植物性饲料中添加胆固醇促虹鳟生长调控机制的代谢组学研究

Our previous studies showed that dietary cholesterol supplementation in plant proteins-based diets generally improved the growth performance of fish (such as Japanese flounder Paralichthys olivaceus, rainbow trout Oncorhynchus mykiss), and the growth-promoting action might be related to the alleviation of the negative effects caused by antinutritional factors in fish feeds and the nutritional regulation of endogenous cholesterol in fish. The finished project, supported by the National Natural Science Foundation of China (30960298), has evaluated the effects of dietary cholesterol supplementation on the deleterious action of soybean antinutritional factors in rainbow trout diet and related mechanisms. However, the present project will focus particularly on the nutritional regulation mechanism of dietary cholesterol supplementation on rainbow trout fed plant-based diet. A 12-week feeding trial will be conducted to investigate the effects of dietary cholesterol supplementation (0.3%, 1.2%) on the serum, muscle and hepatic metabolites in rainbow trout fed plant-based diet (soybean meal- and soybean oil-based diets) via metabolomics technology based on nuclear magnetic resonance (NMR) and gas chromatography mass spectrometry (GC-MS). Metabolite fingerprint of serum, and hydrophilic and lipophilic extracts of fish liver and muscle samples will be obtained. Pattern recognition based on multivariate statistical analysis, such as principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA), will be used to detect differential metabolites, and their structures can be identified by NMR and MS spectral data, aided by comparison with metabolites database, standard spectrum and literature data. The ultimate objective of the present study is to find out the key metabolic enzymes and regulatory factors affecting the characteristic metabolites by using the known metabolic pathways, and thereby to illuminate the key metabolic pathways of the growth-promoting action of cholesterol in rainbow trout fed plant-based diet and its regulatory mechanisms (improving cholesterol metabolism, fatty acid metabolism, amino acid metabolism, energy metabolism, antioxidant capacity or other mechanisms ?). The results obtained in the course of the study are expected to have significant academic contributions and practical implications.

前期研究表明，植物性饲料中添加胆固醇对鱼类具有明显促生长效应，且该效应与胆固醇抑制植物蛋白源中抗营养因子的抗营养作用和胆固醇自身的营养调控作用有关。已结题国家自然科学基金(30960298)探讨了胆固醇对虹鳟饲料中抗营养因子的抑制效应及其机制；本项目拟采用核磁共振(NMR)和气质联用(GC-MS)代谢组学技术，比较分析植物性饲料(以豆粕/豆油为代表)中添加不同剂量胆固醇对虹鳟血清、肝脏和肌肉代谢产物的影响，获取代谢产物指纹图谱；采用模式识别技术对代谢组数据进行多元统计分析，筛选出差异性代谢产物，并经代谢物数据库检索、标准品比对和文献对照等进行指认；通过已知代谢通路逆推找出胆固醇促生长的关键代谢酶或调控因子，阐明植物性饲料中添加胆固醇促虹鳟生长的关键代谢通路及其调控机制(改善胆固醇代谢、脂肪酸代谢、氨基酸代谢、能量代谢、抗氧化能力或其他？)。预期研究成果将具有重要的理论价值和广泛的应用前景。

前期研究表明，植物性蛋白源高比例替代鱼粉可致鱼类低胆固醇血症，外源添加胆固醇具有明显促生长效应，但其作用机制尚不清楚。本项目研究了植物性饲料中添加胆固醇对虹鳟生长性能、生理表型和代谢表型的影响；动物性饲料中添加不同降血脂药物对虹鳟生长性能、生理表型和关键代谢酶的影响，以阐述植物性饲料中添加胆固醇促虹鳟生长的代谢调控机制。结果表明：⑴植物性饲料中添加0.3或1.2%胆固醇明显提高虹鳟生长率，影响生理和代谢表型；①激活Asp和Glu等NEAA分解代谢用于糖异生和氧化供能，抑制His、Val和Ile等EAA氧化分解，有利于机体蛋白质沉积；②激活胆汁酸和牛磺酸的生物合成，有利于饲料脂肪高效利用；③改善维生素D和钙磷利用，有利于机体骨骼发育；④改善血液胆固醇水平，有利于提高机体抗氧化能力和免疫力，从而提高虹鳟生长和饲料利用率；⑵动物性饲料中添加不同降血脂药物(依折麦布、烟酸、非诺贝特)降低了虹鳟生长和饲料利用率，且其降血胆固醇效应与其抑制生长效应密切相关；其中，①添加依折麦布抑制肠道胆固醇吸收，降低血液胆固醇水平；激活BCAA和Thr氧化分解，不利于机体蛋白质合成；抑制牛磺酸和脂肪生物合成，激活脂肪氧化分解，不利于机体脂肪沉积，从而抑制虹鳟生长和饲料利用率；②添加辛伐他汀对虹鳟生长和饲料利用率无明显影响；③添加烟酸降低虹鳟抗氧化能力；同时促进胆固醇合成和转运，而对血液胆固醇水平无明显影响；激活BCAA和Thr氧化分解，不利于机体蛋白质沉积；抑制牛磺酸和脂肪生物合成、脂肪氧化分解，不利于饲料脂肪利用，从而降低虹鳟生长和饲料利用率；④添加非诺贝特同时抑制胆固醇吸收和排出，而对血液胆固醇水平无明显影响；激活BCAA和Thr氧化分解，不利于机体蛋白质沉积，从而抑制虹鳟生长和饲料利用率。本项目为提高鱼类对豆粕等植物性蛋白源的利用提供了一条新的解决思路和途径，具有重要的理论价值和广泛的应用前景。