Mechanism of Diabetic Enteric Neuropathy

描述（由申请人提供）：糖尿病（DM）是一种常见疾病，在美国有1550万患者患病（1）。2007年，糖尿病引起的并发症花费了美国医疗保健系统580亿美元（1）。DM的一个主要并发症是神经病变。便秘是肠神经病变的关键后果，在DM患者中明显更常见，患病率为60%，而在一般人群中为15%。糖尿病相关的肠神经病变是由高血糖诱导的神经元凋亡引起的。然而，糖尿病高血糖驱动神经细胞凋亡的机制仍然是个谜。鉴于Toll样受体（TLR）在驱动各种慢性疾病过程中的作用越来越受到重视，特别是与氧化应激和晚期糖基化产物（AGEs）相关的慢性疾病过程，这是已知的高血糖症的结果，我们假设TLR在DM相关的肠神经病变中发挥作用。为了支持这一观点，我们已经观察到一种特定TLR，即TLR 4的上调与高血糖诱导的神经元凋亡相关。此外，TLR 4的遗传和药理学消融可预防高血糖诱导的神经元凋亡和DM相关的结肠动力障碍。因此，我们假设高血糖诱导的TLR 4激活导致神经元凋亡，从而驱动与DM相关的结肠动力障碍。为了验证这一假设并进一步研究高血糖诱导的神经元凋亡的潜在机制，我们将进行实验以确定TLR 4在介导高血糖诱导的肠神经元凋亡中的作用并阐明所涉及的机制。利用WT/TLR 4-/-小鼠的原代肠神经元和本实验室开发的肠神经元细胞系中的敲低/过表达策略，我们将建立TLR 4在调节高血糖诱导的肠神经元损伤中的必要性和充分性。高血糖诱导的TLR 4激活的机制将集中于晚期糖基化终产物（AGES）和氧化应激的作用进行检查。此外，我们将表征高血糖诱导的TLR 4激活肠神经元凋亡中涉及的信号传导事件，重点关注TLR 4的下游靶点，包括NF-：B。最后，我们将确定TLR 4在体内肠神经元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导致肠神经元凋亡和小鼠结肠肌间神经元的损失。这些变化在TLR 4-/- DM小鼠中得到改善。总之，这些研究不仅将阐明一种新的机制，涉及TLR 4在高血糖诱导的肠神经元凋亡的发病机制，但也提供了“原则的证据”的靶向治疗，可用于治疗或预防与DM相关的运动障碍。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，因此治疗这种使人衰弱的疾病对临床医生来说是一个挑战。在许多患者中，糖尿病的持续时间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。在这个建议中，我们将研究高血糖症如何导致肠道神经元的损失的机制。本提案中概述的实验不仅有助于了解糖尿病肠道神经元的变化，而且还可能为糖尿病中观察到的胃肠动力改变提供新的治疗靶点。

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.