Mechanism of Enteric Neuropathy

 描述（由申请人提供）：高脂肪饮食摄入，特别是糖尿病患者，可能导致便秘相关问题，这是门诊胃肠病就诊的主要原因。我们的实验室专注于糖尿病等疾病中胃肠动力的变化，最近还专注于高脂肪饮食的影响。肠神经变性可导致肠动力障碍，见于慢传输型便秘、先天性巨结肠和糖尿病。本实验室建立了高脂饮食（HFD-34.5%千卡脂肪）诱导的肠神经退行性变小鼠模型。我们的初步数据显示，与常规饮食（RD）的小鼠相比，喂食HFD的小鼠具有延迟的胃肠运动、肠神经节中TLR 4表达增加以及过量的循环和粪便脂多糖（LPS）。此外，饱和脂肪酸如棕榈酸酯与中枢神经元毒性有关。我们假设，HFD喂养小鼠肠道菌群的改变与高棕榈酸盐水平的结合可导致TLR 4信号传导增加，从而驱动结肠运动障碍。为了研究HFD诱导的胃肠道运动延迟的潜在机制和微生物群的作用，我们将给常规（正常和糖尿病）和无菌小鼠喂食HFD 12周，并评估胃肠道运动和肠神经系统的变化。我们将评估特定的神经调节细菌代谢产物，如GABA，组氨酸和谷氨酰胺的饮食诱导的变化。我们将进行粪便移植实验，以确定HFD诱导的结肠运动性变化是否可以通过微生物群转移。最后，通过用益生元修饰肠道细菌，我们将尝试改善HFD诱导的内毒素血症和延迟的胃肠动力。我们的数据表明，棕榈酸酯增加TLR 4的表达和诱导肠神经元凋亡。使用原代肠神经元和肠神经元细胞系，我们将研究TLR 4介导的Myd 88依赖的信号转导通路在棕榈酸诱导的神经元凋亡中的作用。初步数据表明，HFD诱导TLR 4表达及其下游靶点JNK 1（MAPK 8）和C/EBP？。使用肠神经嵴特异性TLR 4和Myd 88敲除模型，我们将评估TLR 4在介导HFD诱导的运动性变化中的必要性和充分性。HFD对TLR 4表达的相互作用和涉及的信号转导将被剖析，重点是JNK 1和C/EBP信号通路的作用。我们发现，与RD喂养的小鼠相比，HFD以TLR 4依赖的方式诱导肠神经节中促凋亡miRNA miR 375的增加。我们将通过与TLR 4-/-小鼠联合评估HFD-在体内和棕榈酸酯在体外对肠神经元的作用来确定TLR 4在调节miR 375中的作用。为了了解HFD诱导的微生物变化对miR 375的影响，我们将评估喂食HFD的无菌小鼠和喂食或不喂食益生元的小鼠中的miR 375表达。了解HFD诱导的运动障碍和神经元丢失的机制可以帮助我们确定治疗延迟胃肠动力的新靶点。

 DESCRIPTION (provided by applicant): High fat diet intake, particularly in diabetic patients, can lead to problems related to constipation, a leading cause for outpatient gastroenterological visits. Our laboratory focuses on the changes in gastrointestinal motility in diseases such as diabetes and recently on the effects of a diet high in fat. Enteric neurodegeneration can lead to intestinal dysmotility seen in slow-transit constipation, Hirschsprung's disease and diabetes. We have established a high-fat diet (HFD-34.5% kcal from fat) induced enteric neurodegeneration mouse model in our laboratory. Our preliminary data shows that mice fed a HFD have delayed gastrointestinal motility, increase TLR4 expression in enteric ganglia and excess circulating and stool lipopolysaccharide (LPS) compared to mice on a regular diet (RD). Furthermore, saturated fatty acids such as palmitate have been implicated in central neuronal toxicity. We hypothesize that altered gut microbiota in HFD fed mice in conjunction with the high palmitate levels can result in increased TLR4 signaling and consequently, drives the colonic dysmotility. To investigate the underlying mechanism(s) and the role of microbiota in HFD-induced delayed gastrointestinal motility we will feed conventional (normal and diabetic) and germ free mice a HFD for 12 weeks and assess changes in gastrointestinal motility and the enteric nervous system. We will assess the changes induced by diet of specific neuromodulatory bacterial metabolites such as GABA, histidine and glutamine. We will perform fecal transplantation experiments to determine if the HFD induced colonic motility changes can be transferred through microbiota. Finally, by modifying the intestinal bacteria with prebiotics we will attempt t ameliorate HFD induced endotoxemia and delayed gastrointestinal motility. Our data indicate that palmitate increases TLR4 expression and induces enteric neuronal apoptosis. Using primary enteric neurons and an enteric neuronal cell line, we will examine the role of TLR4-mediated Myd88 dependent signal transduction pathways in palmitate-induced neuronal apoptosis. Preliminary data indicate that HFD induces TLR4 expression and its downstream targets JNK1 (MAPK8) and C/EBP¿. Using enteric neural crest specific TLR4 and Myd88 knock out models we will assess the necessity and sufficiency of TLR4 in mediating HFD-induced changes in motility. The interaction of HFD on TLR4 expression and the signal transduction involved will be dissected focusing on the role of JNK1 and C/EBP pathway. We found that HFD induces an increase in the proapoptotic miRNA, miR375 in a TLR4 dependent fashion in enteric ganglia compared to RD fed mice. We will determine the role of TLR4 in regulation of miR375 by assessing the effects of HFD- in vivo and palmitate on enteric neurons in vitro in conjunction with TLR4-/- mice. To understand the effects of HFD-induced microbial changes on miR375 we will assess miR375 expression in germ free mice fed HFD and in mice fed with or without prebiotics. Understanding the mechanisms underlying HFD-induced dysmotility and neuronal loss can help us identify new targets to treat delayed gastrointestinal motility.