腸道共生菌叢失衡與精神壓力在內臟高敏感性與腸道功能失調中所扮演之角色探討

Abstract

背景：腸躁症為一種功能性異常之腸胃道疾病，症狀包含腹脹、腹痛及腹瀉或便秘等，但是並無法檢測到腸道結構上的損傷或病原體的存在。遺傳因子、微生物或寄生蟲感染、抗生素的使用以及精神壓力等危險因子皆被懷疑參與腸躁症的致病機轉。近年來在腸躁症病人中發現腸道共生細菌增生、腸道通透性增加及低程度發炎現象等。過去動物實驗研究指出精神壓力會造成腹部疼痛增加以及造成腸道屏障功能缺損。本研究的目的在於探討抗生素引起之腸道共生菌叢失衡與梨形蟲感染是否會加劇精神壓力所引發的內臟高敏感性與腸道功能失調。 材料方法與結果：第一部分實驗，分別給予SPF BALB/c小鼠正常水（NW）或抗生素水（AW）10天，一部分小鼠在第10天犧牲作分析，另一部分小鼠則是移除飲水中抗生素後，給予每天1小時的避水壓力（WAS）或無處理（NH）為期10天。首先分析口服抗生素對腸道共生菌叢數目與腸道電生理參數之影響。飲用抗生素水10天之小鼠其體重下降33.29 ± 0.06 %，而移除抗生素後體重恢復。相較於飲用正常水，抗生素水處理使盲腸與大腸的需氧菌和厭氧菌總量顯著減少，而移除抗生素後總體細菌量則回升。同時發現抗生素處理造成小腸與大腸的迴路電流上升，暗示腸道離子分泌增加；dextran探針從腸腔面到漿液面的流量與腸道組織結構皆沒有改變，顯示飲用抗生素水後上皮屏障功能依然完好。接著探討抗生素引起之腸道共生菌叢失衡是否會加重精神壓力所引發的內臟高敏感性與腸道上皮屏障失能。首先觀察到受避水壓力刺激的小鼠，其血清中腎上腺皮質酮含量明顯增加，確定感受到精神壓力。低體積結直腸撐張刺激的內臟動器反應實驗中，只有AW-WAS組的內臟動器反應比NW-NH小鼠高，顯示抗生素加精神壓力的雙重因子會造成內臟痛覺異常。而高體積結直腸撐張刺激實驗中， NW-WAS、AW-NH和AW-WAS小鼠相較於NW-NH小鼠之反應都有顯著增加，顯示單一因子（抗生素或精神壓力）會造成內臟痛覺過度但無加成效果。細菌總量方面，NW-NH和NW-WAS小鼠之細菌量很接近，但AW-WAS組盲腸共生菌數目比AW-NH組更高，表示雙重因子下細菌回彈的程度更大，且有過度增生的現象。螢光原位雜合分析發現只有AW-WAS小鼠之小腸線窩有細菌入侵的現象。四個組別中，腸道組織型態皆正常且沒有觀察到細菌移位到肝臟或脾臟。腸道上皮功能指標（如迴路電流及dextran通透性）方面，AW-WAS 小鼠與NW-WAS小鼠無明顯差異。第二部分實驗將SPF C57BL/6小鼠灌食磷酸鹽緩衝液（PBS）或10e7之Giardia lamblia strain GS/M品系之滋養體後的第35天即Giardia後排除期，進行避水壓力（WAS）或無處理（NH）。在低體積結直腸撐張刺激的內臟動器反應實驗中，Giardia-NH和Giardia-WAS兩者與PBS-NH組相比都較高，而PBS-WAS與PBS-NH組相比無差異，顯示G. lamblia感染排除單一因子會造成痛覺異常。高體積結直腸撐張刺激實驗中，PBS-WAS和Giardia-WAS小鼠之反應皆比PBS-NH高，但Giardia-NH卻與PBS-NH無差異，顯示單一因子下精神壓力會造成痛覺過度而G. lamblia感染排除不會，且雙重因子對痛覺過度無加成效果。此外，Giardia-NH與Giardia-WAS小鼠之小腸通透性皆有上升但組織結構正常。雖然四個組別的腸道總體共生菌量相仿，但是Giardia-WAS小鼠之小腸有細菌入侵上皮細胞與固有層並伴隨腸道上皮細胞緊密連結ZO-1結構破壞與occludin片段化的現象。 結論：雙重因子組合（抗生素加精神壓力、後感染加精神壓力）都會引起內臟痛覺異常，但是對痛覺過度無加成效果。而雙重因子組合皆會造成腸道共生菌數目或位置的改變，因此推測可能與內臟痛覺敏感化有關。

