Supplementary Material for: Analysis of Neuropeptides in the Intestinal Mucus of Patients with Ulcerative Colitis Using RNA Sequencing

Introduction
Inflammation in ulcerative colitis (UC) originates in the colorectal mucosa. Transcriptome sequencing analysis of the colorectal mucosa allows the identification of potential neuropeptides related to local neurotransmission. The intestinal mucus lining the surface of the mucosa may harbor biomarkers of mucosal inflammation; however, this has not been sufficiently investigated, given the difficulty in obtaining human samples. We previously reported the feasibility of obtaining mucin samples for proteomic analysis by brushing during colonoscopy. Herein, we aimed to investigate the composition of the intestinal mucus and detect neuropeptides characteristic of UC.

Methods
Mucus and mucosal samples were collected from patients with UC from the colorectum in areas showing remission or active UC using a brush catheter and biopsy forceps during colonoscopy. RNA sequencing findings of mucus samples of active and remission areas were compared. RNA and protein expression levels of significantly upregulated neuropeptides were analyzed.

Results
Of the neuropeptides associated with UC, somatostatin (SST) was significantly elevated in areas of remission, according to RNA sequencing results of mucus and expression levels in mucus RNA and proteins. Conversely, SST expression in the mucosa was increased in the inflamed areas. Flow cytometry revealed that the fluorescence intensity of SST-positive cells in the remission zone was higher in the mucus than in the mucosa.

Conclusion
SST expression in the mucus is considered to be an important factor associated with UC activity.

引言
溃疡性结肠炎（ulcerative colitis, UC）的炎症始发于结直肠黏膜。对结直肠黏膜进行转录组测序分析，可识别与局部神经传递相关的潜在神经肽。覆盖于黏膜表面的肠黏液可能携带有黏膜炎症的生物标志物，但由于人类样本获取难度较大，该方向尚未得到充分研究。我们此前曾报道，在结肠镜检查中通过刷取方式获取黏蛋白样本用于蛋白质组分析的可行性。本研究旨在探究肠黏液的组成，并识别溃疡性结肠炎特征性神经肽。

方法
本研究在结肠镜检查中，通过刷拭导管和活检钳，从溃疡性结肠炎患者结直肠的缓解期或活动期病变区域采集黏液及黏膜样本。对比活动期与缓解期区域黏液样本的转录组测序结果，并对显著上调的神经肽的RNA及蛋白质表达水平进行分析。

结果
基于黏液样本的转录组测序结果及黏液中RNA与蛋白质的表达水平分析，在与溃疡性结肠炎相关的神经肽中，生长抑素（somatostatin, SST）在缓解期区域的表达量显著升高。与之相反，黏膜内的生长抑素表达在炎症区域出现升高。流式细胞术检测结果显示，缓解区域中生长抑素阳性细胞的荧光强度在黏液中高于黏膜组织。

结论
肠黏液中生长抑素的表达被认为是与溃疡性结肠炎活动度相关的重要影响因素。