小窝蛋白1介导黄曲霉毒素肠道转运的作用机制研究

Aflatoxin-food contamination is an important risk factor for the occurrence of hepatocellular carcinoma, and is also one of the greatest food safety issues concerned by the public. AFT is mainly absorbed in the intestine. The intestinal epithelial cells (IECs) are main transport sites of AFT and the first defense against AFT crossing the intestine barrier, however, the mechanisms of toxin transport remains unknown. A small interfering RNA library was used in our early study to screen 140 host proteins with membrane trafficking functions. Caveolin1 (Cav1) was identified as the key molecule in mediating AFT transmembrane transportation. Our previous work shows that AFT can specifically utilize Cav1-mediated transport to across the plasma membrane of IECs. Cav1 is located in the cell plasma membrane and endosome membrane, mainly involved in cholesterol transport and biological molecules endocytosis. This applying project plans to illustrate the molecular and cellular mechanisms of Cav1-mediated AFT transmembrane transportation in IECs through exploring the role of Cav1 in transmission of AFT into cells and regulation of the tight junctions between cells, on the bases of established intestinal barrier model in vitro and in mouse. The implementation of this project helps not only identify the key factors and mechanisms of AFT breaking through the intestinal barrier, but also provide new targets for the prevention of AFT transport and the development of drugs for blocking the intestinal uptake of AFT.

黄曲霉毒素（AFT）污染是导致肝癌发生的重要危险因素，也是公众最为关心的食品安全热点问题之一。AFT主要经肠道吸收，其中肠道上皮细胞是AFT经肠道转运的主要场所，也是AFT突破肠道屏障的第一道防线，但其转运机制仍不清楚。我们前期利用siRNA文库筛选了140个具有膜运输功能的蛋白分子，发现介导AFT跨肠上皮细胞转运的关键分子-小窝蛋白1（Cav1）。Cav1位于细胞膜和内体膜，主要参与胆固醇转运、大分子内吞等过程。前期工作显示，AFT特异性利用Cav1介导的胞吞途径实现跨细胞转运。本课题拟在上述基础上，利用已建立的体外肠道屏障模型和小鼠模型，通过探究Cav1在转运AFT入胞和调控细胞间紧密连接结构及屏障通透性的特点，阐明其参与AFT跨肠上皮细胞转运的分子和细胞机制。课题实施不仅有助于确定介导AFT突破肠道屏障的关键细胞因子及作用机制，还可为阻断AFT肠道摄取药物的研发提供新靶点。

黄曲霉毒素（aflatoxin，AFT）广泛存在于被霉菌污染的食品中，是导致肝癌发生的重要危险因素，也是公众最为关心的食品安全热点问题之一。AFT主要经肠道吸收，其中肠道上皮细胞是AFT经肠道转运的主要场所，但其转运机制仍不清楚。我们前期通过基因文库筛选发现介导AFT跨肠道上皮细胞转运的关键分子-小窝蛋白1（Cav-1）。通过项目开展，我们系统分析了Cav-1分子在黄曲霉毒素B1（AFB1）肠道吸收中的作用以及对肠道屏障功能的影响，在此基础上研究了Cav-1分子在AFB1吸收后的急性肝损伤中的作用与机制。研究发现：（1）AFB1可经体外肠屏障模型进行转运且在转运过程中引起了跨肠上皮细胞电阻值的降低以及紧密连接分子OCLN和CLDN1表达的降低，AFB1在转运过程中与Cav-1分子共定位并引发细胞肌动蛋白重排；下调Cav-1分子后可显著抑制AFB1的吸收，抑制率达到71.74％；下调Cav-1分子显著提升跨上皮电阻值、降低屏障通透性，促进OCLN和CLDN1的表达和膜分布；动物实验显示，Cav-1基因敲除小鼠血清和肝组织中AFB1的含量显著低于野生型，且能够降低AFB1引起的肠道损伤，提示Cav-1在AFB1肠吸收与肠损伤中发挥关键作用。（2）Cav-1为参与AFB1引发肝细胞损伤的关键分子，主要发挥了两项重要的调节作用： Cav-1分子一方面通过与Nrf2直接作用以及抑制Nrf2分子表达，从而抑制细胞的抗氧化应激水平，促进细胞活力下降和凋亡；另一方面，Cav-1分子通过激活EGFR-PI3K-mTOR这一非经典的自噬调节通路，抑制细胞的自噬作用，从而促进毒素引发的细胞活力下降和细胞凋亡。课题实施明确了Cav-1为介导AFB1突破肠道屏障的关键细胞因子，进一步解释AFB1致肝脏损伤的作用机制，为阻断黄曲霉毒素肠吸收以及治疗黄曲霉毒素损伤药物的研发提供了新靶点。