摘要
目的研究低蛋氨酸（methionine restriction，MetR）饮食对葡聚糖硫酸钠（DSS）诱导的大鼠结肠炎的结肠黏膜病理组织学、肠黏膜通透性以及结肠上皮紧密连接蛋白表达的影响，并探讨其可能机制。方法SD大鼠随机分为常规饮食正常组（AA组）、低蛋氨酸饮食正常组（MetR组）、常规饮食模型组（DSS＋AA组）、低蛋氨酸饮食模型组（DSS＋MetR组），每组15只。DSS建模后第21天腹主动脉采血，分析血常规、肝肾功能和电解质水平，取结肠组织行HE染色分析肠黏膜病理学变化，检测肠组织髓过氧化物酶（MPO）活性，免疫组化染色检测增殖细胞核抗原（PCNA）分析肠上皮细胞的增殖状况，采用尤斯灌流室（Ussing chamber）检测肠黏膜通透性；采用蛋白质印迹法分析肠上皮紧密连接蛋白的表达。结果结肠炎造模大鼠出现腹泻、便血、体重下降，炎症集中在远端结肠，表现为隐窝脓肿，炎性细胞的浸润。与DSS＋AA组比较，MetR饮食干预可显著降低模型大鼠结肠的组织病理学评分[（10．55±3．62）分比（15．00±4．89）分，P=0．003]。DSS＋MetR组和DSS＋AA组大鼠血白细胞计数、肠组织MPO活性以及PCNA免疫组化结果的差异均无统计学意义。Ussing chamber检测显示，DSS＋AA组的跨膜电阻抗显著低于AA组[（28．40±6．78）Ω·cm。比（46．53±4．03）Ω·cm^2，P〈0．05]，MetR组显著高于AA组[（60．64±8．40）Ω·cm^2比（46．53±4．03）Ω·cm^2，P〈0．05]，DSS＋MetR组的短路电流值显著高于DSS＋AA组[（35．01±2．19）μA／cm^2比（29．61±1．19）μA／cm^2，P〈0．05]。蛋白质印迹结果显示，AA组和MetR组未见claudin2蛋白表达，MetR组的结肠上皮claudin3蛋白表达量明显高于AA组；与DSS＋AA组比较，DSS＋MetR组claudin2的表达量更高，claudin3表达量更高。结论MetR饮食对DSS诱导的结肠炎模型大鼠有明显的治疗作用，其机制可能不是通过调节炎性细胞浸润以及促进肠细胞生长的途径来缓解炎症损伤，而可能是通过改变紧密连接蛋白结构和功能而改善其肠黏膜屏障的功能，促进受损肠黏膜的修复。
Objective To study the impact of methionine restriction （MetR） on mucosal histopathology, permeability and tight junction composition in a dextran sulfate sodium （DSS）-induced colitis model, and to explore its underlying mechanism. Methods SD rats were randomly divided into 4 groups ： normal rats fed by a complete amino acid （ AA group ） diet, normal rats fed by MetR diet （ MetR group） , DSS treated rats fed by a complete amino acid （DSS ＋ AA group） and DSS treated rats fed by MetR diet （ DSS ＋ MetR group） , each group had 15 rats. Abdominal aorta blood sampling was taken at day 21 after DSS model been established to analyze blood routine examination, liver and kidney function and level of electrolyte. Morphological changes in colonic mucosa were evaluated and scored by light microscopy. Myeloperoxidase （MPO） activity was measured. The effect of MetR on mucosal permeability of colon strips was detected by Ussing chamber. Claudin2, occludin, claudin3, ZO-1 expression were quantified by Western blot. Results The early clinical manifestation in the DSS treated rats were loose stool or diarrhea,hematochezia positive and bleeding, and weight losing. HE observation showed prominent colitis in distal colon with manifestations of crypt abscess and infihration of inflammatory cells. Although MPO activity and WBC account between the DSS ＋ MetR and DSS ＋ AA group did not significantly changed, treatment with MetR diet significantly decreased the extent and severity of epithelial injury of DSS ＋ MetR group （ 10. 55±3.62 vs 15.00 ± 4. 89, P = 0. 003 ）. There were no significant difference in PCNA immunohistocbemical result between the DSS ＋ MetR group and DSS ＋ AA group. Compared to the rats on AA diet, transepithelial result between the DSS ＋ MetR group and DSS ＋ AA group. Compared to the rats on AA diet, transepithelial electrical resistance（TEER） in DSS ＋ AA group was obvious lower [ （28.40 ±6. 78 ）Ω· cm^2 vs （46. 53 ± 4. 03 ） Ω· cm^2, P 〈 0. 05 ], and TEER in MetR group were obviously higher [ （60. 64± 8.40） Ω· cm^2 vs （46. 53 ± 4. 03）Ω· cm^2, P 〈 0. 05 1. However, short-circuit current （Isc） in DSS ＋ MetR group was obviously higher that of DSS ＋ AA group [ （ 35.01 ± 2. 19） μA/cm^2 vs （29. 61 ± 1.19 ） μA/cm^2, P 〈 0.05 ]. Western blot suggested that colon claudin2 expression was not found in colon epithelium of normal rats, and an obviously increase expression of claudin3 protein was found in the MetR group, compared to AA group ; and an significantly increase in the abundance of claudin3 was found in the DSS ＋ MetR group, but amount of claudin2 was decreased, compared with the DSS ＋ MetR group. Conclusion The MetR diet has obvious therapeutic effect on ulcerative colitis model rats induced by DSS, and its mechanism may not by regulating inflammatory cell infiltration and the way of promoting intestinal cell growth to alleviate inflammatory injury, but probably by changing the structure and function of tight junction protein and improve the intestinal mucosal barrier function, and promote the repair of damaged intestinal mucosa.
出处
《中华内科杂志》
CAS
CSCD
北大核心
2013年第6期503-509,共7页
Chinese Journal of Internal Medicine
基金
浙江省自然科学基金（LY12H03009）
关键词
结肠炎
紧密连接部
硫酸葡聚糖
肠屏障功能
低蛋氨酸
Colits
Tight junctions
Dextran sulfate
Intestinal barrier function
Methioninere striction
作者简介
通信作者：陈沽，Email：hzcjie@zju．edu．cn
