Peroxynitrite decomposition catalyst:diabetic neuropathy

描述(申请人提供)：氧化/亚硝化应激是周围糖尿病神经病变(PDN)发病机制的主要因素，最近的数据表明，过氧亚硝酸盐(与一氧化氮反应的产物)是糖尿病神经中的主要氧化剂。传统的抗氧化剂分解过氧亚硝酸盐的能力很差，这至少部分解释了它们在临床神经病试验(如硫辛酸)中的低疗效。我们在两种1型糖尿病动物模型上获得的初步数据表明，有效和特异的过氧亚硝酸盐分解催化剂FP15在非常低的剂量下(每天3-5 mg/kg)可以抵消PDN的某些表现，P.O.这项建议的总体目标是使用研究良好的STZ-糖尿病大鼠模型和培养的内皮细胞和雪旺细胞，提供早期PDN中FP15的详细评估。其具体目的是1)评估FP15对STZ糖尿病大鼠周围神经氧化/亚硝化应激和聚腺苷二磷酸核糖聚合酶-1(PARP-1)激活的影响；2)评估FP15对STZ糖尿病大鼠的神经传导速度、神经血管功能、热和机械性痛觉以及关键代谢变量的影响。在动物实验中，我们将评估运动神经和感觉神经传导速度，热和机械痛觉，触觉异常痛感，坐骨神经内营养血流和神经内血管传导，神经外膜小动脉的血管反应性，代谢指标，如神经胞浆和线粒体NAD+/NADH氧化还原状态，能量状态，山梨醇途径中间体，以及氧化应激指标，如硝基酪氨酸，聚(ADPribose)，脂质过氧化产物，周围神经中的非酶类抗氧化剂和抗氧化性防御酶，以及血管中的超氧化物和硝基酪氨酸。在细胞培养实验中，我们将使用DCF荧光探针/流式细胞术来评估细胞内的氧化应激，并通过蛋白质印迹分析来评估硝基酪氨酸、PARP-1和聚(ADP-核糖)。这些发现将产生关于亚硝化应激在PDN发病机制中的作用的新信息，并可能为FP15的长期研究和进一步开发这种化合物治疗PDN提供理论基础。

DESCRIPTION (provided by applicant): Oxidative/nitrosative stress is a leading factor in the pathogenesis of peripheral diabetic neuropathy (PDN), and recent data suggest that peroxynitrite (a product of superoxide reaction with nitric oxide) is a major oxidant in diabetic nerve. Conventional antioxidants have a poor capacity to decompose peroxynitrite which at least partially explains their low efficacy in clinical neuropathy trials (e.g., lipoic acid). Our preliminary data obtained in two animal models of Type 1 diabetes indicate that the potent and specific peroxynitrite decomposition catalyst FP15 counteracts some manifestations of PDN at the very low doses of 3-5 mg/kg per day, p.o. THE OVERALL OBJECTIVE of this proposal is to provide detailed evaluation of FP15 in early PDN using the well-studied STZ-diabetic rat model and cultured endothelial and Schwann cells. The SPECIFIC AIMS are 1) to evaluate FP15 on oxidative/nitrosative stress and poly(ADP-ribose) polymerase-1 (PARP-1) activation in peripheral nerve in STZ-diabetic rats and high glucose-exposed human endothelial and Schwann cells; 2) to assess FP15 on nerve conduction velocity, neurovascular function, thermal and mechanical algesia and key metabolic variables in STZ-diabetic rats. In animal experiments, we will assess motor and sensory nerve conduction velocities, thermal and mechanical algesia, tactile allodynia, sciatic endoneurial nutritive blood flow and endoneurial vascular conductance, vascular reactivity of epineurial arterioles, metabolic indices such as nerve cytosolic and mitochondrial NAD+/NADH redox state, energy state, sorbitol pathway intermediates, and measures of oxidative stress such as nitrotyrosine, poly(ADPribose), lipid peroxidation products, non-enzymatic antioxidants and antioxidative defense enzymes in peripheral nerve, and superoxide and nitrotyrosine in vasa nervorum. In cell culture experiments we will assess intracellular oxidative stress using DCF fluorescent probe/flow cytometry, and nitrotyrosine, PARP-1 and poly(ADP-ribose) by western blot analysis. The findings will generate new information regarding the role for nitrosative stress in the pathogenesis of PDN and may provide rationale for long-term studies of FP15 and further development of this compound for treatment of PDN.