在兩個腸躁症動物模式上觀察腸道神經可塑性及內臟高敏感性

Abstract

研究背景：腸躁症 (Irritable bowel syndrome, IBS) 是症狀包括反覆性腹痛並伴隨排便習慣改變的功能性腸胃道疾病。IBS的危險因子包含精神壓力、遺傳基因、慢性發炎以及過去的腸道感染病史等。然而，關於IBS的致病機轉細節仍未明。過去臨床證據顯示：IBS患者的腸道血清素 (serotonin, 5-HT) 含量或代謝改變、結直腸黏膜的神經纖維 (nerve fiber) 密度較高。我們利用兩個內臟高敏感性 (visceral hypersensitivity) 的動物模式，研究腸道黏膜中的神經纖維延長 (neurite outgrowth) 的機制，及探討血清素是否參與其中機制。材料與方法：我們利用兩個動物模式：雙重因子的梨形蟲 (Giardia) 感染後排除期加上避水壓力 (water avoidance stress, WAS) 之後感染性腸躁症小鼠模式(簡稱GW小鼠)，以及化學劑三硝基苯磺酸(2,4,6-trinitrobenzene sulfonic acid)誘導腸炎後復原的後發炎性腸躁症小鼠模式(簡稱TNBS小鼠)。透過結直腸撐張 (colorectal distension, CRD) 引起的內臟動器反應 (visceromotor response, VMR) 來評估腸道的疼痛程度。以生長相關蛋白-43 (growth-associated protein 43, Gap-43) 的免疫螢光染色法觀察小鼠腸道組織內的神經纖維分布，並且RT-PCR 量測組織內的Gap-43 mRNA表現量。此外，我們製備小鼠腸組織無菌上清液或外加血清素來刺激人類神經瘤母細胞 (human neuroblastoma, SH-SY5Y cells) 以研究涉及腸道神經可塑性和神經纖維延長的生物活化調控因子。研究結果：在後感染壓力雙因子組(GW)及後發炎組(TNBS)的兩個動物模式中皆可看到內臟高敏感性；經口給予小鼠特定的血清素受體的配體 (CYY1005) 可有效降低其內臟敏感性；然而在雙重藥物給予下 (如CYY1005和Alosetron (臨床用腹瀉型腸躁症用藥，5-HT3R拮抗劑)、 CYY1005和Loperamide (臨床用止瀉劑，μ型鴉片受器促進劑)，對內臟高敏感性的減緩無加成作用。Gap-43在腸道黏膜中的mRNA表現量與疼痛程度有正相關增加。口服高量的CYY1005在14天內對小鼠無急毒性 (acute toxicity)。而在人類神經瘤母細胞中，經兩種動物腸道組織上清液或外加血清素的刺激之後，皆可以觀察到神經細胞纖維延長的結果；若給予長期口服CYY1005的兩種動物腸道組織上清液刺激，則不會造成神經細胞纖維延長。此外，血清素受器或膽囊收縮素受器之抑制劑預處理可以降低細胞實驗的神經纖維延長現象。結論：在兩個動物模式中皆可看到內臟高敏感性與黏膜神經纖維延長的結果。腸道黏膜中的調控因子血清素，可能參與影響神經可塑性。

Backgrounds: Irritable bowel syndrome (IBS) is a functional gastrointestinal disease, characterized by recurrent abdominal pain with bowel habit changes. Risk factors of IBS include psychological stress, hereditary factors, chronic inflammation, history of intestinal infection, etc. However, mechanistic details of IBS pathogenesis remain unclear. Accumulating evidence showed that abnormal levels or metabolism of intestinal serotonin (5-HT) and increased nerve fiber density in colorectal mucosal biopsies of IBS patients. Our aim is to investigat the mechanism of neurite outgrowth in intestinal mucosa of two mouse models of visceral hypersensitivity and the role of serotonin in neurite outgrowth. Materials and Methods: Two animal models were used, including mice that received dual trigger of post-giardiasis combined with water avoidance stress, and mice post resolution of chemical-induced colitis. Visceromotor response to colorectal distension was measured to quantify intestinal pain. Immunofluorescent staining of growth-associated protein 43 (Gap-43, a nerve fiber marker) was used to detect neurite distribution in mouse colonic tissues. The mRNA expression of Gap-43 in mouse colon was also measured by RT-PCR. The human neuroblastoma SH-SY5Y cells were stimulated by bacteria-free mouse colonic supernatants to investigate the bioactive mediators involved in neuroplasticity. Results: Visceral hypersensitivity was shown in mouse colon of post-infectious with stress and of post-inflammation. Oral administration of a serotonin receptor ligand (CYY1005) reduced the visceral hypersensitivity in the two IBS-like animal models. But with the use of double agents (CYY10005 and Alosetron (a 5-HT3R antagonist clinical used to treat IBS-diarrhea), or CYY1005 and Loperamide (a μ-opioid receptor agonist as anti-diarrheal agent)), there was no additive effects on the reduction of visceral pain. The mRNA levels of Gap-43 were also increased in the colonic mucosa of mice. Moreover, high dose CYY1005 did not cause acute toxicity in mice. In SH-SY5Y cells, incubation with colonic supernatant from IBS-like mouse models or with exogenous serotonin stimulated the increase of nerve fiber length; the addition of mouse colonic supernatant obtained from multiple dose CYY1005 groups did not cause neurite outgrowth. Lastly, pretreatment with receptor antagonists to serotonin or cholecystokinin blocked the neurite outgrowth in cell cultres. Conclusion: Visceral hypersensitivity and mucosal neurite outgrowth were found in two mouse models of post-infectious with stress and of post-inflammation. Gut-derived mediators such as serotonin may be responsible for neuroplasticity.