Piezo2/TRPA1介导的结肠EC细胞机械传感机制在SNS治疗慢性便秘中的作用

Chronic constipation is a common and distressing condition with no desire of defecation and difficulty in evacuation due to colorectal hyposensitiviey. Sacral nerve stimulation(SNS), regulating visceral sensitivity, is a promising treatment for chronic constipation with an unclear mechanism. Enterochromaffin (EC) cells are mechanosensors that respond to physical forces by translating the mechanical stimulus into an intracellular biochemical response leading to 5-HT release. Effect of SNS on EC cells mechanotransduction function is of great value for chronic constipation study. It has been proved that TRPA1 act as the sensor molecules of EC cells. Our previous study has revealed that the expression of TRPA1 mRNA and protein is significantly attenuated in the colon of patients with slow transit constipation. Recent studies have shown that the mechanosensitive ion channel Piezo2 is the primary mechanosensor in several specialized mechanosensory cells, including EC cells. Previous researches showed that Piezo2 alone appeared not sufficient to mediate the multitude of mechano-evoked phenomena in mechanotransduction. It might function as a low-threshold mechanical sensor in viscera physiologically. Based on the exploration of synergistic effect of Piezo2 and TRPA1 on the mechanotransduction of EC cells, we will use EC model cells, chronic constipation rat model and Piezo2/TRPA1 knockout mice to study the effect of SNS on the colonic sensitivity and motility that illuminate the role of SNS in the Piezo2/TRPA1 mediated EC cells mechanotransduction pathway, which promising a new perspective on the chronic constipation research.

结直肠低敏感是慢性便秘患者无便意、直肠坠胀而排便困难的主要原因。骶神经刺激（SNS）可调节内脏敏感性，是慢性便秘很有前景的治疗方法，但机制不清。肠嗜铬（EC）细胞是肠粘膜感受器，具有机械传感功能，可通过释放5-HT将机械信号转变为化学信号。研究SNS对EC细胞机械传感功能的影响对优化SNS治疗及慢性便秘的发病研究有重要意义。TRPA1是EC细胞的感受器分子，前期研究发现TRPA1在慢性便秘结肠的表达降低。Piezo2是新近发现的机械敏感性离子通道，也表达于EC细胞，与TRPA1负责不同阈值的机械刺激。本课题拟在探讨Piezo2与TRPA1协同调节EC细胞机械传感的基础上，以EC细胞模型、慢性便秘大鼠、Piezo2与 TRPA1基因敲除小鼠为对象，研究SNS对结肠敏感性、结肠动力的影响，明确Piezo2/TRPA1介导的EC细胞机械传感途径在SNS干预中的作用，可望开启慢性便秘研究的新视野。

慢传输型便秘（Slow transit constipation, STC）突出表现为无便意及结肠动力障碍。肠嗜铬细胞（Enterochromaffin Cells,EC）是肠黏膜的感受器，EC细胞上的感受器分子TRPA1、Piezo2感受肠腔内的机械或化学信号后释放5-HT，这一感觉信号传入机制在肠动力调控中发挥关键作用。本项目以临床病人标本、STC小鼠模型、基因敲除小鼠、EC细胞模型为研究对象，利用离体肠动力检测、Western Blot、免疫荧光双标等技术，研究Piezo2、TRPA1介导的结肠感觉信号传入机制在STC发病中的作用及机制。主要研究内容及结果：（1）证实了结肠EC细胞表达感受器分子Piezo2、TRPA1；（2）通过Western Blot发现STC结肠TRPA1、5-HT4R低表达，Piezo2高表达；（3）离体结肠动力监测发现TRPA1、5-HT4R抑制剂显著抑制结肠收缩；（4）在体药物干预实验发现TRPA1激动剂可明显改善STC小鼠排便，这种改善作用被5-HT4R抑制剂逆转；（5）通过Western Blot发现TRPA1基因敲除小鼠结肠Piezo2高表达，Piezo2抑制剂处理后小鼠结肠TRPA1高表达。以上结果提示：肠黏膜感受器分子Piezo2、TRPA1在结肠动力调控中发挥重要作用，5-HT4受体是EC细胞感觉信号传入的主要靶点。STC的发病与TRPA1、Piezo2介导的感觉信号传入障碍有关，二者在STC发病中可能存在相互代偿机制。