基于Keap1-Nrf2-ARE通路的海洋高F值寡肽中肽类激动剂的发现及作用机制研究

Oxidative stress (OS), initiated by superfluous reactive oxygen species (ROS), is the collective pathophysiological mechanism of many hepatopathies, nephropathy, cardiovascular and cerebrovascular diseases, and diabetes mellitus, which plays a significant role in the form and development of diseases on OS. Nuclear factor erythroid-2 related factor 2 (Nrf2) is an important nuclear transcription factor which protects cells against OS injury. Upon exposure to ROS or electrophilic stress, Nrf2 can separate from Keap1 and translocate into the nucleus, and then bind to the antioxidant response element (ARE), regulating the expression of antioxidant and phase II detoxifying enzymes which aim at the detoxification and elimination of harmful exogenous chemicals. Therefore, the Keap1-Nrf2-ARE pathway is extremely important in the progression of prevention and therapy of many diseases related to OS and has been implicated as a new therapeutic target for screening antioxidant drugs.. Oligopeptide is usually composed of 3 to 9 amino acids. High Fischer ratio oligopeptide is one kind of peptide mixture with a molar ratio of branched-chain amino acids (Leu, Ile and Val) to aromatic amino acids (Phe, Trp and Tyr) higher than 20. At present, high Fischer ratio oligopeptides have been reported to have multi-physiological functions, including protecting liver, antioxidation, fatigue resistance and anti-aging. Our previous research indicated that high Fischer ratio oligopeptide from skipjack tuna (Katsuwonus pelamis) could increase the expression level of Nrf2 gene in human embryonic kidney cell line HEK293 models, and two peptide agonists (IELVW and IIGLVP) were prepared from high Fischer ratio oligopeptides of skipjack tuna and Mustelus griseus. Therefore, the antioxidant function of high Fischer ratio oligopeptides might be related to the exciting of the Keapl-Nrf2-ARE pathway.. In our previous experiment, the hepatocyte HL7702 model and human embryonic kidney epithelial cell line Hek293 model of Keapl-Nrf2-ARE on ARE-luciferase reporter have been established. In the following research, these models will be used for screening the active peptides with exciting ability on Keap1-Nrf2-ARE pathway. As expected results, 60-80 peptide agonists, including 3-5 highly reactive agonists, will be isolated from the high Fischer ratio oligopeptides of tuna, squid and cartilaginous fish muscles, and their structures will be elucidated on the basis of extensive spectroscopic data, including IR, NMR, MS, SRCD and X-ray. The exciting abilities and action mechanism of the isolated peptide agonists will also be researched on the hepatocyte HL7702 model. In addition, the in vivo effects on protecting liver of peptide agonists will be researched using the acute and chronic liver injury model of mice. Based on the structure and activity information, the structure-activity relationship and exciting mechanism on Keap1-Nrf2-ARE pathway of peptide agonists will be illuminated, and the dependency of physiological function of marine high Fischer ratio oligopeptides with their peptide agonists will be clarified.. The expected achievements will provide 3-5 kinds of candidates for research and development of high-efficiency antioxidant drugs, which will also provide significant technical support for high-value utilization of marine biological resources.

氧化应激是肝病、肾病等发病的共同病理生理基础，Keapl-Nrf2-ARE通路是迄今发现的最重要的内源性抗氧化应激通路。因此，筛选Keap1-Nrf2-ARE通路的高效激动剂成为海洋抗氧化药物的研究热点。课题组研究发现海洋高F值寡肽具有显著的抗氧化和保肝护肝等生理活性，可促进Nrf2蛋白的表达，并制备了Keap1-Nrf2-ARE通路肽类激动剂IELVW和IIGLVP。基于此，本课题以Keap1-Nrf2-ARE通路抗氧化药物筛选模型为导向，从海洋高F值寡肽中制备60～80种肽类激动剂，其中高活性激动剂3～5个，并利用HL7702细胞和小鼠肝损伤模型研究肽类激动剂体内外抗氧化应激的作用机制，阐明肽类激动剂激动Keap1-Nrf2-ARE通路的作用机制及其构效关系，剖析肽类激动剂与高F值寡肽生理活性之间的内在联系。本项目的研究将为海洋抗氧化新药研发提供候选药物，促进海洋生物资源的高值化利用。

氧化应激是肝病、肾病和糖尿病等发病的共同病理生理基础，Keapl-Nrf2-ARE通路是最重要的内源性抗氧化应激通路。因此，筛选Keap1-Nrf2-ARE通路的高效激动剂成为海洋抗氧化药物的研究热点。海洋高F值寡肽具有显著的抗氧化、保肝护肝、抗疲劳等活性。基于此，本课题完善了适合海洋肽类激动剂的Keapl-Nrf2-ARE 通路药物筛选模型；建立了海洋高F 值寡肽和Keapl-Nrf2-ARE 通路海洋肽类激动剂（Nrf2-MPA）的制备关键技术；利用带鱼、金枪鱼、鱿鱼、灰星鲨和南极磷虾等海洋生物资源制备了6种高F值寡肽，297个抗氧化肽和62 个Nrf2-MPA。构效关系研究表明分子量、疏水性氨基酸组成与比值、氨基酸侧链氨基、羧基和芳香基团对Nrf2-MPA的活性具有显著影响；抗氧化分子机制研究表明Nrf2-MPA通过激活Keap1-Nrf2-ARE通路，启动体内抗氧化系统，提高抗氧化酶，如超氧化物歧化酶（SOD）、过氧化氢酶（CAT）、谷胱甘肽过氧化物酶（GSH-Px）等的水平，清除过量活性氧（ROS），降低ROS对细胞生物大分子的损伤。体内作用机制研究表明：鳕鱼皮胶原肽可显著升高肝组织抗氧化酶SOD、GSH-Px、CAT水平，降低小鼠血清中谷丙转氨酶（ALT）、谷草转氨酶（AST）、碱性磷酸酶（ALP）、γ-谷氨酰转肽酶（γ-GT）和丙二醛（MDA）水平，有效抑制肝组织的脂质过氧化；通过下调P-JNK表达增加抑细胞凋亡基因Bcl-2蛋白表达量，降低促细胞凋亡基因Bax蛋白的表达量，减少Cytosolic Cyt c的释放而抑制Caspase 家族的活化，最终减少肿瘤坏死因子TNF-α的产生，达到保护肝细胞的效果；RVAPEEHPVEGRYLV通过激活Keapl-Nrf2-ARE通路增加肝脏中抗氧化酶的水平、降低ALT、AST、TG、MDA、IL-1β、IL-6和TNF-a水平，调控TLR4介导的NF-κB通路和细胞凋亡信号蛋白水平，显著减轻环磷酰胺（CTX）的肝毒性；高F值寡肽通过激活Keapl-Nrf2-ARE通路显著提高抗氧化酶和乳酸脱氢酶的活力，降低MDA、血清尿素氮和乳酸含量，增加小鼠酒精耐受度，缩短醒酒时间，增加糖原含量而延长力竭游泳时间，实现抗醉酒、醒酒、抗疲劳和抗氧化功能。本课题为海洋抗氧化新药研发提供候选药物，促进了海洋生物资源的高值化利用。