腸道神經內分泌物質對神經纖維延長和腸道痛覺異常的影響

Abstract

背景:腸躁症(Irritable bowel syndrome)是一種常見的功能性腸胃道失調疾病，特徵為長期反覆性腹痛和排便習慣的改變，卻無特定病因或病原菌的存在。近期研究發現，血清素（serotonin, 5-hydroxytryptamine, 5-HT）、膽囊收縮素(cholecystokinin;CCK)、神經生長因子(nerve growh factor;NGF)與腸道痛覺敏感有其相關性。然而，腸躁症與其內臟痛覺敏感的病理機制仍不明。因此，我們實驗室利用人類母細胞瘤SH-SY5Y細胞探討CCK是否導致神經纖維延長，並利用後感染及精神壓力之雙重刺激因子來建立腸道痛覺敏感之小鼠模式和評估血清素受器配體對內臟高敏感性和腸道功能的影響。 方法:細胞實驗部分，利用人類SH-SY5Y神經母細胞株加入視黃酸(retinoic acid;RA)促使分化後，給予硫酸化的CCK-8S (sulphated CCK octapeptide)培養，而後測量其神經纖維長度，觀察神經纖維生長變化。動物實驗部分，利用後感染及精神壓力雙重因子的小鼠，測試血清素受器配體對其內臟敏感與腸胃道蠕動狀況的影響。藉由內臟動器對結直腸撐張來評估腹痛敏感程度。 結果:神經母細胞經RA作用分化後給予CCK-8S作用，結果神經纖維生長為3倍。若預先給予中和性anti-NGF或 CCK-A 或CCK-B receptor inhibitors (AIH或BIH)均能顯著降低神經纖維的延長。透過西方轉漬法分析發現，經CCK-8S作用後的神經細胞，其NGF蛋白的表現量會提高。此外，血清素受器配體不論以腹腔注射（單次性）或經口給予（單次或連續性），皆可有效降低小鼠的內臟敏感性，但惟有連續性經口給予可以減緩腸胃道蠕動。 結論:本篇細胞實驗證實了，CCK-8S能促使神經纖維的延長，藉由結合至CCK受器以及增加NGF的表現來達成。此外，血清素受器配體能降低小鼠的內臟敏感性。因此推論，CCK和5-HT有參與腸道痛覺高敏感性之機制。

Background: Irritable bowel syndrome (IBS) is a functional gastrointestinal disorder characterized by chronic recurrent abdominal pain and altered bowel habits, in the absence of identifiable organic cause and macroscopic lesions. Recent findings indicate links between serotonin (5-hydroxytryptamine, 5-HT), cholecystokinin (CCK) and nerve growth factor (NGF) with intestinal hyperalgesia. However, the mechanism of visceral hypersensitivity in IBS remains unclear. The aim is to evaluate whether CCK may cause neurite outgrowth in human neuroblastoma SH-SY5Y cells and the effecst of 5-HT7 receptor ligand on visceral hypersensitivity and gut dysfunctions in a mouse model of dual triggers of post-infection and psychological stress. Method: SH-SY5Y cells were treated with retinoic acid (RA) to induce cell differentiation, and then cultured with sulphated CCK octapeptide (CCK-8S) for measurement of nerve fiber length. In some experiments, 5-HT7 receptor ligands were intraperitoneally administered to mice with dual triggers of post-infection and psychological stress. Abdominal pain was evaluated by visceromoter response to colorectal distension. Result: SH-SY5Y cells displayed 3-times increase of nerve fiber length after incubation with exogenously CCK-8S compared to control group. Moreover, pretreatment with neutralizing anti-NGF or with antagonists to CCK receptors decreased the nerve fiber length in cells incubated with CCK-8S. Western blots showed increased level of NGF in cells after culturing with CCK-8S. In addition, intraperitoneal (one dose) and peroral (one or multiple doses) administration of 5HT7 receptor ligands inhibited visceral hypersensitivity; however, only multiple peroral dose reduced intestinal motility. Conclusion: Exogenously CCK-8S induces neurite outgrowth via CCK receptors and induction of NGF synthesis. Moreover, 5HT7 receptor ligands inhibited visceral hypersensitivity. In sum, CCK and 5-HT are involved in the mechanisms of intestinal hyperalgesia.