酪蛋白糖肽调控脂质代谢及相关慢性炎症反应的效应与作用机制

Obesity has become the leading metabolic disease in both developed and developing countries during recent years. A pathologic increase in the accumulation of fat are its main characteristics at cellular level. Food components with the ability to suppress intracellular lipid accumulation may be beneficial to body weight control and prevention of obesity. Casein glycomacropeptide (GMP) is the C-terminal amino acid fragment of κ-casein released by chymosin. It has been shown that GMP could inhibit gastric acid secretion in calves. It also has been shown to be associated with reduced fat mass in rats. It can stand the further degradation of chymosin and pepsin, but it can not resist the further action of trypsin. Our previous study shows that compared with GMP, the degradation fragment of GMP (which is named casein glycopeptides) possessed higher inhibition activity to proliferation and differentiation of SD preadipocytes. However, the regualtion pattern, mechanism and the active sites of casein glycopeptides need further investigation. Therefore, this study will solve the following issues. The effect of casein glycopeptide on the fat metabolism and inflammation of 3T3-L1 preadipocytes and obese rats will be investigated. The inhibitory pattern and mechanism of casein glycopeptide on fat deposition and related chronic inflammation will also be explored. Endocellular lipid content, free glycerol, GPDH activity, fat metabolism-related enzymes and adipocytokines will be determined. The mRNA and expression of adipocyte differentiation transcriptional factors (PPARγ, C/EBPα, ADD-1/SREBP-1c), adipocytokines (leptin, adiponectin) and inflammatory cytokines (TNF-α, IL-6, MCP-1, CRP, IL-10）will also be determined using RT-PCR and Western blot method. The structure-activity relationship of casein glycopeptides will also be explored. The aim of this study is to revail the inhibition mechanisms of casein glycopeptide on fat deposition and related chronic inflammation on cellular and molecular level, which can provide scientific basis for the development of novel fat metabolism regulators based on bioavtive peptides.

从充分利用酪蛋白糖肽影响糖脂代谢的特性出发，采用3T3-L1前脂肪细胞和肥胖大鼠脂肪细胞研究其调节脂质代谢及相关炎症反应的作用模式和分子机制。研究酪蛋白糖肽对脂肪代谢关键酶活性、炎症因子分泌、巨噬细胞浸润等的影响；采用实时定量RT-PCR、蛋白质印迹和蛋白质组学技术研究其添加与否及糖链组成对脂肪细胞分化转录因子（PPARγ, C/EBPα, ADD-1/SREBP-1c）、脂肪细胞因子（脂联素等）和炎症因子（TNF-α、IL-6、MCP-1、CRP、IL-10）mRNA及蛋白表达水平的影响，鉴定差异蛋白的肽指纹图谱。研究其通过MAPK和Wnt/β-catenin以及IKKβ/NF-κB和JNK信号通路调控脂质代谢及炎症反应的作用。确定活性酪蛋白糖肽的结构特性，从细胞和分子水平揭示酪蛋白糖肽调节脂质代谢的生物学机制，为蛋白肽类物质降脂抗炎的基础研究和新型脂代谢调节因子的研发提供科学依据。

肥胖相关慢性炎症是胰岛素抵抗以及脂质代谢类疾病发病的关键因素，如何调控脂质代谢与慢性炎症成为治疗肥胖的重要靶点。酪蛋白糖巨肽（casein glycomacropeptide, GMP）是一种具有多种生理活性的C-末端亲水性糖肽，前期研究结果表明，GMP能够降低肥胖大鼠血脂水平，轻微改善大鼠机体的炎症反应状态，证明GMP具有缓解肥胖相关疾病的潜在效果。本课题利用蛋白酶解技术水解酪蛋白糖巨肽制备酪蛋白糖肽（GHP）并进一步分离纯化得到活性肽段，旨在探究酪蛋白糖肽调控脂质代谢及慢性炎症的效应及作用机制。. 首先以RAW264.7巨噬细胞为模型，评价了GHP对细胞氧化损伤和炎症反应的干预作用。结果表明，GHP通过活性氧（ROS）介导的细胞内核转录因子Nrf2和血红素氧合酶-1（HO-1）蛋白表达的激活，提高细胞内抗氧化酶水平，降低过氧化氢（H2O2）诱导细胞内ROS生成以及细胞凋亡水平。GHP能直接结合内毒素（LPS），抑制LPS结合巨噬细胞表面TLR4/MD2复合物，抑制 TLR4/MyD88/NF-κB信号通路的激活，发挥干预炎症反应的生物学活性。. 以HepG2细胞及高脂饮食小鼠模型探究了GHP对于肝脏胰岛素抵抗的效果及作用机制。结果表明，GHP降低了高脂饮食小鼠的体重增加、空腹葡萄糖及血清胰岛素水平，增加肝脏糖原含量，改善葡萄糖耐受，增加胰岛素敏感性，降低肝脏脂质积累和巨噬细胞浸润。GHP通过增加胰岛素抵抗HepG2肝细胞AMPK磷酸化，调控胰岛素受体底物（IRS-1）/蛋白激酶B（Akt）信号，促进糖原合成，增加葡萄糖的摄取能力，从而缓解胰岛素抵抗。. 最后，以3T3-L1脂肪细胞和高脂饮食小鼠模型探究了GHP抑制脂肪分化的效果及作用机制。结果表明，GHP能降低脂肪形成关键转录因子C/EBPα、PPARγ、FAS的表达，促进AMPK活化，激活Wnt/β-catenin信号通路，增加β-catenin的核移位，同时上调总β-catenin的蛋白表达量，进而抑制脂质积累。. 综上，本研究从动物及细胞分子水平证明了GHP通过缓解慢性炎症状态，调控脂质代谢紊乱，改善胰岛素抵抗，为GHP应用于预防和缓解肥胖及相关代谢性疾病奠定理论基础。