中华围产医学杂志
中華圍產醫學雜誌
중화위산의학잡지
CHINESE JOURNAL OF PERINATAL MEDICINE
2015年
4期
290-295
,共6页
苏衡%吕回%周伟%李美雪%黄龙光%李晶%袁伟明
囌衡%呂迴%週偉%李美雪%黃龍光%李晶%袁偉明
소형%려회%주위%리미설%황룡광%리정%원위명
小肠结肠炎,坏死性%二裂菌属%Toll样受体2%Toll样受体4%动物,新生%大鼠
小腸結腸炎,壞死性%二裂菌屬%Toll樣受體2%Toll樣受體4%動物,新生%大鼠
소장결장염,배사성%이렬균속%Toll양수체2%Toll양수체4%동물,신생%대서
Enterocolitis,necrotizing%Biifdobacterium%Toll-like receptor 2%Toll-like receptor 4%Animals,newborn%Rats
目的：探讨双歧杆菌防治新生大鼠坏死性小肠结肠炎（necrotizing enterocolitis， NEC）的可能分子机制。方法出生2 h内的新生Sprague-Dawley大鼠75只，随机分为5组，每组15只：（1）NEC模型组：人工喂养+脂多糖；（2）双歧杆菌干预组：人工喂养+脂多糖+双歧杆菌；（3）人工喂养对照组：仅人工喂养；（4）双歧杆菌对照组：人工喂养+双歧杆菌；（5）母乳喂养对照组：与母鼠同笼鼠乳喂养。脂多糖按30 mg/kg每天灌胃1次，共3 d。双歧杆菌微囊按1×1010菌落形成单位/ml加入到代乳品中灌胃，每天1次。喂养72 h，末次喂养后空腹12 h，断头法处死大鼠。取末段回肠组织光镜下观察形态学改变并对肠道损伤评分，免疫组织化学法检测回肠组织 Toll 样受体（Toll-like receptor，TLR）2、TLR4、核转录因子（nuclear transcription factor，NF）-κB p65蛋白表达。采用Kruskal-Wallis检验、方差分析、校正χ2检验或Fisher精确概率法进行统计学分析。结果 NEC模型组、双歧杆菌干预组、人工喂养对照组、双歧杆菌对照组及母乳喂养对照组的NEC发生比例分别为11/15、4/15、3/15、2/15与0/15；肠道损伤评分分别为（3.37±0.27）、（1.53±0.44）、（1.75±0.37）、（0.92±0.39）与（0.30±0.18）分；TLR2蛋白表达分别为0.35±0.05、0.30±0.03、0.32±0.04、0.30±0.02与0.29±0.03；TLR4蛋白表达分别为0.48±0.05、0.34±0.03、0.36±0.03、0.37±0.04与0.35±0.02；NF-κB p65蛋白表达分别为0.43±0.03、0.29±0.03、0.35±0.02、0.32±0.02与0.30±0.02。5组的NEC发生比例、肠道损伤评分、TLR2、TLR4及NF-κB p65蛋白表达量比较，差异均有统计学意义（χ2、H或F值分别为23.836、70.290、8.803、38.599和75.076，P值均＜0.05），NEC模型组均高于其他4组（P值均＜0.05）。双歧杆菌干预组的NEC发生比例与3个对照组比较，差异均无统计学意义（P值均＞0.05）；肠道损伤评分高于双歧杆菌对照组及母乳喂养对照组（P值均＜0.01），但与人工喂养对照组比较，差异无统计学意义（P＞0.05）；TLR4和NF-κB p65蛋白表达量均低于人工喂养对照组和双歧杆菌对照组（P值均＜0.05），但与母乳喂养对照组比较，差异均无统计学意义（P值均＞0.05）。双歧杆菌干预组TLR2蛋白表达量与3个对照组比较，差异均无统计学意义（P值均＞0.05）。结论双歧杆菌可能通过抑制致病菌或通过TLR2负反馈调节等方式，减少肠黏膜细胞TLR2及TLR4的蛋白表达，并进一步抑制NF-κB通路，使炎症反应得以缓解，发挥对NEC的防治作用。
