精氨酸与谷氨酸对呕吐毒素所致猪肠上皮细胞损伤的保护机制研究

Vomitoxin(VT) contamination, which is widespread among swine feed ingredients is known to cause several adverse effects in pigs, including negative structural and functional changes in the intestinal porcine epithelial cells, resulting in cell apoptosis. Arginine and glutamic acid are two functional amino acids whose requirements are increased for pigs under stressful conditions, and are known to promote protein synthesis in intestinal epithelial cells and to regulate cell survival. Our previous animal studies showed that arginine and glutamic acid can relief VT-induced damage in the intestines and promote pig growth. However, the mechanisms by which arginine and glutamic acid ameliorates VT-induced intestinal injury in pigs still remains unclear. This project will focus on the mechanisms through which arginine and glutamic acid relievesthe injurious effects in intestinal pig epithelial cells following VT exposure. Response criteria to be measured includes, cell morphological changes, cell proliferation and apoptosis, cell protein synthesis, cell tight junction proteins, expression of amino acids transporters, and NF-κB, p38 MAPKs and mTOR signaling pathway or inhibition before or after downstream cytokine expression, using phosphoproteomic methods, bioinformatics analysis and experimental verification to investigate the protective mechanisms for arginine and glutamic acid regulation of VT-induced intestinal porcine epithelial cells damage on the system level. It is anticipated that our results will provide a novel theoretical basis for exploring intestinal damage related to nutrition and possible control technologies, including the adjustment of feed formulation for pigs exposed to mycotoxins contamination, thus improving pig performance.

呕吐毒素广泛污染饲料，是制约猪生长的主要问题，能引起猪肠道上皮细胞形态结构与功能的变化，造成细胞凋亡。精氨酸与谷氨酸是应激条件下猪必需的功能性氨基酸，可促进猪肠上皮细胞蛋白质合成而调控细胞存活。我们前期的动物试验表明，精氨酸与谷氨酸能减轻呕吐毒素对猪肠道的损伤且促进猪的生长。但目前精氨酸与谷氨酸对呕吐毒素所致猪肠道损伤的保护机制尚不清楚。据此，本项目拟以猪肠上皮细胞为模型，分析精氨酸与谷氨酸对呕吐毒素所致猪肠上皮细胞损伤前后细胞的形态结构、增殖与凋亡、细胞内外蛋白质合成、紧密连接蛋白与氨基酸转运载体表达、NF-κB、p38 MAPKs与mTOR通路调控其下游细胞因子表达的影响，并通过磷酸化蛋白质组学、生物信息学分析与试验验证来深入揭示精氨酸与谷氨酸对呕吐毒素所致猪肠上皮细胞损伤的保护机制，为探索猪肠道霉菌毒素损伤的营养调控技术与饲料配方调整技术提供理论依据，对提高猪的生产性能具有重要意义。

呕吐毒素(DON)污染饲料，是影响猪生长的主要问题，能引起猪肠道细胞凋亡。精氨酸等能促进猪肠道细胞蛋白质合成而调控细胞存活。本项目完成以下三个试验：(1)1%精氨酸对断奶仔猪DON损伤的保护作用.1%精氨酸组的血液生化指标，空肠形态，血清、空肠与回肠游离氨基酸浓度均提升。营养素转运载体SGLT-1，GLUT-2和y+LAT-1基因表达水平在6mg/kg DON组下降，但在6mg/kg DON + 1%精氨酸组表达水平却上升。表明精氨酸能保护仔猪肠道健康减轻由日粮中DON带来的损伤;(2)不同浓度DON对肥育猪生长性能，血清生化指标，空肠形态与营养素转运载体的影响.试验发现12mg/kg DON与对照组相比，日增重和肝脏相对器官重有较大差异。在三个DON组中，血清生化BUN,ALP,ALT和AST指标升高，而血清游离氨基酸Valine,Glycine,Serine,Glutamine水平下降、绒毛高度均变短与杯状细胞数量均升高、EAAC-3，SGLT-1，PepT-1，CAT-1，LAT-1营养素载体基因表达水平均升高。表明DON能诱导剂量增加的毒性作用;(3)高浓度(200ng/mL)与低浓度(2000ng/mL)DON作用IPEC-J2细胞24小时内凋亡、肠道屏障功能、营养素转运载体、游离氨基酸，以及线粒体合成与功能基因表达水平的影响.低浓度DON能显著抑制细胞活率和促进细胞从G2/M期到S期。肠道屏障功能ZO-1、OCLN、CLDN1基因表达显著降低。高低浓度DON能显著降低营养素转运载体B0,+AT、SGLT1基因表达水平，而显著提升ASCT2和GLUT2基因表达水平。高低浓度DON能导致线粒体生物合成与功能TFAM、NRF、mt SSB、mt polr、ND4、CcOX、Cyt c、SIRT-1、CS基因表达水平显著升高。表明高低浓度DON能影响细胞增殖、促进细胞周期从G2/M期到S期、调控肠道屏障功能、营养素转运载体、线粒体生物合成与功能基因的表达水平。上述结果可为探索猪肠道DON损伤的营养调控与配方调整提供理论依据，对提高猪生产性能具有重要意义。在项目的资助下共计发表SCI文章12篇(其中第一标注论文7篇)，中文核心期刊2篇，中文会议壁报论文1篇，申报中国专利1项和参加学术会议6次。