Identification of potential visceral pain biomarkers in colon exudates from mice with experimental colitis: an exploratory in vitro study

Chronic visceral pain (CVP) is extremely difficult to diagnose, and available analgesic treatment options are quite limited. Identifying the proteins secreted from the colonic nociceptors, or their neighbor cells within the tube walls, in the context of disorders that course with visceral pain, might be useful to decipher the mechanism involved in the establishment of CVP. Addressing this question in patients with gastrointestinal disorders entails multiple difficulties, as there is not a clear classification of disease severity, and colonic secretion is not easy to manage.We propose using of a murine model of colitis to identify new algesic molecules and pathways that could be explored as pain biomarkers or analgesia targets.Descending colons from naïve and colitis mice with visceral hyperalgesia were excised and maintained ex vivo. The proteins secreted in the perfusion fluid before and during acute noxious distension were evaluated using high-resolution mass spectrometry (MS). Haptoglobin (Hp), PZD and LIM domain protein 3 (Pdlim3), NADP-dependent malic enzyme (Me1), and Apolipoprotein A-I (Apoa1) were increased during visceral insult, whilst Triosephosphate isomerase (Tpi1), Glucose-6-phosphate isomerase (Gpi1), Alpha-enolase (Eno1), and Isoform 2 of Tropomyosin alpha-1 chain (Tpm1) were decreased.Most identified proteins have been described in the context of different chronic pain conditions and, according to gene ontology analysis, they are also involved in diverse biological processes of relevance.Thus, animal models that mimic human conditions in combination with unbiased omics approaches will ultimately help to identify new pathophysiological mechanisms underlying pain that might be useful in diagnosing and treating pain.

慢性内脏痛（Chronic visceral pain, CVP）诊断难度极高，现有镇痛治疗方案也十分有限。在伴内脏痛的疾病进程中，鉴定结肠伤害感受器及其肠管壁内邻近细胞分泌的蛋白质，或有助于阐明慢性内脏痛发生的潜在分子机制。在胃肠道疾病患者中开展此类研究存在诸多难题：一方面缺乏统一的疾病严重程度分级标准，另一方面结肠分泌物的采集与处理难度较大。为此，我们提出采用结肠炎小鼠模型，鉴定可作为疼痛生物标志物或镇痛靶点的新型致痛分子与信号通路。我们对未造模正常小鼠及伴内脏痛觉过敏的结肠炎小鼠的降结肠进行切除，并开展体外维持培养。采用高分辨率质谱（high-resolution mass spectrometry, MS）对急性有害扩张刺激前及刺激过程中灌注液内的分泌蛋白进行检测分析。结合珠蛋白（Haptoglobin, Hp）、PZD与LIM结构域蛋白3（LIM domain protein 3, Pdlim3）、NADP依赖性苹果酸酶（NADP-dependent malic enzyme, Me1）以及载脂蛋白A-I（Apolipoprotein A-I, Apoa1）在内脏伤害性刺激过程中表达上调；而磷酸丙糖异构酶（Triosephosphate isomerase, Tpi1）、葡萄糖-6-磷酸异构酶（Glucose-6-phosphate isomerase, Gpi1）、α-烯醇化酶（Alpha-enolase, Eno1）以及原肌球蛋白α-1链亚型2（Isoform 2 of Tropomyosin alpha-1 chain, Tpm1）的表达则出现下调。本次鉴定出的多数蛋白质已在多种慢性疼痛相关研究中被报道，且通过基因本体分析（gene ontology analysis）发现，这些蛋白质参与了多种与之相关的生物学过程。综上，模拟人类疾病状态的动物模型结合无偏倚组学策略，将有助于鉴定疼痛相关的新型病理生理机制，从而为疼痛的诊断与治疗提供新的思路。