利用小鼠模型研究肠道免疫功能紊乱在肠易激综合征致病机制中的作用

Pathogenic effect of intestinal immune dysfunction on irritable bowel syndrome in mouse model

目的研究肠道免疫功能在肠易激综合征（IBS）的作用机制及丁酸杆菌对肠道免疫系统紊乱的调节作用。方法C57BL／6雄性6周龄小鼠共50只，随机分为3组。实验组（n=20）正丁酸钠结肠灌注，诱导C—IBS小鼠模型，每次200μl（浓度500mmol／L）每天2次，连续3d。对照组（n=20）小鼠等体积生理盐水灌肠。丁酸杆菌组（n=10）正丁酸钠造模前2h，丁酸杆菌灌胃每次500μl（活菌浓度1／10^9CFU／ml）每天1次，连续6d。检测生理指标、结肠高敏状态、肠道病理损伤程度，评价C-IBS模型。分离肠道上皮内淋巴细胞（IELs）和固有层单核细胞（LPMC），流式分析，进一步评价C—IBS模型小鼠肠道免疫功能异常激活和紊乱状态及丁酸杆菌对肠道免疫系统紊乱的调节作用。结果（1）实验组小鼠生理指标改变明显，实验组肠道出现低度炎症状态结肠膨胀试验（CRD）和最低阈值明显降低（t=8．926、6．103，P均〈0．001），提示C—IBS模型造模成功；（2）与对照组相比，实验组肠道IELs中树突状细胞（DCs）减少（t=2．878、3．086，P均〈0．05）而巨噬细胞增加（t=3．191，P〈0．05），记忆性T细胞增加（t=3．071，P〈0．05）；（3）与对照组相比，固有层（LP层）树突状细胞（DCs）减少（t=2．880、2．664，P均〈0．05），记忆性T细胞增加（t=3．732、2．682，P均〈0．05）；（4）与对照组相比，丁酸杆菌组生理指标无变化；IELs中巨噬细胞、记忆性T细胞及DCs均接近正常水平（造模第6天，t=1．103、0．0213、0．418，P均〉0．05），LP层中巨噬细胞和DCs（造模第6天，t=0．782、0．347，P均〉0．05），与对照组相比均无显著性差异；（5）与实验组相比，丁酸杆菌组肠道LP层记忆性T细胞数量也显著下降（造模第6天，t=2．346，P=0．0470，P〈0．05），但仍高于对照组（t=2．233，P=0．0476，P〈0．05）。丁酸杆菌干预后，肠道免疫系统功能恢复并接近正常水平。结论IBS病理生理机制的产生可能与肠道免疫功能异常激活和紊乱状态相关，肠道免疫功能异常激活和紊乱状态是诱导IBS出现的重要因素之一。丁酸杆菌能够调节肠道免疫系统，恢复胃肠道功能。

Objective To investigate the role of intestinal immune dysfunction in the pathogenesis of irritable bowel syndrome（IBS） and to study the effects of Clostridium butylicum on the regulation of intes- tinal immune disorders. Methods A total of 50 male 6-week-old C57BL/6 mice were randomly divided into three groups, including the experimental group （ n = 20） , the control group （ n = 20 ） and the Clostridium buty- licum group （ n = 10 ）. A mouse model of constipation-predominant IBS （C-IBS） was established by perfusing sodium butyrate solution（200 μl, concentration of 500 mmol/L） into the mouse colon twice a day for three consecutive days. The mice in control group were intrarectally perfused with normal saline enema （200 μl）. Two hours before the perfusion of sodium butyrate into colon, the mice in Clostridium butylicum group were given Clostridium butylicum 500 μl（ viable cell concentration of 1×109 CFU/ml） by oral garage once a day for six days. The colorectal distention test（CRD） was carried out for evaluation of clinical parameters. HE staining of intestinal tissue section was performed for histopathological assessment of colonic mucosal inflam- mation. Intestinal intraepithelial lymphocytes （IELs） and lamina propria mononuclear cells （LPMCs） were isolated and analyzed by flow cytometry to evaluate the correlation between IBS and intestinal immune dys- function/abnormal activation of intestinal immune cells in mouse model of C-IBS, and to assess the regulato- ry effects of Clostridium btttylicum on the intestinal immune disorder. R^ults （1）Compared with the con- trol group, the mice in experimental group showed a significant change in physiological parameters, histologi- cal structure of colon, inflammatory ceils infiltration and low-grade inflammatory state. There was a signifi- cant increase in scores of CRD and a decrease in lowest sensory threshold （t= 8. 926 and t = 6. 103, both P 〈0.001） ; （2）There was a decrease in the numbers of DC in IELs （t=2. 878 and t=3. 086, both P〈 0.05 ） , but an increase in the numbers of macrophage （t = 3. 191, P〈0.05 ） and the memory T cells in mice with IBS （ t = 3.071, P〈0.05 ） as compared with that in control group; （3） DCs were decreased （ t = 2.880 and t=2.664, both P〈0.05）, but memory T cells were increased （t=3.732 and 2.682, P〈0.01 and P〈 0.05 ） in the LPMCs of mice in experimental group; （g） There was no significant difference in the physiolog- ical index between the mice in control group and the Clostridium butylicum group. Levels of memory T cells, macrophages and DCs in the IELs were close to the normal level （6 d, t = 1. 103, 0. 0213, 0.418, all P〉 0.05）, and levels of macrophages and DCs in the LPMCs of mice in the Clostridium butylicum group were also similar to that in the control group （6 d, t=0. 782, 0. 347, both P〉0.05） ; （5） Compared with the mice in experimental group, the level of memory T ceils in LPMCs of mice treated with Clostridium butylicum was dramatically declined （6 d, t=2. 346, P=0.0470, P〈0.05）, however, which was still higher than that of mice in control group （6 d, t = 2. 233, P = 0.0476, P〈0.05 ）. The intestinal immune function was restored to normal level with Clostridium butylicttm intervention. Conclusion The pathophysiologic mecha- nism of IBS might be closely related to the abnormal activation of intestinal immune cellsand disordered func- tional state in the intestinal mucosa. Clostridium butylicum could regulate the intestinal immune homeostasis and restore the physiological function of gastrointestinal tract.