梨形蟲感染排除後之小鼠腸道呈現持續性上皮屏障失常及黏膜發炎的現象：探討「後感染腸躁症」之致病機轉

Abstract

腸躁症是一種功能性缺損的腸道疾病，症狀包括腹部疼痛以及排便習慣的改變，然而卻無法偵測到明顯腸道組織病變或病原體之存在。最新研究發現在腸躁症病人身上有低程度腸道組織發炎，黏膜屏障失能和腸腔細菌增生的現象。許多文獻指出感染性急性腸胃炎之後可能會誘發腸躁症，此種類型特稱為「後感染腸躁症」。梨形蟲Giardia lamblia是一種常見以水作傳染媒介之腸道寄生蟲。近年在挪威之流行病學研究發現，高達80 %受梨形蟲感染的病人在受感染後的12-30個月，且在已排除體內之寄生蟲至少6個月之後出現腸躁症的症狀。儘管腸躁症造成了相當嚴重的醫療問題，但苦於無適當之實驗動物模式，消化醫學界對腸躁症之致病機轉及原理仍不清楚。因此本實驗的目的為利用梨形蟲感染小鼠建立「後感染腸躁症」的動物模式，並探討梨形蟲感染期間及後排除期是否會引發腸道屏障失常及發炎現象。本實驗將Balb/c小鼠灌食10e7隻Giardia lamblia GS/M 品系之滋養體，或給予無菌之磷酸鹽緩衝液，並在感染後第0天至第49天每週分別計數小腸中滋養體的數目以建立出感染曲線。由感染曲線得知，梨形蟲寄生於小腸之高峰期約在感染第4天到第7天，本實驗中稱為「感染期」；而在感染第14天則完全無法偵測到寄生蟲，因此本實驗將第21天到第49天視為「後排除期」。本實驗接著分析腸道黏膜層中內吞至細胞內的細菌數作為腸道屏障功能的指標。分析小腸及大腸之黏膜層細菌量發現，在感染第7天（中位數分別為164.2及253.4 CFU/g）及第35天（中位數分別為118.4及98.1 CFU/g）時皆明顯高於感染前即第0天之黏膜細菌量（中位數分別為0.0及0.3 CFU/g）。緩衝液灌食組第35天時，其小腸及大腸黏膜層細菌量與第0天相似。在第7天時，感染組之小腸及大腸總細菌量比起緩衝液組顯著增加 4-10倍，表示腸道細菌在感染期有增生現象; 但第35天，感染組和緩衝液組並無差別。此外，在感染第7和35天時，在小腸之上皮表層細菌量相較於個別之緩衝液組皆有升高，顯示出梨形蟲感染期至後排除期會有持續性腸道細菌附著的現象。而在感染第7天時，黏膜之occludin有片斷化的情形。此外，小腸組織的骨髓過氧化酶活性在第7天及第35天時也提高。在感染第7天時，血液中嗜中性白血球之百分比增加，並在第7天及第35天的小腸組織切片中皆有看到大量嗜中性白血球進到腺窩附近。此外，在感染第35天，小腸中之腫瘤壞死因子α （TNFα），細胞激素-1β(IL-1β) 及巨噬細胞炎性蛋白-1α（MIP-1α）總量增加，並伴隨著誘導型一氧化氮合成酶（iNOS）表現增加的情形。由上述之實驗結果顯示，小鼠在梨形蟲感染之「後排除期」，會有腸道上皮屏障失常引發腸腔細菌持續進入腸組織的現象，並伴隨著腸道發炎及嗜中性白血球的活化情形產生。而此梨形蟲感染「後排除期」之動物模式或許將來可用來探討「後感染腸躁症」之致病機轉。

Irritable bowel syndrome (IBS) is a functional gastrointestinal disorder characterized by abdominal pain and changes in bowel habits, for which neither apparent structural lesion nor presence of pathogen could be found. Recent data indicated that low grade intestinal inflammation, impaired mucosal barrier function, and enteric bacterial overgrowth were identified in IBS patients. Consistent evidence showed that IBS may be the adverse outcome of an acute episode of infectious gastroenteritis, termed post-infectious (PI) IBS. Giardia lamblia is the most frequently identified etiologic agent of waterborne disease worldwide. Recent epidemiological data from Norway indicated that more than 80 % of patients from an outbreak of giardiasis showed IBS symptoms after 12-30 months post-onset of Giardia infection and at least 6 months after the clearance of parasites. Despite that IBS represents a substantial clinical problem, its mechanism and pathogenesis remain poorly understood mainly due to the lack of animal models. The aim of the current study is to establish a PI-IBS model using Giardia-infected mice and to evaluate gut barrier dysfunction and mucosal inflammation during infection and after eradication of parasites. Balb/c mice were inoculated with 10e7 trophozoites of Giardia lamblia strain GS/M or pair-fed with phosphate-buffered saline (PBS). The trophozoites in the small intestines were enumerated weekly from PI day 0 to 49 to determine the time course of parasitic infection. Giardia colonization peaked at PI day 4-7 and this period was termed the 'infection phase'; the parasites were cleared by PI day 14, and therefore, day 21-49 was denoted the 'post-clearance phase'. Intestinal epithelial barrier dysfunction was evidenced by increased bacterial colony forming units (CFU) in the gut mucosa. The medium of mucosal endocytosed bacterial counts in the small and large intestines on PI day 7 (164.2 and 253.4 CFU/g) and on PI day 35（118.4 and 98.1 CFU/g）were respectively higher than the values on PI day 0（0.0 and 0.3 CFU/g）. In mouse pair-fed with PBS, the mucosal bacterial counts in small and large intestines on day 35 were comparable with day 0. Total bacterial counts in small and large intestines on PI day 7 in mouse infected with G. lamblia was 4 to 10 times higher than those pair-fed with PBS, suggesting bacterial overgrowth during giardiasis; whereas no difference was seen between the two groups on PI day 35. The numbers of superficial bacteria in small intestines of Giardia-infected mice increased on day 7 and day 35 compared to those pair-fed with PBS, suggesting bacterial adherence during infection and after clearance of Giardia. Increased tight junctional occludin cleavage was noticed in small intestinal mucosa during infection. Heightened intestinal myeloperoxidase activity was found on day 7 and 35. Increased percentage of neutrophils in white blood cells was seen on PI day 7, and augmented neutrophil infiltration into the crypt area in small intestinal tissues was observed on PI day 7 and 35. Elevated intestinal TNFα, IL-1βand MIP-1α levels were associated with increased epithelial iNOS expression on PI day 35. In conclusion, Giardia-infected mice showed persistent enteric bacterial influx accompanied by mucosal inflammation and neutrophil activation after parasite clearance. The post-giardiasis animals may be suitable models for investigating gut barrier dysfunction underlying the mechanism of PI-IBS.