內臟高敏感性機制中神經營養因子與神經內分泌之交互作用

Abstract

背景: 腸躁症 (Irritable bowel syndrome, IBS) 為伴隨反覆性腹痛之功能性腸胃道疾病。腸躁症的危險因子包含遺傳因素、精神壓力、腸道慢性發炎、腸道感染病史等。過去研究發現腸躁症患者結直腸黏膜層檢體中高密度的神經纖維分布及較高表現量的神經營養因子，如:神經生長因子 (Nerve Growth Factor, NGF) 及腦源性神經營養因子 (Brain-derived neurotrophic factor, BDNF)，與腸躁症患者腹痛程度有關。此外，神經內分泌物質的失調，如血清素 (Serotonin, 5-HT) 與膽囊收縮素 (cholecystokinin, CCK)，也被指出參與在腸道痛覺機制中。本篇研究目的為探討血清素受器在神經纖維延長機制中扮演之角色，以及神經內分泌物質與神經營養因子間之交互作用。 材料與方法: 兩種類腸躁症動物模式包含使用2,4,6-三硝基苯磺酸誘發的後腸炎性類腸躁症之PT小鼠，以及寄生蟲後排除期/慢性避水壓力試驗之GW/WAS小鼠。藉由結直腸撐張刺激所引發的內臟動器反應來測定其腹痛程度，並以腹腔注射方式給予PT小鼠第七型血清素受器化合物CYY073-b，測試其對PT小鼠腸道疼痛感覺之效用。另外也收集動物大腸黏膜、腸段全組織以及背根神經節 (胸椎第九節至腰椎第一節以及腰椎第五節至薦椎第一節) 作核醣核酸分析。神經纖維長度、mRNA及蛋白表現量則是在SH-SY5Y人類神經細胞株 (human neuroblastoma) 中探討，以無菌小鼠腸道組織上清液或外加血清素及神經營養因子NGF、BDNF，觀察神經纖維延長及細胞中與神經內分泌物質及神經營養因子相關之基因和蛋白表現量。 結果: 在兩種類腸躁症動物模式皆發現有內臟高敏感性的現象，給予PT小鼠第七型血清素受器化合物CYY073-b可抑制其腸道疼痛感覺。PT小鼠的大腸全組織中，tryptophan hydroxylase 2 (TPH2)、5-HT7R、BDNF基因表現量上升，而TPH1、NGF基因表現量無改變；PT小鼠的大腸黏膜和背根神經節中，這些基因皆無改變。GW小鼠的大腸全組織中，p75 neurotrophin receptor (p75NTR) and Tropomyosin-related kinase receptor B (TrkB)基因表現量上升，而其餘基因有上升趨勢但無統計差異。在SH-SY5Y人類神經細胞株的實驗方面，以無菌之小鼠腸道組織上清液刺激會使神經細胞的神經內分泌物質及神經營養因子相關之基因表現量上升，但PT組與Sham組小鼠之間並無差異。在神經細胞實驗中，外加血清素使其神經纖維延長之效果可受5-HT7R拮抗劑抑制。以血清素或第七型血清素受器促進劑LP-211刺激之神經細胞，其NGF、BDNF及其受器p75NTR、TrkB 和CCK及其受器CCK-AR、CCK-BR mRNA 表現量上升，且此現象可受5-HT7R拮抗劑抑制。以NGF或BDNF刺激之神經細胞，其TPH2、5-HT7R mRNA表現量增加，而TPH1、CCK及其受器CCK-AR、CCK-BR mRNA表現量則無顯著差異。 結論: 本篇研究證實，類腸躁症動物模式小鼠之腸道痛覺異常可受第七型血清素受器拮抗劑所抑制。而神經內分泌物質與神經營養因子間存在交互作用，血清素透過第七型血清素受器與神經營養因子形成之正回饋反應可促進神經纖維延長。

Background: Irritable bowel syndrome (IBS) is a functional gastrointestinal disorder with recurrent abdominal pain. Risk factors related to IBS include hereditary factors, psychological stress, chronic inflammation, and history of intestinal infection etc. Higher nerve fiber density and neurotrophin levels such as nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) were found in colorectal biopsies associated with pain scores in IBS patients. In addition, dysregulation of neuroendocrines such 5-hydroxytryptamine (5-HT) and cholecystokinin (CCK) was involved in intestinal pain. Our aim is to investigate the interaction between neurotrophins and neuroendocrines in the mechanisms of intestinal neurite outgrowth and visceral hypersensitivity. Materials and Methods: Two animal models were used, including mice post-inflammatory of Trinitrobenzene sulfonic acid-induced colitis (PT) and mice that received post-giardiasis and chronic water avoidance stress (GW/WAS). Visceromotor response (VMR) to colorectal distension was measured to quantify intestinal pain. PT mice were used to test the effect of newly synthesized 5-HT7 receptor (5-HT7R) ligand―CYY073-b on visceral hypersensitivity. Colonic mucosa, colonic whole tissue, and Thoracic vertebra 9 to Lumbar vertebra 1 and Lumbar vertebra 5 to Sacral vertebra 1 (T9 - L1 and L5-S1) of dorsal root ganglia (DRG) collected from animals are used for measurement of mRNA expression. The human neuroblastoma SH-SY5Y cells were used for analysis of nerve fiber length, real-time PCR and Western blotting. Results: Higher VMR was found in the two animal models, and the visceral hypersensitivity in PT mice was inhibited by the newly synthesized 5-HT7R ligand―CYY073-b. Higher tryptophan hydroxylase 2 (TPH2), 5-HT7R, and BDNF mRNA expression were found in the colonic whole tissues of PT mice, whereas no change was seen in the TPH1 and NGF levels; no difference of these gene transcription was observed in mucosal tissues and DRG samples between PT and Sham mice. Higher levels of p75 neurotrophin receptor (p75NTR) and Tropomyosin-related kinase receptor B (TrkB) were found in the colonic whole tissues of GW mice but only increased trend was seen in other molecules without statistical significance. For the experiments in differentiated SH-SY5Y neural cells, all neuroendocrine- and neurotrophin-related gene expression were increased following incubation with bacteria-free mouse colonic supernatant; however, no difference was seen between PT and sham groups of colonic supernatants. In the differentiated SH-SY5Y neural cells, neurite outgrowth that are induced by 5-HT were inhibited by 5-HT7R antagonists. Treatment with 5-HT and 5-HT7R agonist LP-211 increased the NGF, BDNF, p75NTR, TrkB, CCK and CCK receptor mRNA expression, but not TrkA level, in neural cells. Treatment with recombinant NGF and BDNF increased the TPH2 and 5-HT7R mRNA expression, but not TPH1, CCK or CCK receptor levels. Conclusion: Colonic hyperalgesia of IBS-like animal model was inhibited by 5-HT7R antagonists. A positive feedback loop was observed between neuroendocrine and neurotrophin expression. Serotonin contributed to neurite outgrowth by promoting neurotrophin expression via 5-HT7R.