T-2毒素原型与其隐蔽型T2-3G致IPEC-J2细胞屏障功能损伤的联合毒性作用及机理研究

Co-occurrence of T-2 toxin and its masked T-2 toxin-3-glucoside (T2-3G) in the cereal food seriously interfere in the accurate evaluation of single T-2 toxin toxicity. Thus, there is a need for giving an insight to combined toxic effects and mechanism of T-2 toxin and T2-3G, which will provide more accurate dates for toxic assessment and risk reduction of T-2 toxin in food. The aim of this research proposal is to explore simultaneously toxic effects and mechanism of T-2 toxin and T2-3G on the damage of barrier function in IPEC-J2 cell. Firstly, the dose-response relationship of T-2 toxin and T2-3G single or combined on cytotoxicity of IPEC-J2 cell will be studied, and the toxicological interactions between T-2 toxin and T2-3G will be quantitatively characterized by the method of isobologram and combination index (CI). Secondly, the molecular mechanism and regulatory pathways of toxicological interactions of T-2 toxin and T2-3G on IPEC-J2 cell will be elucidated in the field of cell toxicology and molecular biology, which is based on the determination of cell ultrastructure, permeability, and the expression of tight junction proteins (Claudins, ZO-1, Occludin) and the proteins involved in MARK/ERK signaling pathway, as well as analysis results from transcriptome and bioinformation. The development of this proposal will provide a theoretical reference for risk evaluation on co-contamination of parent mycotoxin and masked mycotoxin, and for standard formulation about the maximum levels of mycotoxins in food.

T-2毒素与其隐蔽型T2-3G常联合存在于谷物食品中，严重干扰了T-2毒素毒性的准确评估。深入了解两种毒素的联合毒性机制，是发展符合真实情况下评价食品中T-2毒素风险的基础和降低其危害的关键。项目拟以T-2/T2-3G为研究对象，以致肠道细胞屏障功能损伤为突破点，利用敏感靶动物猪肠道上皮细胞IPEC-J2模型，探讨两种毒素联合作用的毒性机制。采用等效应线图法和联合指数模型（CI）定量表征T-2/T2-3G单独、联合毒性的剂量-效应关系，探明二者共存时交互作用的类型及程度；通过测定细胞结构、通透性、紧密连接蛋白（Claudins、ZO-1、Occludin）与其上游MARKs/ERK信号通路蛋白表达，结合转录组分析结果，以期从细胞毒理学和分子生物学角度阐明T-2/T2-3G联合毒性的作用机制。旨在为准确评价食品中真菌毒素原型与其隐蔽型联合污染的危害以及为真菌毒素限量标准的研制提供理论依据。

首先，基于T2-3G毒素标准品的空白，以T-2毒素作为研究对象，通过酵母菌（B.muscicola）的生物转化，中压制备液相色谱仪分离纯化获得隐蔽型毒素T2-3G。然后采用液相色谱串联四极杆时间质谱（LC-QTOF）进行初步鉴定，最后以核磁共振波谱仪（NMR）结果确定产物，T-2毒素转化为T2-3G的生物转化效率为51.9%，产物纯度达99.99%。. 其次，以IPEC-J2细胞作为研究对象，CCK-8法检测细胞活率，采用等效应线图法和联合指数模型（Combination index，CI）定量表征T-2/T2-3G单独、联合毒性的剂量-效应关系，研究发现T-2及T2-3G二元联合作用于猪IPEC-J2细胞时，细胞活性整体呈剂量-依赖性降低，且T-2和T2-3G在低剂量联合时呈现协同作用，在高剂量联合时呈现拮抗作用。. 然后，T-2和T2-3G单独或联合作用均可导致猪小肠上皮细胞试验组LDH释放含量均升高，促炎因子IL-1、IL-6、TNF-a含量均显著升高，抗炎因子IL-10的含量降低，联合组变化显著大于单独剂量组；紧密连接蛋白Claudin-1 Claudin-1、Occludin、ZO-1mRNA和蛋白表达降低，联合组紧密连接蛋白也显著降低。激光共聚焦观察发现三种紧密连接蛋白的形态均发生一定的破裂，联合组尤为明显，说明T-2与T2-3G两种毒素联合呈现一定的协同作用，能显著破坏IPEC-J2细胞的屏障功能。. 最后，用T-2+HT-2G联合剂量为25%细胞活率时作用于猪IPEC-J2细胞。组学结果显示，与对照组相比，T-2+HT-2G联合处理组对IPEC-J2细胞内有143种代谢物发生显著的差异，主要属于三酰甘油、甘油胆碱、酰肉碱、二酰甘油、肽类、氨基酸、甘油乙醇胺和脂肪酸等。路径分析表明，癌症胆碱代谢途径的变化最明显，通过ROC分析诊断价值评估显示，变化的代谢物包括Leu-Ala/Ile-Ala、3-(4-羟基苯基)丙酸、Ile-Val/Leu-Val、Gly-Val/Val-Gly、天冬氨酸、DG(34:2)_DG(16:0/18:2)、DG(34:1)、DG(16:0/18:1)、Phe-Trp、DG(36:1)、DG (18:0/18:1)、Leu-Leu/Leu-Ile，均具有较高的AUC值，可以作为T-2+