Toll样受体4在糖尿病性胃部痛觉过敏中的作用及其表达调控机制研究

Upper abdominal pain or discomfort, one of the cardinal symptoms of patients with long-term diabetes, has been a challenger for gastroenterologists to treat effectively. Several lines of evidence suggest that the discomfort and pain of gastroparesis is associated with altered function of visceral afferent pathways. We have recently reported that promoted interaction of nuclear factor-κB (NF-κB) with gene of endogenous hydrogen sulfide (H2S) producing enzyme cystathionine β- synthetase (CBS) played an important role in gastric hypersensitivity. However, the precise mechanism about NF-κB activation in gastric-specific primary sensory neurons remains unknown. In this proposal, we hypothesize that (1) Diabetic gastric hypersensitivity is due to an upregulated toll like receptor 4 (TLR4) which resulted in activation of NF-κB signaling pathway and an increased excitability of gastric-specific primary sensory neurons; (2) The epigenetic regulation of TLR4 plays a crucial role in diabetic gastric hypersensitivity. The specific aims to test these hypotheses are: Aim 1: To determine the role of TLR4 in gastric hypersensitivity in diabetic rats; Aim 2: To investigate the mechanisms of TLR4 upregulation by examing miRNA181b in gastric-specific primary sensory neurons in rats with diabetes. EMG(electromyogram) will be used to record gastric distension- induced painful behavior in normal and diabetic rats. DiI will be used to label gastric-specific DRG neurons. Patch clamp recordings will be performed in vitro on single gastric-specific primary sensory neurons acutely isolated from these rats. Expression of key nociceptive gene (i.e., TLR4, NF-κB, miRNA181b) will be examined with immunocytochemistry, western blotting and (or) RT-PCR analyses. We expect that our studies may shed light on epigenetic mechanisms of chronic visceral pain in organic gastrointestinal disorders. This added knowledge would provide new strategies for treatment of chronic visceral pain in patients with diabetes.

糖尿病患者多合并胃部痛觉过敏，其机制不明，临床治疗手段有限。前期研究显示糖尿病大鼠胃特异性脊髓背根节（T7-10 DRG）中NF-κB激活，通过上调CBS-H2S系统，导致糖尿病胃部痛觉过敏。但有关NF-κB激活的机制不清。近期研究显示：糖尿病大鼠T7-10DRG神经元中TLR4表达增加、应用TLR4抑制剂后NF-κB表达下调、可能参与调控TLR4表达的miRNA181b表达下降。由此，我们提出如下假设：高糖环境下，miRNA181水平降低，促进了TLR4的表达，后者通过特定信号通路激活NF-κB，增加T7-10DRG神经元兴奋性，介导产生糖尿病性胃部痛觉过敏。我们将分别应用TLR4抑制剂及miRNA181b过表达慢病毒进行研究，探讨：1.TLR4在糖尿病胃部痛觉过敏中的作用；2. TLR4表达变化的表观调控机制。预期通过本研究初步揭示糖尿病胃部痛觉过敏的形成机理，为治疗提供理论依据。

糖尿病患者多合并胃部痛觉过敏，其机制不明，临床治疗手段有限，且疗效欠佳。前期研究显示糖尿病大鼠胃特异性脊髓背根节（T7-10 DRG）中NF-κB激活，通过上调CBS-H2S系统，导致糖尿病胃部痛觉过敏。但有关NF-κB激活的机制不清。近期研究显示：糖尿病大鼠T7-10DRG神经元中TLR4表达增加、应用TLR4抑制剂后NF-κB表达下调、可能参与调控TLR4表达的miRNA181b表达下降。由此，我们提出如下假设：高糖环境下，miRNA181水平降低，促进了TLR4的表达，后者通过特定信号通路激活NF-κB，增加T7-10DRG神经元兴奋性，介导产生糖尿病性胃部痛觉过敏。我们分别探讨了下列两个研究内容：1.TLR4在糖尿病胃部痛觉过敏中的作用；2. TLR4表达变化的表观调控机制。实验方法包括：给予雌性成年SD大鼠腹腔注射STZ（65mg/kg）诱导糖尿病，空腹血糖>15.5mmol/L，持续两周以上为诱导成功。应用胃内球囊扩张诱发的后颈肌肌电图变化来反映大鼠的胃部痛觉敏感性。应用全细胞膜片钳技术测定大鼠胃特异性DRG神经元兴奋性；应用Western Blot及RT-PCR检测DRG中痛关键基因（TLR4、NF-kB、TRAF6等）的表达；应用双荧光素酶报告基因技术测定相关miRNA对TLR4表达的调控作用。研究结果显示：1.给予 NF-κB p65siRNA慢病毒或者TLR4受体拮抗剂CLI-095鞘内注射均能明显缓解糖尿病大鼠胃部痛觉过敏；2. 给予 NF-κB p65siRNA慢病毒或者TLR4受体拮抗剂CLI-095鞘内注射均能降低糖尿病大鼠T7-10DRG神经元的兴奋性；3. 糖尿病大鼠 T7-10DRG中TLR4，p65及TRAF6蛋白表达明显升高，鞘内注射CLI-095能够降低p65及TRAF6的蛋白表达；4.通过生物信息学软件筛选出最有可能参与调控TLR4表达的miRNA，包括miRNA7a，218，23a，181a等，应用RT-PCR技术检测发现上述miRAN在糖尿病T7-10DRG中表达明显降低。通过双荧光素酶报告基因实验，发现miRNA218，23a，181a对TLR4的表达具有调控作用。预期通过本研究初步揭示糖尿病胃部痛觉过敏的形成机理，为治疗提供理论依据。