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表明，12/15-脂氧合酶(12/15-LO)1参与细胞信号转导和炎症反应受损；2)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有关。本研究的目的是探讨12/15-LO在糖尿病周围神经病变(PDN)中的作用。方法：采用1型和2型糖尿病动物模型及高糖暴露的小鼠雪旺细胞、背根神经节神经元和人雪旺细胞(HSC)共培养。我们的初步数据表明：1)12/15-LO在周围神经中大量表达，其表达和活性增加；2)12/15-LO-/-小鼠发生PDN不如野生型小鼠严重；3)STZ-糖尿病小鼠的PDN的一些表现可被短期12/15-LO抑制剂治疗逆转；4)12/15-LO在短期(24小时)高糖暴露后明显过度表达；5)12/15-LO抑制剂治疗可抵消高糖诱导的HSC中丝裂原活化蛋白激酶(MAPK)的磷酸化。其具体目的是1)评价两种结构无关的12/15-LO抑制剂对1型和2型糖尿病小鼠模型PDN功能、生化和结构指标的影响，即STZ-糖尿病和高脂饮食喂养的小鼠：2)比较12/15-LO-/-小鼠和野生型1型和2型糖尿病小鼠PDN的严重程度；3)研究PDN在12/15-LO上调中的作用，以及12/15-LO在上述动物和细胞培养模型中对周围神经、脊髓和背根节神经元MAPK激活的影响。该项目将结合动物的生理、行为、生化、免疫组织化学和结构研究，以及细胞培养模型中的生化方法(高效液相色谱、Western blotting、荧光分光光度酶分析)和分子方法(实时聚合酶链式反应、siRNA转基因)。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抑制剂预防和治疗糖尿病提供理论依据。

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.