Supplementary Material for: Regulation of the MCHergic Neural Circuit to Dorsal Raphe Nucleus on Emotion-Related Behaviors and Intestinal Dysfunction in Mice Model of Irritable Bowel Syndrome with Diarrhea

Abstract Introduction: Irritable bowel syndrome with diarrhea (IBS-D) is frequently accompanied by depression and anxiety, resulting in a reduced quality of life and increased medical expenditures. Although psychological factors are known to play an important role in the genesis and development of IBS-D, an understanding of the central neural control of intestinal dysfunction remains elusive. Melanin-concentrating hormone (MCH) is a gut–brain peptide involved in regulating feeding, sleep–wake rhythms, and emotional states. Methods: This study investigated the regulation of the MCHergic neural circuit from the lateral hypothalamic area (LHA) to the dorsal raphe nucleus (DRN) on anxiety- and depression-like behaviors, intestinal motility, and visceral hypersensitivity in a mice model of IBS-D. The models of IBS-D were prepared by inducing chronic unpredictable mild stress (CUMS). Results: Chemogenetic activation of the MCH neurons in the LHA could excite serotonin (5-HT) neurons in the DRN and induce anxiety- and depression-like behaviors and IBS-D-like symptoms, which could be recovered by microinjection of the MCH receptor antagonist SNAP94847 into the DRN. The mice model of IBS-D showed a reduction of 5-HT and brain-derived neurotrophic factor (BDNF) expression in the DRN, while an elevation of 5-HT and BDNF was observed in the colon through immunofluorescent staining, ELISA, and western blot analysis. SNAP94847 treatment in the DRN alleviated anxiety- and depression-like behaviors, improved intestinal motility, and alleviated visceral hypersensitivity responses by normalizing the 5-HT and BDNF expression in the DRN and colon. Conclusion: This study suggests that the activation of MCH neurons in the LHA may induce IBS-D symptoms via the DRN and that the MCH receptor antagonist could potentially have therapeutic effects.

摘要与引言：腹泻型肠易激综合征（IBS-D）常伴随抑郁与焦虑症状，可导致患者生活质量下降及医疗支出增加。尽管已知心理因素在腹泻型肠易激综合征（IBS-D）的发生与发展中发挥重要作用，但目前对于肠功能紊乱的中枢神经调控机制仍不甚明确。黑色素聚集激素（MCH）是一种参与调控摄食、睡眠-觉醒节律以及情绪状态的肠-脑肽。
方法：本研究以腹泻型肠易激综合征（IBS-D）小鼠模型为研究对象，探究了从外侧下丘脑区（LHA）至中缝背核（DRN）的黑色素聚集激素能神经环路对抑郁样、焦虑样行为、肠动力及内脏敏感性的调控作用。腹泻型肠易激综合征模型通过慢性不可预见性温和应激（CUMS）诱导构建。
结果：化学遗传学激活外侧下丘脑区的黑色素聚集激素神经元，可激活中缝背核内的5-羟色胺（5-HT）神经元，并诱发抑郁样、焦虑样行为及腹泻型肠易激综合征样症状；向中缝背核显微注射黑色素聚集激素受体拮抗剂SNAP94847可逆转上述异常。免疫荧光染色、酶联免疫吸附实验（ELISA）及蛋白质印迹（western blot）分析结果显示，腹泻型肠易激综合征小鼠模型的中缝背核内5-羟色胺与脑源性神经营养因子（BDNF）的表达水平降低，而结肠组织中二者的表达水平则显著升高。向中缝背核给予SNAP94847干预，可通过使中缝背核与结肠内的5-羟色胺及脑源性神经营养因子表达恢复至正常水平，从而改善抑郁样、焦虑样行为，调节肠动力，并缓解内脏高敏感性反应。
结论：本研究表明，外侧下丘脑区的黑色素聚集激素神经元激活可通过中缝背核诱发腹泻型肠易激综合征样症状，而黑色素聚集激素受体拮抗剂或具有潜在的临床治疗价值。