Introduction: Irritable bowel syndrome (IBS) is a functional gastrointestinal disorders characterized by symptoms such as bloating, abdominal pain, diarrhea and constipation, without structural abnormality and etiological agents. Risk factors such as genetic straits, microbial and parasitic infection, antibiotic usage and psychological stress have been long implicated in the pathogenesis of IBS. Enteric commensal overgrowth, intestinal permeability rise and low grade inflammation were recently identified in IBS patients. Previous animal studies have shown that psychological stress increases abdominal pain perception and causes intestinal barrier disruption. Aim: To investigate whether enteric dysbiosis by antibiotic usage and Giardia lamblia infection potentiates stress-induced visceral hypersensitivity and intestinal dysfunctions. Methods and Results: In the first part, specific pathogen free BALB/c male mice drinking normal water (NW) or antibiotic water (AW) for 10 days were sacrificed immediately, or subsequently nonhandled (NH) or subjected to water avoidance stress (WAS) for 1h/day for 10 days after antibiotic removal. We assessed the effects of oral antibiotics on gut bacterial counts and electrophysiological parameters. Compared to mice drinking NW, the body weight of AW mice dropped 33.29 ± 0.06 % at the end of antibiotic treatment, and recovered to normal weight after antibiotic removal. The total aerobic and anaerobic bacterial counts in the cecum and colon were significantly decreased by antibiotic treatment, and bacterial rebound to normal levels after antibiotic removal. Increased short-circuit current was found in the jejunal and colonic tissues in AW mice compared to NW mice, suggesting increased ion secretion after antibiotic treatment. Neither change in mucosal-to-serosal dextran flux nor histological structural damage was seen in intestinal tissues of AW mice compared to NW mice, suggesting that the epithelial barrier remains intact following antibiotic usage. Then we investigate whether antibiotic dysbiosis potentiates stress-induced visceral hypersensitivity and intestinal epithelial dysfunction. Mice drinking NW and AW were subjected to NH or WAS after antibiotic withdrawal. Perception of psychological stress was confirmed by increased serum corticosterone levels following WAS. The visceromoter responses (VMR) caused by low volume of colorectal distension (CRD) was statistically increased in AW-WAS compared to NW-NH mice, indicating that the combination of antibiotic usage and psychological stress may synergistically trigger allodynia. Moreover, VMR induced by high volume of CRD were augmented in NW-WAS, AW-WAS and AW-NH mice compared to NW-NH mice, suggesting that psychological stress alone causes hyperalgesia but no synergistic effect with antibiotics. The intestinal bacterial counts were similar between NW-NH and NW-WAS mice, whereas statistically higher numbers were found in the cecum of AW-WAS mice compared to that of NW-WAS mice, suggesting an exaggerated bacterial rebound by antibiotic withdrawal upon exposure to psychological stress. Fluorescence in situ hybridization (FISH) revealed bacterial invasion to jejunal crypts in AW-WAS mice only but not in other groups. All four groups of mice displayed normal intestinal histological structures with no sign of bacterial translocation to liver and spleen. No difference in intestinal epithelial functions (i.e. short-circuit current and dextran permeability) were seen between NW-WAS and AW-WAS mice. In the second part, specific pathogen free C57BL/6 male mice were inoculated with phosphate-buffered saline (PBS) or 10e7 trophozoites of Giardia lamblia strain GS/M. On post-infection day 35 in which the trophozoites cannot be detected in the small intestines (post-clearance phase), mice were subjected to NH or WAS. Compared to PBS-NH mice, the VMR caused by low volume of CRD was increased in Giardia-NH and Giardia-WAS groups but not PBS-WAS groups, indicating that post-giardiasis alone may trigger allodynia. Moreover, the VMR induced by high volume of CRD was increased in PBS-WAS and Giardia-WAS mice compared to PBS-NH groups, whereas no difference was seen between Giardia-NH and PBS-NH mice, suggesting that psychological stress but not Giardia infection causes hyperalgesia and the combination of two factors show no synergistic effects. Increased jejunal permeability was seen Giardia-NH and Giardia-WAS mice without histological structural change. Although the total bacterial numbers were comparable in the four groups, bacterial invasion to enterocytes and gut mucosa accompanied with structural disruption of tight junction ZO-1 and occludin was detected in small intestine of Giardia-WAS mice. Conclusions: The combinational risk factors (i.e. antibiotic usage plus psychological stress, and post-infection plus the latter) induced visceral hypersensitivity in the form of allodynia but showed no synergistic effects on hyperalgesia. Moreover, both combinations induced enteric bacterial dysbiosis, which may be involved in the mechanism of visceral hypersensitivity.