阿魏酰麦芽糊精与花色苷-sGLT1/GLUT2的互作机制及其对花色苷跨膜转运的影响

Intestinal absorption is a major barrier of the bioavailability of anthocyanins. Interaction between anthocyanin and a component has potential to regulate its absorption, which is closely related to the role of transporters (sGLT1 and GLUT2). However, The relationship between the interaction of components-transporters and anthocyanin absorption efficiency is still obscure. Our previous study found that feruloyl maltodextrin (FA-g-MD) can improve the stability of anthocyanins, subsequently promoting transmembrane transport and enhancing the antioxidant activity of cells and being verified by the UV-vis spectra. It is found that feruloyl polysaccharide exhibit the properties of regulating the activity of glucose transporter. Therefore, we speculate that FA-g-MD may influence the interaction of anthocyanin and sGLT1/GLUT2, which promotes transmembrane transport of anthocyanins. In this study, by using Caco-2 monolayer cells as model, quantitative analysis of the effect of FA-g-MD on the anthocyanin transmembrane transport was carried out through HPLC. And analysis of the expression and subcellular distribution of transmembrane transporter (sGLT1 and GLUT2) under both of activated or inhibited conditions by molecular biology method was performed. We also investigated the interaction between FA-g-MD, anthocyanin, sGLT1 and GLUT2 via molecular docking method, to illuminate molecular effect mechanism of FA-g-MD on anthocyanin transmembrane transport. It is hoped that this study can provide theoretical guidance for the improvement of the rational development and application of absorption enhancers of anthocyanins.

肠道吸收是限制花色苷生物利用度的主要屏障。花色苷与组分间的相互作用具有调节其吸收的潜力，这与转运载体（sGLT1和GLUT2）的作用密切相关。然而，组分-转运载体的相互作用与花色苷吸收效率的关系尚不清楚。我们前期发现阿魏酰麦芽糊精（FA-g-MD）可提高花色苷稳定性，促进跨膜转运，UV-vis提示二者存在相互作用。在此基础上结合国外发现阿魏酰多糖调节转运载体活性的研究成果，推测FA-g-MD可能参与花色苷-sGLT1/GLUT2的互作，促进花色苷跨膜转运。因此，本课题基于Caco-2单层细胞，利用HPLC研究FA-g-MD对花色苷跨膜转运的影响，通过分子生物学方法分析跨膜转运载体在激活或抑制状态的表达和亚细胞分布，进而采用量子化学方法揭示FA-g-MD、花色苷、sGLT1和GLUT2的相互作用关系，阐明FA-g-MD影响花色苷跨膜转运的分子机制，为花色苷吸收促进剂的开发与应用提供理论。

花色苷类化合物是一类具有广泛生物活性的水溶性天然色素，而肠道吸收是限制花色苷生物利用度的主要屏障。本项目评价了FA-g-MD、花色苷复合物的抗氧化能力，考察了FA-g-MD、花色苷对抗氧化酶的调节功能，分析了二者对sGLT1、GLUT2蛋白表达的影响，进而利用分子对接模型试验阐明了二者与sGLT1/GLUT2结合的稳定性及其相互作用机制。花色苷跨膜转运试验表明花色苷自身的转化效率极低（Papp＜10-6cm/s），但经FA-g-MD孵育后，其转化效率显著提高（P<0.05）。Western blotting试验显示FA-g-MD处理细胞后抗氧化酶GCLC和NQO1的表达分别上调217.5%和229.2%。 siRNA干扰、Western blotting及荧光染色试验表明花色苷、FA-g-MD对sGLT1、GLUT2表达均有促进作用。siRNA试验使sGLT1和GLUT2蛋白表达分别下调了60%和54%，随后加入花色苷、FA-g-MD联合处理，两组蛋白表达均增加了90%。分子对接模型表明花色苷和FA-g-MD均能与sGLT1, GLUT2靶点蛋白存在很好的结合作用且匹配度高(结合能均小于-6 kcal/mol)。总体表明，sGLT1 和GLUT2参与了花色苷的跨膜转运，且FA-g-MD具有促进其吸收的作用，但是否存在其它作用方式还需进一步研究。本项目采用量子化学方法揭示了FA-g-MD、花色苷、sGLT1和GLUT2的相互作用关系，阐明了FA-g-MD影响花色苷跨膜转运的作用机制，对提高花色苷生物利用度具有重要意义。