谷氨酰胺对大豆皂甙致大菱鲆肠道黏膜屏障损伤的保护作用及机制研究

The influence of soybean meal (SBM) on intestinal healthy status of fish, especially carnivorous fish, is the hotspot in the research of fishmeal replacement. It has been demonstrated that soya-saponins (SAP), a thermal stable anti-nutrient in SBM, could significantly reduce the intestinal healthy status of carnivorous fish by damaging the intestinal mucosal barrier. As an essential nutrient of intestinal mucosal enterocytes, glutamine (Gln) has the potential to protect the intestinal mucosal barrier from the damage caused by SAP. The present project is to work on the protection of Gln against SAP inducing intestinal mucosal barrier injury of turbot Scophthalmus maximu, one of the most important mariculture species in China. Intestinal histology, diamino oxidase activity and the D-lactate level will be used to evaluate the protective effects of Gln on the intestinal mucosal barrier injury induced by SAP. Furthermore, in the presence of SAP, the effects of Gln on the proliferation and differentiation of intestinal mucosal cells, on the immunity and antioxidant capacity of intestinal mucosa will be investigated by testing the proliferating cell nuclear antigen (PCNA) protein using immunohistochemical technology (IHC) and by enzyme-linked immunoabsorbent assay (ELISA), enzyme and biochemical analysis, respectively. High-throughput RNA sequencing technology will be applied to analyze the differences of gene expression profiles of intestinal mucosa induced by SAP and combination of Gln and SAP to screen the key genes contribute to the effects of Gln. The present project will clarify the mechanisms of the protective effects of Gln on the intestinal mucosal barrier injury of turbot Scophthalmus maximu induced by SAP and provide the theoretical basis on the safe use of SBM and the healthy cultivation of carnivorous marine fish.

豆粕对肉食性鱼类肠道健康的影响是鱼粉替代研究中的热点。研究表明，豆粕中的抗营养因子大豆皂甙主要通过损伤肠道黏膜屏障引起鱼类肠道健康水平的下降。谷氨酰胺（Gln）作为肠道黏膜细胞代谢必需的营养物质，具有保护鱼类肠道黏膜屏障的潜力。本项目拟以我国重要的海水养殖鱼类大菱鲆作为研究对象，通过肠道黏膜屏障组织结构观察、二氨氧化酶（DAO）活性和D-乳酸水平检测，评估Gln对大豆皂甙致大菱鲆肠道黏膜屏障损伤的保护作用。采用IHC方法检测大豆皂甙以及添加Gln后对肠道黏膜增殖细胞核抗原蛋白分布和表达量的影响；采用IHC、ELISA、酶学及生物化学方法检测其对肠道黏膜免疫力和抗氧化力的影响；采用转录组高通量测序技术筛选出Gln缓解大豆皂甙损伤肠道黏膜屏障的关键功能基因。从而阐明Gln对大豆皂甙致大菱鲆肠道黏膜屏障损伤的保护机制。本项目能够为豆粕在水产饲料中的安全使用以及肉食性海水鱼的健康养殖提供理论依据。

本项目以我国重要的海水养殖鱼类大菱鲆作为研究对象，通过肠道黏膜组织光镜结构、电镜结构观察，血清二氨氧化酶（DAO）活性及D-乳酸水平检测，发现谷氨酰胺对大豆皂甙引起的大菱鲆肠道黏膜屏障损伤具有保护作用。本研究采用免疫组织化学、酶联免疫吸附法、酶学及生物化学等方法检测发现谷氨酰胺对大菱鲆肠道溶菌酶活力、IgM水平、补体C3水平、补体C4水平、超氧化物歧化酶活力、过氧化氢酶活力、谷胱甘肽过氧化物活力等指标具有提高作用，说明谷氨酰胺对大菱鲆肠道黏膜免疫力和抗氧化力具有保护作用。本项目采用实时定量PCR的方法研究表明谷氨酰胺具有降低肠道炎性细胞因子IL-1β、IL-8、TNF-α、TGF-β的表达量水平的作用。本项目采用转录组高通量测序技术研究发现谷氨酰胺能够改善大菱鲆肠道菌群结构，增加有益菌的丰度。本研究的结果阐明了谷氨酰胺对大豆皂甙致大菱鲆肠道黏膜屏障损伤的保护机制。能够为豆粕在水产饲料中的安全使用以及肉食性海水鱼的健康养殖提供了理论依据。