Mechanism of Diabetic Enteric Neuropathy

描述(申请人提供)：糖尿病(DM)是一种常见的疾病，在美国有1550万患者(1)。2007年，糖尿病引起的并发症使美国医疗系统损失了580亿美元(1)。糖尿病的一个主要并发症是神经病变。便秘是肠道神经病变的一个关键后果，在糖尿病患者中更为常见，其患病率为60%，而普通人群的患病率为15%。糖尿病相关肠神经病变是由高血糖诱导的神经细胞凋亡引起的。然而，高血糖导致糖尿病神经细胞凋亡的机制仍然是个谜。鉴于Toll样受体(Toll-like Receptor，TLR)在推动多种慢性疾病过程中的作用日益受到重视，尤其是与氧化应激和晚期糖基化产物(AGEs)相关的过程，我们假设Toll样受体在糖尿病相关肠神经病变中发挥作用。为了支持这一观点，我们观察到一种特定的TLR，即TLR4的上调与高血糖诱导的神经元凋亡有关。此外，基因和药物消融TLR4可防止高血糖诱导的神经细胞凋亡和DM相关的结肠运动障碍。因此，我们假设高血糖诱导的TLR4激活导致神经细胞凋亡，从而驱动与DM相关的结肠运动障碍。为了验证这一假说，并进一步研究高血糖诱导神经细胞凋亡的潜在机制(S)，我们将进行实验，以确定TLR4在高血糖诱导的肠神经细胞凋亡中的作用并阐明相关机制。利用WT/TLR4-/-小鼠的原代肠神经细胞和我们实验室建立的肠神经细胞系中的下调/过表达策略，我们将确定TLR4在调节高血糖诱导的肠神经细胞损伤中的必要性和充分性。高血糖诱导TLR4激活的机制将集中在晚期糖基化终产物(AGEs)和氧化应激的作用上。进一步，我们将围绕包括核因子-B在内的TLR4下游靶点，研究TLR4在高血糖诱导的肠神经细胞凋亡中所涉及的信号转导事件。最后，我们将在活体内确定TLR4在肠神经细胞凋亡和结肠动力障碍中的作用。我们的初步数据表明，链脲佐菌素(STZ)诱导的DM导致小鼠结肠肌间神经细胞的丢失和肠神经细胞的凋亡。这些变化在TLR4-/-DM小鼠中得到改善。综上所述，这些研究不仅将阐明TLR4在高血糖诱导的肠神经细胞凋亡发病机制中的作用，而且还将为可用于治疗或预防糖尿病相关运动障碍的靶向治疗提供“原则证据”。公共卫生相关性：糖尿病胃肠道并发症的病因和发病机制在很大程度上是未知的，因此这种虚弱的疾病的治疗对临床医生来说是一个挑战。在许多患者中，糖尿病病程与糖尿病自主神经病变的发展以及糖尿病胃肠道并发症(如糖尿病便秘)的存在相关。在这项建议中，我们将研究高血糖如何导致肠神经元丢失的机制。这项提案中概述的实验不仅将有助于了解糖尿病患者肠道神经元的变化，还可能为糖尿病患者胃肠动力改变带来新的治疗靶点。

DESCRIPTION (provided by applicant): DM mellitus (DM) is a common disorder with a prevalence of 15.5 million sufferers in the United States (1). In 2007, complications arising from DM cost the US healthcare system $58 billion (1). One major complication of DM is neuropathies. Constipation is a key consequence of enteric neuropathy and is significantly more common in DM patients with a prevalence of 60% compared to 15% in the general population. DM-associated enteric neuropathy results from hyperglycemia-induced neuronal apoptosis. However, the mechanism by which hyperglycemia drives neuronal apoptosis in DM remains enigmatic. In light of the increasing appreciation of the role of toll-like receptors (TLR) in driving a variety of chronic disease processes, especially those associated with oxidative stress and advanced glycation products (AGEs), which is known to result from hyperglycemia, we hypothesized that TLRs play a role in DM-associated enteric neuropathy. In support of this notion, we have observed that upregulation of one particular TLR, namely TLR4 is associated with hyperglycemia-induced neuronal apoptosis. Moreover, genetic and pharmacological ablation of TLR4 prevents both hyperglycemia-induced neuronal apoptosis and DM-associated colonic dysmotility. Thus, we hypothesize that hyperglycemia-induced activation of TLR4 results in neuronal apoptosis and, consequently, drives the colonic dysmotility associated with DM. To test the hypothesis and further investigate the underlying mechanism(s) of hyperglycemia-induced neuronal apoptosis, we will perform experiments to establish the role of TLR4 in mediating hyperglycemia-induced enteric neuronal apoptosis and elucidate the mechanism involved. Using primary enteric neurons from WT/TLR4-/- mice and knock down/overexpression strategies in the enteric neuronal cell line developed in our laboratory, we will establish the necessity and sufficiency of TLR4 in modulating hyperglycemia-induced enteric neuronal damage. The mechanism of hyperglycemia induced TLR4 activation will be examined focusing on the role of Advanced Glycation end products (AGES) and oxidative stress. Further, we will characterize the signaling events involved in hyperglycemia-induced TLR4 activation in enteric neuronal apoptosis focusing on the downstream targets of TLR4 including NF-:B. Finally, we will determine the role of TLR4 on enteric neuronal apoptosis and colonic dysmotility in vivo. Our preliminary data indicate that streptozotocin (STZ) -induced DM results in enteric neuronal apoptosis and loss of mouse colonic myenteric neurons. These changes are ameliorated in TLR4-/- DM mice. Taken together these studies will not only elucidate a novel mechanism involving TLR4 in the pathogenesis of hyperglycemia-induced enteric neuronal apoptosis but also provide "proof of principle" for targeted therapies that could be used for the treatment or prevention of dysmotility associated with DM. PUBLIC HEALTH RELEVANCE: The etiology and pathogenesis of the gastrointestinal complications of diabetes is largely unknown and thus treatment of this debilitating condition is a challenge to clinicians. In many patients the duration of diabetes correlates with the development of diabetic autonomic neuropathy as well as the presence of diabetic gastrointestinal complications such as diabetic constipation. In this proposal we will examine the mechanism of how hyperglycemia leads to loss of enteric neurons. Experiments outlined in this proposal will not only contribute to the understanding of the changes in enteric neurons in diabetes, but may also lead to new therapeutic targets for the altered gastrointestinal motility seen in diabetes.