD3+ T淋巴细胞的减少，外周血和脾脏淋巴细胞活力降低，降低猪血清IL-2含量和增加TNF-α含量，增加猪肾脏和脾脏GRP78的表达、p38和ERK1/2的磷酸化和LC3II和Atg5蛋白表达。体内外首次证明，PCV2感染可通过p38介导的自噬加剧OTA引起的猪肾毒性。OTA可通过p38 MAPK信号通路引起猪肾细胞毒性，ERK MAPK信号通路引起免疫细胞毒性。OTA通过DNMT1介导的SOCS3/JAK2/STAT3信号通路引起猪肾细胞毒性和DNA损伤，而免疫毒性是OTA通过DNMT1介导的JAK2/STAT3信号通路引起的，与SOCS3表达无关。富硒益生菌可改善OTA引起的猪肾损伤。硒蛋氨酸可以通过激活AKT/mTOR信号通路抑制自噬，进而阻断OTA对PCV2复制的促进作用。SelS可通过调节氧化应激和p38磷酸化影响OTA引起的肾细胞毒性和凋亡。硒化黄芪多糖通过激活PI3k/AKT信号通路抑制自噬来阻断过氧化氢对PCV2复制的促进作用。这些结果不仅对阐明OTA的中毒机理有重要的理论意义，而且对OTA中毒以及猪圆环病毒病防控有重要的应用意义，为有机硒（硒化黄芪多糖、富硒益生菌）在畜牧生产上的应用提供科学依据。