TCF4调控DRG神经元Nav1.8表达影响慢性术后疼痛的机制研究

Chronic post-surgical pain (CPSP) impedes the patients’ recovery and decreases the quality of life after the surgery. Previous studies have demonstrated that it was effective to prevent and treat CPSP by inhibiting the transduction of peripheral electrical signals induced by noxious surgical stimulation. However, at present it remains unclear how to effectively inhibit it. In our previous study, we found that sodium channel Nav1.8 in DRG neurons played an important role in the transduction of peripheral electrical signals and could significantly affect human pain sensitivity. In addition, in our pilot study the up-regulation of Nav1.8 was found in the rat DRG neurons of CPSP model. And recently in another study we found that an enhancer named EnhA in SCN10A gene, which encoded Nav1.8, could regulate the expression of Nav1.8 and bias human pain. Furthermore, based on the ENCODE database and CHIP strategy we found that transcription factor 4 (TCF4) can interact with SCN10A, and what's interesting is that the binding site is right in EnhA. Based on the above information, we hypothesized that TCF4 can regulate the Nav1.8 expression of DRG neurons and thus can prevent CPSP. The current study aims to use dual luciferase experiment and electrophysiological experiments to validate the interaction effect between TCF4 and EnhA. Then transgenic mice are used for regulation of TCF4 expression, and a combined CPSP model is established. Thereafter, behavioral assessment, qPCR, western blot and electrophysiologic techniques are performed to explore the effect of TCF4 on Nav1.8 expression in DRG neurons and chronic post-surgical pain. In addition, TCF4 gene screening and clinical investigation for CPSP in cardiac surgery patients are used to investigate the association between TCF4 genotype and CPSP. The current study may clarify the effect of TCF4 in Nav1.8 expression and reveal its role in CPSP. And this may eventually provide a potential intervention target for CPSP in the future.

慢性术后疼痛影响手术患者康复，目前认为从外周阻断疼痛电信号传导有助于其防治，但如何有效干预仍有待研究。申请者前期研究发现外周背根神经节（DRG）神经元Nav1.8疼痛电信号传导功能减弱可降低人体疼痛程度，且Nav1.8表达上升参与慢性术后疼痛的发生，另外，Nav1.8编码基因SCN10A增强子EnhA可影响其表达活性，而TCF4与SCN10A存在结合作用且作用区域与EnhA重合。据此我们提出假设：TCF4可调控DRG神经元Nav1.8表达影响慢性术后疼痛的发生。本课题拟利用双荧光素酶实验等在细胞水平证实TCF4与EnhA的相互作用；利用沉默/过表达技术等在动物水平验证TCF4对DRG中Nav1.8表达及慢性术后疼痛的调控；并通过TCF4基因筛查结合临床调查探讨TCF4在人体慢性术后疼痛中的作用。本课题有望从新的角度解释Nav1.8表达及慢性术后疼痛调控的机制，并为其防治提供可能的干预策略。

慢性术后疼痛影响手术患者康复，目前认为从外周阻断疼痛电信号传导有助于其防治，但如何有效干预仍有待研究。课题组前期研究发现外周背根神经节（DRG）神经元Nav1.8疼痛电信号传导功能减弱可降低人体疼痛程度，且Nav1.8表达上升参与慢性术后疼痛的发生，另外，Nav1.8编码基因SCN10A增强子EnhA可影响其表达活性，而TCF4与SCN10A存在结合作用且作用区域与EnhA重合。据此我们提出假设：TCF4可调控DRG神经元Nav1.8表达影响慢性术后疼痛的发生。基于国家自然科学基金资助，（1）课题组利用来源人体的数据发现Nav1.8功能减弱的受试者疼痛敏感性显著高于其他受试者，利用双荧光素酶实验证实Nav1.8编码基因SCN10A增强子的作用决定了这种疼痛影响，该部分数据发表论文于《Molecular Pain》；（2）课题组利用双荧光素酶实验证实了TCF4与EnhA的相互作用，证实了TCF-4可以通过SCN10A增强子起作用进而影响SCN10A的表达；接着，课题组建立慢性术后疼痛主要的组成部分，炎性痛大鼠模型（CFA）和神经病理性疼痛模型（SNI），同时利用过表达技术等在动物水平验证了TCF4过表达可降低对DRG中Nav1.8表达，并降低疼痛程度；同时分别取野生型和TCF-4过表达的大鼠DRG神经元进行钙成像实验验证了TCF-4过表达对于DRG神经元电信号的影响，该部分研究数据发表论文《Experimental Neurology》；（3）因为本课题宗旨在于探讨Nav1.8抑制用于疼痛治疗的可能，其潜在心脏作用对于未来该类抑制剂用于临床意义关键，我们意外观察到Nav1.8可能影响心脏心率，于是本课题基于前期现有数据库和Nav1.8敲出动物，结合心率调控药物阿托品发现Nav1.8参与了心脏对阿托品的反应，该部分研究数据发表论文《Frontiers in Pharmacology》；基于上述研究，可帮助我们从新的角度理解Nav1.8在人体疼痛调控中的作用，并为相关镇痛策略提供参考。