Peroxynitrite decomposition catalyst:diabetic neuropathy

描述（由申请人提供）：氧化/亚硝化应激是糖尿病周围神经病变（PDN）发病的主要因素，最近的数据表明，过氧亚硝酸盐（与一氧化氮的超氧化物反应的产物）是糖尿病神经的主要氧化剂。传统抗氧化剂分解过氧亚硝酸盐的能力较差，这至少部分解释了它们在临床神经病变试验中的低疗效（例如硫辛酸）。我们在两种1型糖尿病动物模型中获得的初步数据表明，在3-5 mg/kg /天的极低剂量下，有效且特异性的过氧亚硝酸盐分解催化剂FP15可以抵消PDN的一些表现，p.o。本建议的总体目标是通过充分研究的stz -糖尿病大鼠模型和培养的内皮细胞和雪旺细胞，对FP15在早期PDN中的作用进行详细评估。具体目的是：1)评估FP15对stz -糖尿病大鼠和高糖暴露的人内皮细胞和雪旺细胞周围神经氧化/亚硝化应激和聚（adp -核糖）聚合酶-1 （PARP-1）激活的影响；2)评价FP15对stz -糖尿病大鼠神经传导速度、神经血管功能、热痛、机械痛及关键代谢指标的影响。在动物实验中，我们将评估运动和感觉神经传导速度、热痛觉和机械性痛觉、触觉异常性痛、坐骨神经内膜营养血流和神经内膜血管传导、神经外小动脉血管反应性、代谢指标，如神经细胞质和线粒体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、外周神经中的非酶抗氧化剂和抗氧化防御酶，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/流式细胞术评估细胞内氧化应激，并使用western blot分析硝基酪氨酸、PARP-1和聚adp核糖。这些发现将为亚硝化应激在PDN发病机制中的作用提供新的信息，并可能为FP15的长期研究和进一步开发该化合物治疗PDN提供理论依据。

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.