神經營養因子刺激神經纖維生長在腸道疼痛感覺中所扮演的角色

Abstract

背景：腸躁症(Irritable Bowel Syndrome, IBS)為功能性腸胃道疾病，亦即腸道無明顯的結構或生化異常，而患者卻出現慢性且反覆性的腹痛，以及排便型態改變的臨床症狀。腸躁症的危險因子包含精神壓力、腸道慢性發炎、腸道感染病史等。然而，多因素的腸躁症疾病其潛在的病生理機制並不清楚，其中在神經-內分泌網絡機制指出，內臟高敏感性與神經內分泌物質的作用有關，如血清素(Serotonin, 5-HT)、神經生長因子(Nerve Growth Factor, NGF)和腦源性神經營養因子(Brain-Derived Neurotrophic Factor, BDNF)。過去文獻指出腸躁症患者結直腸黏膜層檢體中NGF和BDNF表現量高，且有高密度的神經纖維分布，與腹痛程度有正相關性。本篇研究目的為利用化學性誘導結腸炎復原之類腸躁症動物模式探討血清素受器對於腸道痛覺的影響，以及利用人類神經細胞模式探討血清素與神經營養因子共同調控腸道神經纖維延長的機制。 方法：實驗室先前建立兩種類腸躁症動物模式，第一種是結合雙重誘導因子即感染後排除合併避水壓力試驗，稱為GW小鼠；第二種則是使用2, 4, 6-三硝基苯磺酸誘發結腸炎復原後，此組為後腸炎性類腸躁症模式，稱為PT小鼠。經口或腹腔注射給予小鼠血清素受器化合物，進行結直腸撐張刺激所引發的內臟動器反應，測定其疼痛程度，並在結束後給予活性碳試驗，評估腸道蠕動性。在小鼠組織中使用神經標誌 PGP9.5以及血清素受器進行免疫螢光染色。利用SH-SY5Y人類神經細胞株(human neuroblastoma)來探討血清素受器在神經纖維延長的效果與機制，將GW及PT小鼠的腸道組織製成無菌上清液或外加血清素及神經營養因子刺激SH-SY5Y神經細胞，觀察神經纖維延長和細胞中特定基因的表現量。最後，由大腸鏡切片取得腸躁症患者及健康者的結腸黏膜檢體，使用免疫螢光染色觀察神經纖維及血清素受器分布位置及表現。 結果：在正常Ctrl組小鼠中，經口給予特定的血清素受器配體(CYY1005)，發現其不會影響生理疼痛感覺；而在PT組小鼠中，則可以有效降低其內臟敏感性。此外，腹腔注射新合成之血清素受器化合物(CYY073、CYY1280-3、CYY1294)於PT組小鼠，只有CYY1280-3在高撐張刺激下能顯著性降低內臟高敏感性反應，且腸道蠕動性也有顯著降低。在免疫螢光染色結果發現GW及PT小鼠結腸黏膜組織之PGP9.5與血清素受器表現量較正常組高，而腸躁症患者降結腸檢體中之PGP9.5與血清素受器表現量比起健康者分布量亦較高。利用類腸躁症動物腸道組織上清液或外加血清素及神經營養因子刺激SH-SY5Y神經細胞後，能使神經細胞纖維延長，而若先給予血清素受器配體(CYY1005)或5-HT7R拮抗劑(SB7)預先處理，則出現抑制神經纖維的延長的效果。同時，以NGF或BDNF刺激SH-SY5Y神經細胞，會促使神經營養因子受器及血清素受器表現量增加。 結論：本篇實驗證實，血清素受器有參與類腸躁症動物模式小鼠中內臟高敏感性的反應。此外，血清素與神經營養因子共同調控神經纖維延長。

Background：Irritable bowel syndrome (IBS) is a functional gastrointestinal disorder, that meaning that there is no obvious structural or biochemical abnormality in the intestine. IBS patient have clinical symptoms of chronic and recurrent abdominal pain associated with bowel habit changes. Risk factors include psychological stress, chronic intestinal inflammation and history of intestinal infection etc. However, the underlying pathophysiological mechanism of multifactorial intestinal disease is unclear. In the neuro-endocrine network, visceral hypersensitivity is related to the action of neuroendocrine substances such as serotonin, brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF). Previous study showed increased NGF and BDNF levels and high density of nerve fiber distribution in colon mucosa of IBS patients, which were correlated to abdominal pain score. Our aim is to investigate the effects of serotonin receptor on intestinal pain sensation in IBS-like mouse model and to explore regulatory mechanisms of nerve fiber elongation caused by serotonin and neurotrophic factors in human neuroblastoma SHSY5Y cells. Method：Two IBS-like mouse models, one is dual trigger of post- infection combined with water avoidance stress (GW), and the other one is post-inflammatory of TNBS-induced colitis (PT). The Ctrl or PT mice were given serotonin receptor compounds by oral or intraperitoneal injection for measurement of colorectal distension-visceromotor response (CRD-VMR) and charcoal meal test. Immunofluorescence (IF) staining of PGP9.5 (a pan-neuron marker) and serotonin receptors were conducted in mouse colonic tissues. Human neuroblastoma SH-SY5Y cells were incubated with GW and PT mouse colonic supernatant, serotonin and neurotrophic factor to stimulate nerve fiber elongation and to observe the gene expression. Lastly, colonic biopsies of IBS patients and healthy controls were collected to examine mucosal nerve distribution and serotonin receptors (5-HT3R, 5-HT4R and 5-HT7R). Result：Oral administration of a specific serotonin receptor ligand (CYY1005) reduced the visceral hypersensitivity in the PT mice, but did not affect normal pain sensation in control mice. PT mice were intraperitoneal administered serotonin receptor compounds (CYY073、CYY1280-3、CYY1294), and only CYY1280-3 can significantly decrease visceral hypersensitivity. By immunofluorescence staining, higher PGP9.5 and serotonin receptor immunoreactivity was found in the colonic mucosa of GW and PT mice compared to control mice, and those in IBS patients were higher than healthy controls. Pretreatment with serotonin receptor ligand (CYY1005) or 5-HT7R antagonist (SB7) inhibited the induction of nerve fiber outgrowth by GW and PT mouse colonic supernatant, serotonin and neurotrophic factor in SH-SY5Y cells. Lastly, the mRNA expression levels of p75NTR, TrkA, TrkB, and serotonin receptor of SH-SY5Y cells were significantly increased by neurotrophic factors. Conclusion：Our data showed that serotonin receptor was involved in the visceral hypersensitivity response in PT mice. In addition, serotonin and neurotrophic factors regulated nerve fiber elongation.