Attenuation of Renovascular Damage in Zucker Diabetic Fatty Rat by NWT-03, an Egg Protein Hydrolysate with ACE- and DPP4-Inhibitory Activity

BackgroundDipeptidyl peptidase 4 (DPP4) and angiotensin-converting enzyme (ACE) are important target enzymes in glycemic control and renovascular protection. Here, we studied the effect of NWT-03, an egg protein hydrolysate with DPP4- and ACE-inhibitory activity, on renovascular damage in Zucker diabetic fatty (ZDF) rats. Comparisons were made to rats treated with vildagliptin (VIL), included as a positive control for the effect of DPP4 inhibition.
MethodsZDF rats received NWT-03 (1 g/kg/day) or VIL (3 mg/kg/day) from 10 to 25 weeks of age. Metabolic and renal functions were assessed; the kidney was removed for histological analysis of glomerulosclerosis and expression of pro-inflammatory/fibrotic markers (RT-PCR and Western blotting); and the aorta was removed for studies of endothelium-dependent relaxation (EDR).
FindingsHyperinsulinemic ZDF rats typically developed signs of type-2 diabetes and renovascular damage, as evidenced by albuminuria, glomerulosclerosis, and impaired EDR. Neither NWT-03 nor VIL improved metabolic parameters; for VIL, this was despite a 5-fold increase in glucagon-like peptide (GLP)-1 levels. NWT-03 and VIL both reduced renal interleukin (Il)-1β/Il-13 mRNA expression and glomerulosclerosis. However, only NWT-03 additionally decreased renal tumor necrosis factor (TNF)-α mRNA and P22phox protein expression, reduced albuminuria, and restored aortic EDR. Indomethacin added to the organ bath instantly improved aortic EDR, indicating a role for cyclooxygenase (COX)-derived contractile prostanoids in opposing relaxation in ZDF rats. This indomethacin effect was reduced by NWT-03, but not by VIL, and coincided with decreased renal COX-1/2 protein expression.
Conclusion and InterpretationLong-term supplementation with the egg protein hydrolysate NWT-03 attenuated renovascular damage in this preclinical rat model of type 2 diabetes. A comparison to the DPP4-inhibitor VIL suggests that the effects of NWT-03 were related to both ACE- and DPP4-inhibitory properties. The development of protein hydrolysates with a multiple-targeting strategy may be of benefit to functional food formulations.

背景：二肽基肽酶4（Dipeptidyl peptidase 4, DPP4）与血管紧张素转换酶（angiotensin-converting enzyme, ACE）是血糖控制与肾血管保护中的重要靶酶。本研究探讨兼具DPP4与ACE抑制活性的鸡蛋蛋白水解物NWT-03对Zucker糖尿病肥胖（Zucker diabetic fatty, ZDF）大鼠肾血管损伤的影响，并以维格列汀（vildagliptin, VIL）作为DPP4抑制作用的阳性对照开展对比研究。
方法：将ZDF大鼠于10至25周龄期间予以NWT-03（1 g/kg/天）或维格列汀（3 mg/kg/天）干预。检测其代谢与肾功能；摘取肾脏用于肾小球硬化的组织学分析，以及促炎/纤维化标志物的表达检测（采用逆转录聚合酶链反应（RT-PCR）与蛋白质免疫印迹（Western blotting））；摘取主动脉开展内皮依赖性舒张功能（endothelium-dependent relaxation, EDR）相关研究。
结果：高胰岛素血症ZDF大鼠通常会出现2型糖尿病与肾血管损伤的相关体征，表现为白蛋白尿、肾小球硬化及内皮依赖性舒张功能受损。NWT-03与维格列汀均未改善代谢指标；即便维格列汀可使胰高血糖素样肽-1（glucagon-like peptide, GLP)-1水平升高5倍，仍未产生代谢改善效果。NWT-03与维格列汀均可降低肾脏白细胞介素（interleukin, IL)-1β/IL-13的mRNA表达水平，并减轻肾小球硬化。但仅NWT-03可进一步降低肾脏肿瘤坏死因子（tumor necrosis factor, TNF)-α的mRNA表达与P22phox蛋白表达，减少白蛋白尿，并恢复主动脉内皮依赖性舒张功能。向器官浴槽中加入吲哚美辛可即刻改善主动脉内皮依赖性舒张功能，提示环氧合酶（cyclooxygenase, COX）衍生的收缩性前列腺素类物质参与拮抗ZDF大鼠的血管舒张。NWT-03可削弱该吲哚美辛干预效应，而维格列汀无此作用，该现象与肾脏COX-1/2蛋白表达降低相一致。
结论与解读：在该2型糖尿病临床前大鼠模型中，长期补充鸡蛋蛋白水解物NWT-03可减轻肾血管损伤。与DPP4抑制剂维格列汀的对比结果提示，NWT-03的作用与其同时兼具ACE与DPP4抑制活性相关。采用多靶点策略开发蛋白水解物，或可为功能性食品配方的研发带来积极价值。