Transcriptional Profiling of Diabetic Neuropathy in BKS-db/db Mouse, a Model for Type 2 Diabetes. Mus musculus

A better understanding of the molecular mechanisms underlying the development and progression of diabetic neuropathy (DN) is essential for the design of mechanism-based therapies. We examined changes in global gene expression to define pathways regulated by diabetes in peripheral nerve. Microarray data for 24 week-old BKS db/db and db/+ mouse sciatic nerve were analyzed to define significantly differentially expressed genes (DEGs); DEGs were further used for functional enrichment analysis and network analysis to identify biological processes and pathways differentially regulated in the db/db nerve. Expression profile clustering was performed to identify co-expressed DEGs. A set of co-expressed lipid metabolism genes was used for promoter sequence analysis.We identified 4,017 DEGs; 2,122 genes were up-regulated and 1,895 genes were down-regulated in the db/db relative to the db+ samples. Over-represented biological processes identified by the functional enrichment analysis include cell cycle, lipid metabolic process, lipid transport, carbohydrate metabolic process, response to stress, apoptosis, axonogenesis and cell adhesion. Pathways regulated in the db/db nerve include lipid metabolism, carbohydrate metabolism, energy metabolism, PPAR signaling, apoptosis, and axon guidance. The majority of DEGs in the glycolysis, TCA cycle, oxidative phosphorylation, fatty acid metabolism, glycerolipid metabolism, mitochondrial fatty acid elongation, lipid transport, adipocytokine signaling, PPAR signaling, and apoptosis pathways are up-regulated, whereas most of the axonogenesis-related genes are down-regulated in db/db nerve. A network of DEGs based on their co-citation in literature identified regulatory relationship between Tnf-α and key genes from the regulated pathways. Promoter sequence analysis identified over-represented transcription factor binding site (TFBS) motifs in the promoter regions of twenty two co-expressed lipid metabolism-related genes suggesting coordinated regulation of these genes by multiple transcription factors (TF). Furthermore, TF binding to these TFBS and differentially regulated in our data are annotated with nervous system development and immune response suggesting possible co-regulation of lipid metabolism, nervous system development and stress response genes. Gene expression changes in our data are consistent with pathological characteristics observed in DN including axon degeneration and demyelination, and support existing hypotheses regarding hyperglycemia mediated nerve damage in DN. Our findings support the role of hyperglycemia-induced oxidative stress and ischemia in nerve injury. Our results also support the hypothesis of oxidized lipid-mediated nerve injury and increased mitochondrial oxidative stress in dyslipidemia. Moreover, our analyses revealed a possible co-regulation mechanism connecting hyperlipidemia, stress response and axonal degeneration. Overall design: Microarray data for 24 week-old BKS db/db and db/+ mouse sciatic nerve were analyzed to define significantly differentially expressed genes

深入阐明糖尿病性神经病（diabetic neuropathy, DN）发生与进展的分子机制，是研发基于机制的治疗手段的核心前提。本研究通过检测全基因表达谱变化，以明确糖尿病在周围神经中调控的信号通路。我们分析了24周龄BKS背景db/db与db/+小鼠坐骨神经的微阵列数据，以筛选得到显著差异表达基因（differentially expressed genes, DEGs）；随后利用这些DEGs开展功能富集分析与分子网络分析，鉴定db/db小鼠坐骨神经中存在差异调控的生物学过程与信号通路。我们通过表达谱聚类鉴定共表达的DEGs，并选取一组共表达的脂质代谢基因进行启动子序列分析。

本研究共鉴定出4017个DEGs，相较于db/+对照样本，db/db小鼠坐骨神经中有2122个基因上调表达，1895个基因下调表达。功能富集分析显示，显著富集的生物学过程包括细胞周期、脂质代谢过程、脂质转运、碳水化合物代谢过程、应激反应、细胞凋亡、轴突发生与细胞黏附。db/db小鼠坐骨神经中差异调控的信号通路涵盖脂质代谢、碳水化合物代谢、能量代谢、过氧化物酶体增殖物激活受体（peroxisome proliferator-activated receptor, PPAR）信号通路、细胞凋亡以及轴突导向。在糖酵解、三羧酸循环、氧化磷酸化、脂肪酸代谢、甘油脂代谢、线粒体脂肪酸延伸、脂质转运、脂肪细胞因子信号通路、PPAR信号通路以及细胞凋亡通路中，绝大多数DEGs呈现上调趋势；而与轴突发生相关的基因在db/db小鼠坐骨神经中大多呈下调状态。

基于文献共引关系构建的DEGs分子网络，揭示了肿瘤坏死因子α（Tnf-α）与各调控通路关键基因之间的调控关联。启动子序列分析在22个共表达脂质代谢相关基因的启动子区域中，鉴定出显著富集的转录因子结合位点（transcription factor binding site, TFBS）基序，提示这些基因可被多种转录因子（transcription factor, TF）协同调控。进一步分析发现，能够结合上述TFBS且在本研究数据中存在差异表达的转录因子，其注释功能涉及神经系统发育与免疫反应，提示脂质代谢、神经系统发育与应激反应相关基因可能存在协同调控机制。

本研究中检测到的基因表达变化，与糖尿病性神经病中已观测到的病理特征（包括轴突变性与脱髓鞘）高度一致，同时支持了高血糖介导糖尿病性神经病神经损伤的现有学术假说。本研究结果证实了高血糖诱导的氧化应激与缺血在神经损伤中的关键作用。此外，本研究结果也支持了氧化脂质介导的神经损伤以及血脂异常中线粒体氧化应激升高的相关假说。此外，本研究的分析还揭示了一条潜在的协同调控机制，可连接高脂血症、应激反应与轴突退行性变。

总体实验设计：本研究通过分析24周龄BKS背景db/db与db/+小鼠坐骨神经的微阵列数据，以筛选得到显著差异表达基因。