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表明，12/15-脂氧合酶（12/15-LO） 1)有助于细胞信号传导和炎症反应受损；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。本研究的目的是通过1型和2型糖尿病动物模型、高糖暴露的小鼠雪旺细胞和DRG神经元共培养以及人类雪旺细胞（HSC）培养来评估12/15-LO在周围型糖尿病神经病变（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，在糖尿病患者中其表达和活性增加；2) 12/15-LO-/-小鼠发生PDN的严重程度低于野生型小鼠；3)短期给予12/15-LO抑制剂可逆转stz -糖尿病小鼠PDN的部分表现；4) 12/15-LO过表达在HSC短期（24小时）暴露于高糖环境后出现；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）磷酸化。具体目的是：1)评价两种结构不相关的12/15-LO抑制剂对1型和2型糖尿病小鼠模型（即stz型糖尿病小鼠和高脂饮食小鼠）PDN功能、生化和结构指标的影响；2)比较12/15-LO-/-小鼠与野生型1型和2型糖尿病小鼠PDN的严重程度；3)评估PDN最重要的“上游”机制，即醛糖还原酶活性增加在12/15-LO上调中的作用；4)在上述动物和细胞培养模型中检测12/15-LO对周围神经、脊髓和DRG神经元中MAPK激活的贡献。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（HPLC、Western blotting、荧光酶测定）和分子（实时PCR、siRNA转染）方法。该研究结果将为12/15-LO在1型和2型糖尿病PDN中的作用提供新的信息，并可能为开发12/15-LO抑制剂以预防和治疗其提供理论依据。

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.