COX-2 促进的TRPA1/TRPV1 基因调控区甲基化修饰在慢传输型便秘发病中的意义

Slow transit constipation (STC) is a common and distressing condition with unknown pathogenesis. The clinical menifestation is prominently no desire of defecation and infrequency of evacuation, which implies that sensory nerve regulation may play a crucial role in the pathogenesis of STC. It has been proved that TRPA1 and TRPV1 act as sensor molecules of the intestinal mucosa. Our previous study has revealed that the expression of TRPA1 and TRPV1 mRNA and protein is significantly attenuated, while the expression of cyclooxygenase (COX)-2 is upregulated in the colon of patientswith STC. Recent study suggested that the presence of a regulatory DNA methylation region in a CpG-island shore of theTRPA1 promoter have an impact on TRPA1 gene expression. Moreover, it has been reported that COX-2 could induce DNA methylation by inflammation processes such asoxidative stress and nitrogen species produced by inflammatory cells. Taken together, we speculate that the upregulation of COX-2 activity in the colon of patients with STC can induce gene methylation modification, which resulted in downreglation of TRPA1 and TRPV1 expression, causing on STC intestinal mucosa sensory signals obstacles. To verify this hypothesis, we will focus on STC patients, STC models of rats and COX-2 knockout mice. By using the immunofluorescence, enzymeimmune detection, quantitative specificity methylation and demethylation PCRtechniques, we are going to explore the role of COX-2 induced TRPA1/TRPV1 methylationmodification in the pathogenesis of STC.

慢传输型便秘（STC）发病机制不清，患者无便意、大便次数减少的临床特征提示结肠感觉功能障碍。瞬时受体电位通道TRPA1与TRPV1是肠粘膜感受器，我们前期的研究发现TRPA1与TRPV1在STC结肠的表达显著降低，而环氧化酶-2（COX-2）表达异常增高。新近的研究发现基因甲基化修饰是TRPA1表达下调的重要原因，而STC结肠的低度炎症可通过氧化应激等造成粘膜损伤促进基因甲基化，异常增高的COX-2可能加速这一过程。基于此，探讨COX-2 在TRPA1和TRPV1基因调控区甲基化修饰中的作用对研究STC的发病机制有重要意义。本项目拟以STC 患者、STC小鼠模型及COX-2基因敲除小鼠STC模型为研究对象，利用免疫荧光、基因表达、去甲基化、定量特异性甲基化PCR 等技术，从临床标本、整体动物模型、细胞等几个层面来验证假说，可望初步阐明TRPA1与TRPV1在STC发病中的作用机制。

本课题阐明了TRPA1/TRPV1基因启动子区甲基化修饰导致了慢传输型便秘（Slow transit constipationg, STC）结肠粘膜感觉信号传入机制障碍，其机制是TRPA1/TRPV1基因启动子区甲基化修饰干扰了TRPA1/TRPV1粘膜感受器的表达，导致 EC 细胞功能障碍、5-HT 释放异常，最终导致结肠感觉功能障碍、肠动力异常。主要方法和结果：1、通过免疫荧光双标观察发现TRPA1与TRPV1在结肠组织粘膜EC细胞上表达；2、通过免疫组化和Western Blot证明STC结肠粘膜TRPA1与TRPV1均表达下调；3、通过urogene网站预测TRPA1基因启动子区存在CpG岛；4、通过PCR方法扩增测序结果与UCSC网站查阅获取的启动子区序列进行比对，完全匹配；5、对TRPA1基因启动子区CpG岛甲基化程度进行测定，发现STC组TRPA1基因启动子区CpG岛处于高甲基化状态；6、通过免疫组化和Western Blot观察发现STC结肠粘膜Dnmt1、Dnmt3a与Dnmt3b甲基转移酶表达显著增高；7、分离提取EC细胞并成功建立STC小鼠模型，研究结果同前。研究结果表明：TRPA1基因启动子区甲基化修饰影响了慢传输型便秘肠粘膜感觉信号传入机制。