CAJ | 학술논문

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·cm2比(46.53 ±4.03)Ω·cm2,P＜0.05],MetR组显著高于AA组[(60.64 ±8.40)Ω·cm2比(46.53 ±4.03)Ω·cm2,P＜0.05],DSS+ MetR组的短路电流值显著高于DSS+ AA组[(35.01 ±2.19) μA/cm2比(29.61±1.19)μA/cm2,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. 目的研究低蛋氨痠(methionine restriction,MetR)飲食對葡聚糖硫痠鈉(DSS)誘導的大鼠結腸炎的結腸黏膜病理組織學、腸黏膜通透性以及結腸上皮緊密連接蛋白錶達的影響,併探討其可能機製.方法 SD大鼠隨機分為常規飲食正常組(AA組)、低蛋氨痠飲食正常組(MetR組)、常規飲食模型組(DSS+ AA組)、低蛋氨痠飲食模型組(DSS+ MetR組),每組15隻.DSS建模後第21天腹主動脈採血,分析血常規、肝腎功能和電解質水平,取結腸組織行HE染色分析腸黏膜病理學變化,檢測腸組織髓過氧化物酶(MPO)活性,免疫組化染色檢測增殖細胞覈抗原(PCNA)分析腸上皮細胞的增殖狀況,採用尤斯灌流室(Ussing chamber)檢測腸黏膜通透性；採用蛋白質印跡法分析腸上皮緊密連接蛋白的錶達.結果結腸炎造模大鼠齣現腹瀉、便血、體重下降,炎癥集中在遠耑結腸,錶現為隱窩膿腫,炎性細胞的浸潤.與DSS+ AA組比較,MetR飲食榦預可顯著降低模型大鼠結腸的組織病理學評分[(10.55 ±3.62)分比(15.00±4.89)分,P=0.003].DSS+ MetR組和DSS+ AA組大鼠血白細胞計數、腸組織MPO活性以及PCNA免疫組化結果的差異均無統計學意義.Ussing chamber檢測顯示,DSS+ AA組的跨膜電阻抗顯著低于AA組[(28.40±6.78)Ω·cm2比(46.53 ±4.03)Ω·cm2,P＜0.05],MetR組顯著高于AA組[(60.64 ±8.40)Ω·cm2比(46.53 ±4.03)Ω·cm2,P＜0.05],DSS+ MetR組的短路電流值顯著高于DSS+ AA組[(35.01 ±2.19) μA/cm2比(29.61±1.19)μA/cm2,P＜0.05].蛋白質印跡結果顯示,AA組和MetR組未見claudin2蛋白錶達,MetR組的結腸上皮claudin3蛋白錶達量明顯高于AA組；與DSS+ AA組比較,DSS+ MetR組claudin2的錶達量更高,claudin3錶達量更高.結論 MetR飲食對DSS誘導的結腸炎模型大鼠有明顯的治療作用,其機製可能不是通過調節炎性細胞浸潤以及促進腸細胞生長的途徑來緩解炎癥損傷,而可能是通過改變緊密連接蛋白結構和功能而改善其腸黏膜屏障的功能,促進受損腸黏膜的脩複.
목적 연구저단안산(methionine restriction,MetR)음식대포취당류산납(DSS)유도적대서결장염적결장점막병리조직학、장점막통투성이급결장상피긴밀련접단백표체적영향,병탐토기가능궤제.방법 SD대서수궤분위상규음식정상조(AA조)、저단안산음식정상조(MetR조)、상규음식모형조(DSS+ AA조)、저단안산음식모형조(DSS+ MetR조),매조15지.DSS건모후제21천복주동맥채혈,분석혈상규、간신공능화전해질수평,취결장조직행HE염색분석장점막병이학변화,검측장조직수과양화물매(MPO)활성,면역조화염색검측증식세포핵항원(PCNA)분석장상피세포적증식상황,채용우사관류실(Ussing chamber)검측장점막통투성；채용단백질인적법분석장상피긴밀련접단백적표체.결과 결장염조모대서출현복사、편혈、체중하강,염증집중재원단결장,표현위은와농종,염성세포적침윤.여DSS+ AA조비교,MetR음식간예가현저강저모형대서결장적조직병이학평분[(10.55 ±3.62)분비(15.00±4.89)분,P=0.003].DSS+ MetR조화DSS+ AA조대서혈백세포계수、장조직MPO활성이급PCNA면역조화결과적차이균무통계학의의.Ussing chamber검측현시,DSS+ AA조적과막전조항현저저우AA조[(28.40±6.78)Ω·cm2비(46.53 ±4.03)Ω·cm2,P＜0.05],MetR조현저고우AA조[(60.64 ±8.40)Ω·cm2비(46.53 ±4.03)Ω·cm2,P＜0.05],DSS+ MetR조적단로전류치현저고우DSS+ AA조[(35.01 ±2.19) μA/cm2비(29.61±1.19)μA/cm2,P＜0.05].단백질인적결과현시,AA조화MetR조미견claudin2단백표체,MetR조적결장상피claudin3단백표체량명현고우AA조；여DSS+ AA조비교,DSS+ MetR조claudin2적표체량경고,claudin3표체량경고.결론 MetR음식대DSS유도적결장염모형대서유명현적치료작용,기궤제가능불시통과조절염성세포침윤이급촉진장세포생장적도경래완해염증손상,이가능시통과개변긴밀련접단백결구화공능이개선기장점막병장적공능,촉진수손장점막적수복.
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 infiltration 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 immunohistochemical 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 · cm2 vs (46.53 ± 4.03)Ω · cm2,P ＜0.05],and TEER in MetR group were obviously higher[(60.64 ± 8.40)Ω · cm2 vs (46.53 ±4.03)Ω · cm2,P ＜0.05].However,short-circuit current (Isc) in DSS + MetR group was obviously higher that of DSS + AA group [(35.01 ± 2.19) μA/cm2 vs (29.61 ± 1.19) μA/cm2,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 regnlating 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.