TRPA1/TRPV1介导的肠粘膜感觉信号传入在去盆腔神经支配结直肠动力适应性恢复中的作用机制

Injury to pelvic nerve(PN) from surgical and obstetrical trauma has long been cited as a cause for abnormal colorectal motility in humans, which is characterized by constipation,urgency,incomplete and difficult defecation. We studied the effects of PN denervation on colorectal motility using a rat model. Our results showed that transection of the PN causes an acute decrease in rectal motility and colonic transit.However, These changes normalize over time implicating a compensatory mechanism within the bowel itself.The mechanism underlying this adaptive recovery remains unclear. If we can elucidate the adaptive mechanisms of colorectal motility recovery after PN sever, we may find a way to facilitate this process..Recent neurobiological insights into the gut-brain crosstalk have revealed a complex, bidirectional communication system that ensures the proper maintenance of gastrointestinal homeostasis and digestion. Luminal signals do not influence afferent nerve terminals directly, but act through intermediate cells in the lamina propria including enterochromaffin(EC) cells. Recent study showed that the transient receptor potential (TRP) channels TRPA1 was highly expressed in EC cells, and that stimulation of TRPA1 by agonists evoked the release of 5-HT from EC cells. It is suggested that EC cells can act as sensors for detecting the chemical makeup of the luminal contents. Our preliminary studies indicated that TRPA1 and TRPV1 agonists augmented the recovery process while 5-HT3 and 5-HT4 receptor antagonists can attenuated the motility recovery following PN denervation. Taken together, these results implied that TRPA1/TRPV1 regulates mucosal sensory afferent through 5-HT release from EC cells is one of the compensatory pathway for colorectal motility recovery after PN sever. .We hypothesized that the expression of TRPA1 and TRPV1 are down-regulated in the colon and rectum,which in turn attenuates the 5-HT release from EC cells resulting in decreased colorectal motility following PN cut. Over time, their expression are restored to facilitate the adaptive recovery of colorectal motility. To verify this hypothesis, we will construct the PN denervation model in rats and TRPA1/TRPV1 knockout mice. We will investigate the sensory afferent mechanisms underlying TRPA1/TRPV1 activation of the EC cells to release 5-HT. We will also investigate the influence of TRPA1/TRPV1, 5-HT3 and 5-HT4 receptors upon the colorectal motility recovery. The objective of this project is to determine the adaptive mechanisms underlying colorectal motility recovery following PN denervation, and to provides new strategies for clinical treatment and rehabilitation of this dysfunction.

盆腔神经损伤导致结直肠动力紊乱的恢复是一临床难题。我们前期研究发现切断盆腔神经后大鼠结直肠动力存在适应性恢复趋势，瞬时受体电位通道TRPA1与TRPV1激动剂可促其恢复，而5-HT3和5-HT4受体拮抗剂减弱这一作用，但详细的调控机制不清。新近的研究发现TRPA1是肠嗜铬(EC)细胞的感受器分子，通过激活EC细胞释放5-HT调控肠动力。上述结果提示，TRPA1(TRPV1?)介导EC细胞释放5-HT的肠粘膜感觉信号传入是调控肠动力恢复的有效途径之一。本项目采用免疫荧光、酶免疫检测、器官培养及siRNA等技术，从细胞、组织、器官层面初步阐明TRPA1/TRPV1介导的肠粘膜感觉信号传入机制。建立大鼠及TRPA1/TRPV1基因敲除小鼠去盆腔神经支配模型，进一步探讨TRPA1/TRPV1介导的肠粘膜感觉信号传入在去盆腔神经支配结直肠动力恢复中的调控作用，为临床治疗和康复研究提供新思路和新靶点。

本课题以盆腔神经损伤导致结直肠动力紊乱存在适应性的恢复，其机制是肠粘膜TRPA1表达上调介导EC细胞释放更多的5-HT，激活肠壁内神经丛调控肠动力代偿恢复。主要方法和结果：（1）成功建立去盆腔神经支配动物模型。（2）通过放射性同位素51Cr几何中心分布法检测模型大鼠结肠传输功能发现术后第1天结肠传输功能减弱，随着时间推移结肠传输功能逐渐恢复。（3）证明TRPA1与TRPV1在肠EC细胞上表达。（4）通过qPCR、免疫组化和Western Blot检测发现去盆腔神经支配模型大鼠远端结肠粘膜感觉信号分子（TRPA1、TRPV1、5-HT3R和5-HT4R）术后第1天表达显著下降，随着时间推移出现表达逐渐上调的趋势。（5）成功建立去盆腔神经支配小鼠模型。（6）通过盲肠埋管注射美兰检测发现模型小鼠术后第1天结肠传输功能显著减弱，随着时间推移结肠传输功能逐渐恢复。（7）通过直肠敏感性试验发现模型小鼠术后第1天直肠内脏敏感性显著下降，随着时间推移直肠内脏敏感性逐渐恢复。（8）建立TRPA1-/-小鼠去盆腔神经支配模型，完成了结肠传输试验和直肠敏感性试验，发现术后随时间推移的恢复趋势消失。研究结果表明：TRPA1/TRPV1介导的肠粘膜感觉信号传入在去盆腔神经支配结直肠动力恢复中的调控作用。