12/15-Lipoxygenase and Diabetic Neuropathy

描述（由申请人提供）：花生四烯酸代谢在糖尿病并发症中的重要作用的证据正在出现。最近的体内和细胞培养研究，包括我们小组的研究，揭示了12/15-脂氧合酶（12/15-LO）1）有助于受损的细胞信号传导和炎症反应;和2）涉及与糖尿病相关的内皮功能障碍，肾病和视网膜病变的发病机制。本提案的目的是使用1型和2型糖尿病的动物模型以及小鼠雪旺细胞和DRG神经元的高糖暴露共培养物和人雪旺细胞（HSC）培养物来评估12/15-LO在周围糖尿病神经病变（PDN）中的作用。我们的初步数据表明：1）12/15-LO在周围神经中大量表达，并且其表达和活性在糖尿病条件下增加; 2）12/15-LO-/-小鼠比野生型小鼠发展更轻的PDN; 3）STZ-糖尿病小鼠中的PDN的一些表现被短期12/15-LO抑制剂治疗逆转; 4）在HSC短期（24小时）暴露于高糖后，12/15-LO过表达是明显的;和5）12/15-LO抑制剂治疗抵消HSC中高糖诱导的促分裂原活化蛋白激酶（MAPK）磷酸化。具体目的是：1）评价两种结构上不相关的12/15-LO抑制剂对1型和2型糖尿病小鼠模型中PDN的功能、生化和结构指数的影响，即，2）比较患有1型和2型糖尿病的12/15-LO-/-小鼠和野生型小鼠中PDN的严重程度; 3）评估PDN的最重要的“上游”机制，即增加的醛糖还原酶活性在12/15-LO上调中的作用，和4）检查12/15-LO对MAPK活化的贡献，在上述动物和细胞培养模型中的外周神经、脊髓和DRG神经元中。该项目将结合联合收割机的生理，行为，生物化学，免疫组织化学和结构的研究与生物化学（HPLC，蛋白质印迹，荧光分光光度酶测定）和分子（实时PCR，siRNA转染）的方法在细胞培养模型的动物。这些发现将产生关于12/15-LO在1型和2型糖尿病PDN中作用的新信息，并可能为开发12/15-LO抑制剂以预防和治疗糖尿病提供理论基础。

DESCRIPTION (provided by applicant): Evidence for the important role of arachidonic acid metabolism in diabetes complications is emerging. Recent in vivo and cell culture studies including those from our group revealed that 12/15-lipoxygenase (12/15-LO) 1) contributes to impaired cell signaling and inflammatory response; and 2) is implicated in the pathogenesis of endothelial dysfunction, nephropathy and retinopathy, associated with diabetes. The objective of this proposal is to evaluate the role of 12/15-LO in peripheral diabetic neuropathy (PDN) using animal models of Type 1 and Type 2 diabetes and high glucose-exposed co-cultures of mouse Schwann cells and DRG neurons and cultures of human Schwann cells (HSC). Our preliminary data indicate that 1) 12/15-LO is abundantly expressed in the peripheral nerve and its expression and activity increase in diabetic conditions; 2) 12/15-LO-/- mice develop less severe PDN than wild-type mice; 3) some manifestations of PDN in STZ-diabetic mice are reversed by a short-term 12/15-LO inhibitor treatment; 4) 12/15-LO overexpression is manifest after a short-term (24h) exposure of HSC to high glucose; and 5) a 12/15-LO inhibitor treatment counteracts high glucose-induced mitogen-activated protein kinase (MAPK) phosphorylation in HSC. The SPECIFIC AIMS are 1) evaluate two structurally unrelated 12/15-LO inhibitors on functional, biochemical and structural indices of PDN in mouse models of Type 1 and Type 2 diabetes, i.e., STZ-diabetic and high-fat diet fed mice; 2) compare severity of PDN in 12/15-LO-/- mice and wild-type mice with Type 1 and Type 2 diabetes; 3) assess the role for the most important, "upstream", mechanism of PDN, i.e. increased aldose reductase activity, in 12/15-LO upregulation, and 4) examine the contribution of 12/15-LO to MAPK activation, in peripheral nerve, spinal cord and DRG neurons in the afore-mentioned animal and cell culture models. The project will combine physiological, behavioral, biochemical, immunohistochemical and structural studies in animals with biochemical (HPLC, Western blotting, spectrofluorometric enzymatic assays) and molecular (real-time PCR, siRNA transfections) approaches in cell culture models. The findings will generate new information on the role for 12/15-LO in PDN of Type 1 and Type 2 diabetes, and may provide rationale for development of 12/15-LO inhibitors for its prevention and treatment.