Peroxynitrite, protein nitration and advanced diabetic neuropathy

描述（由申请人提供）：过氧亚硝酸盐（与一氧化氮的超氧化物反应产物）在糖尿病并发症中的重要作用的证据正在出现。利用新型药物（过氧亚硝酸盐分解催化剂和蛋白质硝化抑制剂）和inos敲除小鼠，我们获得了过氧亚硝酸盐损伤toto和蛋白质硝化在早期实验性糖尿病周围神经病变（PDN）中的相关发现。我们发现1)硝基酪氨酸（NT）是过氧亚硝酸盐损伤的足迹，在链脲佐菌素(STZ)-糖尿病和ob/ob小鼠的周围神经、脊髓和DRG神经元中积累；2)与糖尿病野生型小鼠相比，stz -糖尿病inos缺陷小鼠未出现神经传导缺损，感觉神经病变程度较轻；3)过氧亚硝酸盐分解催化剂，在较小程度上，蛋白质硝化抑制剂，纠正1型和2型糖尿病小鼠的神经传导缺陷和感觉神经病变。我们还发现，2型糖尿病患者在外周血单核细胞中积累了不同数量的硝化蛋白，2型糖尿病患者的单核细胞NT浓度比非糖尿病组高75%，吡格列酮治疗可减少皮肤中一氧化氮（过氧亚硝酸盐的前体）的产生。其他研究表明，血浆NT含量增加与糖尿病患者内皮功能障碍和压迫反应的再分配相关，这是交感神经功能障碍的早期迹象。本研究的总体目标是分析过氧亚硝酸盐和蛋白质硝化在晚期实验性PDN的功能和形态学变化中的作用，并确定血清、外周血单核细胞和皮肤中的NT水平是否可以作为糖尿病患者PDN存在、严重程度和进展的生物标志物。具体目的是：1)评价过氧亚硝酸盐分解催化剂和蛋白质硝化抑制剂是否能逆转STZ-糖尿病小鼠和秋田小鼠晚期PDN的功能、行为和形态学变化；2)评估血糖控制对PDN、皮肤和循环组织部位NT积累和消失的影响；3)确定血清、外周血单核细胞和皮肤NT水平作为糖尿病患者PDN存在、严重程度和进展的生物标志物的潜在价值。这些发现将促进我们对PDN发病机制的理解，并可能为开发新的治疗方法提供理论依据。它们也可能导致PDN具有诊断和预后价值的新生物标志物的鉴定。

DESCRIPTION (provided by applicant): Evidence for important role of peroxynitrite, a product of superoxide reaction with nitric oxide, in diabetic complications is emerging. Using new pharmacological agents i.e., peroxynitrite decomposition catalysts and protein nitration inhibitor, and iNOS-knockout mice, we obtained findings implicating peroxynitrite injury in toto and protein nitration in early experimental peripheral diabetic neuropathy (PDN). We found that 1) nitro- tyrosine (NT), a footprint of peroxynitrite injury, accumulates in peripheral nerve, spinal cord, and DRG neurons of streptozotocin (STZ)-diabetic and ob/ob mice; 2) STZ-diabetic iNOS-deficient mice do not develop nerve conduction deficits and have less severe sensory neuropathy compared with diabetic wild-type mice; and 3) peroxynitrite decomposition catalysts and, to a lesser extent, a protein nitration inhibitor, corrected nerve conduction deficits and sensory neuropathy in mice with Type 1 and Type 2 diabetes. We also found that Type 2 diabetic patients accumulate different amounts of nitrated proteins in peripheral blood monocytes, that monocyte NT concentration is ~ 75% greater in Type 2 diabetic subjects compared with non-diabetic group, and that skin production of NO, a precursor of peroxynitrite, is reduced by pioglitazone treatment. Others showed that increased plasma NT content correlated with endothelial dysfunction and redistribution of sudomotor responses, an early sign of sympathetic nerve dysfunction, in diabetic patients. The overall objective of this proposal is to dissect the roles of peroxynitrite and protein nitration in functional and morphological changes of advanced experimental PDN, and to determine if NT levels in serum, peripheral blood monocytes, and skin can be used as biomarkers of the presence, severity, and progression of PDN in human subjects with diabetes. The specific aims are: 1) evaluate if peroxynitrite decomposition catalyst and protein nitration inhibitor reverse functional, behavioral, and morphological changes of advanced PDN in STZ- diabetic and Akita mice; 2) assess the effect of blood glucose control on accumulation and disappearance of NT in tissue-sites of PDN, skin, and circulation; and 3) determine potential values of serum, peripheral blood monocyte, and skin NT levels as biomarkers of the presence, severity, and progression of PDN in human subjects with diabetes mellitus. The findings will advance our understanding of the pathogenesis of PDN, and may provide rationale for development of new therapeutics. They may also lead to identification of a new biomarker(s) of PDN with diagnostic and prognostic value. PUBLIC HEALTH RELEVANCE: Peripheral diabetic neuropathy (PDN) is the most devastating complication of diabetes mellitus, and a leading cause of foot amputation. Evidence for important role of peroxynitrite (a product of superoxide reaction with nitric oxide) in PDN is emerging and is supported by our preliminary data obtained in both diabetic mouse models and human subjects with diabetes mellitus. The overall objective of this proposal is to dissect the roles of peroxynitrite and one of its components, protein nitration, in functional and morphological changes of advanced experimental PDN, and to determine if nitrotyrosine levels in serum, peripheral blood monocytes, and skin can be used as biomarkers of the presence, severity, development and rate of progression of PDN in human subjects with diabetes. The specific aims are: 1) evaluate if peroxynitrite decomposition catalyst or protein nitration inhibitor reverse functional, behavioral, and morphological manifestations of advanced PDN in two mouse models of Type 1 diabetes; 2) assess the effect of blood glucose control on accumulation and disappearance of nitrotyrosine in tissue-sites of PDN, skin, and circulation; and 3) determine potential values of serum, peripheral blood monocyte, and skin nitrotyrosine levels as biomarkers of the presence, severity, development, and rate of progression of PDN in human subjects with diabetes mellitus. The findings will advance our understanding of the pathogenesis of PDN, and may provide rationale for development of new therapeutics. They may also lead to identification of a new biomarker(s) of PDN with diagnostic and prognostic value.