腸躁症小鼠模式中rifaximin對於腸道痛覺反應與神經可塑性的影響

Abstract

腸躁症(Irritable Bowel Syndrome, IBS)是一種功能性腸胃道疾病，患者有長期的反覆性腹痛、及排便型態的改變，但在內視鏡及糞便的篩檢卻沒有出現腸胃道結構上的損害及病原體的存在。腸躁症的危險因子包含遺傳因子、心理壓力、慢性發炎及腸道感染病史等。治療方面，2015年美國食品藥品管理局(FDA)核准Rifaximin (RFX)用於治療腸躁症腹瀉型患者，但機轉尚未明確。另外先前研究發現，腸躁症患者結直腸黏膜層檢體中的神經纖維密度高。因此我的研究目的是利用兩種腸躁症動物模式去探討RFX對於腸道痛覺反應及神經可塑性的影響。實驗包含兩種類腸躁症動物模式，第一種是結合雙重誘導因子，包括寄生蟲感染(Giardia)以及避水壓力試驗(WAS)，此組稱為GW小鼠；另一種則是使用2, 4, 6-三硝基苯磺酸誘導成腸炎，此組稱為TNBS小鼠。經口給予RFX及LWX（血清素受器配體）後，在GW及TNBS小鼠模式進行結直腸撐張刺激測定內臟敏感性，並進行腸道組織神經標定物PGP9.5及血清素受器的螢光免疫染色。利用腸道組織製成上清液並添加至人類神經瘤SH-SY5Y細胞中，以探討不同動物處理組對於神經纖維延長的調控。最後，收集腸躁症患者及健康者的結腸黏膜檢體測定神經纖維分布。GW及TNBS小鼠經口給予RFX、LWX及RFX+LWX後，均有顯著下降內臟高敏感性反應，但腸道蠕動性並無差異。利用螢光免疫染色技術發現GW及TNBS小鼠結腸黏膜組織之PGP9.5表現較正常組別量高，而腸躁症患者降結腸檢體中之PGP9.5表現比起健康者分布量亦較高。GW小鼠經口給予RFX或LWX組別之結腸黏膜組織PGP9.5表現量均有顯著降低。GW及TNBS小鼠給予RFX後，腸道黏膜層的血清素受器mRNA和蛋白質表現量均無顯著差異。此外，將GW及TNBS小鼠腸道組織上清液或5-HT添加至人類神經瘤SH-SY5Y細胞後，會引起神經纖維延長，而GW＋RFX或TNBS+RFX小鼠上清液組別則無法引起纖維延長。本篇實驗證實，RFX單一給予或與新合成之血清素受器配體合併使用皆可透過抑制神經纖維延長來降低內臟高敏感性的反應。在類腸躁症動物模式及腸躁症患者結腸檢體中觀察到黏膜神經纖維延長，且神經纖維的延長可能由血清素調控。

Irritable bowel syndrome (IBS) is a functional gastrointestinal disorder with recurrent abdominal pain associated with bowel habit changes, but neither apparent structural lesion nor presence of pathogen could be found. Risk factors include hereditary factor, psychological stress, chronic inflammation and history of infection etc. Rifaximin (RFX) was approved by FDA in USA in 2015 for the treatment of IBS with diarrhea; however, the mechanism is not clear. Previous study documented high density of neurites in colon mucosa of IBS patients. Our aim is to investigate the effect of RFX on intestinal nociception and neuroplastisity in IBS-like mouse models. Two mouse models were used, including post-giardiasis with water avoidance stress (GW) and post resolution of 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis. Mice were given RFX or LWX (a 5-HT receptor ligand) and tested for colorectal distension-visceromotor response (CRD-VMR). Immunofluorescence (IF) staining of PGP9.5 (a pan-neuron marker) and 5-HT receptors were conducted. Mouse colonic supernatant was added to human neuroblastoma SH-SY5Y cells to evaluate nerve fiber outgrowth. Lastly, colonic biopsies of IBS patients and healthy controls were collected to examine mucosal nerve distribution. Decrease in visceral hypersensitivity was observed in the GW and TNBS mice given RFX, LWX, or RFX+LWX; however, no difference in intestinal transit was found. By immunofluorescence staining, higher PGP9.5 immunoreactivity was found in the colonic mucosa of GW and TNBS mice compared to control mice, and those in IBS patients were higher than healthy controls. Moreover, the PGP9.5 fluorescent intensity was lower in the colonic mucosa of GW+RFX and GW+LWX mice compared to GW-veh mice. After given RFX, the mRNA and protein levels of 5-HT receptors in the mucosal layers were similar among mouse groups. Lastly, GW and TNBS mouse colonic supernatant or exogenous 5-HT induced nerve fiber outgrowth in SH-SY5Y cells, whereas the nerve fiber length was reduced in cells incubated with RFX-treated GW and TNBS mouse supernatant. RFX alone or in combination with LWX decreased visceral hypersensitivity partly via inhibition of mucosal neurite outgrowth in IBS-like mouse models. Extension of nerve fiber length in the mucosa was found in IBS-like mice and IBS patients, which may be mediated by 5-HT.