目的：探討雙歧桿菌防治新生大鼠壞死性小腸結腸炎（necrotizing enterocolitis， NEC）的可能分子機製。方法齣生2 h內的新生Sprague-Dawley大鼠75隻，隨機分為5組，每組15隻：（1）NEC模型組：人工餵養+脂多糖；（2）雙歧桿菌榦預組：人工餵養+脂多糖+雙歧桿菌；（3）人工餵養對照組：僅人工餵養；（4）雙歧桿菌對照組：人工餵養+雙歧桿菌；（5）母乳餵養對照組：與母鼠同籠鼠乳餵養。脂多糖按30 mg/kg每天灌胃1次，共3 d。雙歧桿菌微囊按1×1010菌落形成單位/ml加入到代乳品中灌胃，每天1次。餵養72 h，末次餵養後空腹12 h，斷頭法處死大鼠。取末段迴腸組織光鏡下觀察形態學改變併對腸道損傷評分，免疫組織化學法檢測迴腸組織 Toll 樣受體（Toll-like receptor，TLR）2、TLR4、覈轉錄因子（nuclear transcription factor，NF）-κB p65蛋白錶達。採用Kruskal-Wallis檢驗、方差分析、校正χ2檢驗或Fisher精確概率法進行統計學分析。結果 NEC模型組、雙歧桿菌榦預組、人工餵養對照組、雙歧桿菌對照組及母乳餵養對照組的NEC髮生比例分彆為11/15、4/15、3/15、2/15與0/15；腸道損傷評分分彆為（3.37±0.27）、（1.53±0.44）、（1.75±0.37）、（0.92±0.39）與（0.30±0.18）分；TLR2蛋白錶達分彆為0.35±0.05、0.30±0.03、0.32±0.04、0.30±0.02與0.29±0.03；TLR4蛋白錶達分彆為0.48±0.05、0.34±0.03、0.36±0.03、0.37±0.04與0.35±0.02；NF-κB p65蛋白錶達分彆為0.43±0.03、0.29±0.03、0.35±0.02、0.32±0.02與0.30±0.02。5組的NEC髮生比例、腸道損傷評分、TLR2、TLR4及NF-κB p65蛋白錶達量比較，差異均有統計學意義（χ2、H或F值分彆為23.836、70.290、8.803、38.599和75.076，P值均＜0.05），NEC模型組均高于其他4組（P值均＜0.05）。雙歧桿菌榦預組的NEC髮生比例與3箇對照組比較，差異均無統計學意義（P值均＞0.05）；腸道損傷評分高于雙歧桿菌對照組及母乳餵養對照組（P值均＜0.01），但與人工餵養對照組比較，差異無統計學意義（P＞0.05）；TLR4和NF-κB p65蛋白錶達量均低于人工餵養對照組和雙歧桿菌對照組（P值均＜0.05），但與母乳餵養對照組比較，差異均無統計學意義（P值均＞0.05）。雙歧桿菌榦預組TLR2蛋白錶達量與3箇對照組比較，差異均無統計學意義（P值均＞0.05）。結論雙歧桿菌可能通過抑製緻病菌或通過TLR2負反饋調節等方式，減少腸黏膜細胞TLR2及TLR4的蛋白錶達，併進一步抑製NF-κB通路，使炎癥反應得以緩解，髮揮對NEC的防治作用。
목적：탐토쌍기간균방치신생대서배사성소장결장염（necrotizing enterocolitis， NEC）적가능분자궤제。방법출생2 h내적신생Sprague-Dawley대서75지，수궤분위5조，매조15지：（1）NEC모형조：인공위양+지다당；（2）쌍기간균간예조：인공위양+지다당+쌍기간균；（3）인공위양대조조：부인공위양；（4）쌍기간균대조조：인공위양+쌍기간균；（5）모유위양대조조：여모서동롱서유위양。지다당안30 mg/kg매천관위1차，공3 d。쌍기간균미낭안1×1010균락형성단위/ml가입도대유품중관위，매천1차。위양72 h，말차위양후공복12 h，단두법처사대서。취말단회장조직광경하관찰형태학개변병대장도손상평분，면역조직화학법검측회장조직 Toll 양수체（Toll-like receptor，TLR）2、TLR4、핵전록인자（nuclear transcription factor，NF）-κB p65단백표체。채용Kruskal-Wallis검험、방차분석、교정χ2검험혹Fisher정학개솔법진행통계학분석。결과 NEC모형조、쌍기간균간예조、인공위양대조조、쌍기간균대조조급모유위양대조조적NEC발생비례분별위11/15、4/15、3/15、2/15여0/15；장도손상평분분별위（3.37±0.27）、（1.53±0.44）、（1.75±0.37）、（0.92±0.39）여（0.30±0.18）분；TLR2단백표체분별위0.35±0.05、0.30±0.03、0.32±0.04、0.30±0.02여0.29±0.03；TLR4단백표체분별위0.48±0.05、0.34±0.03、0.36±0.03、0.37±0.04여0.35±0.02；NF-κB p65단백표체분별위0.43±0.03、0.29±0.03、0.35±0.02、0.32±0.02여0.30±0.02。5조적NEC발생비례、장도손상평분、TLR2、TLR4급NF-κB p65단백표체량비교，차이균유통계학의의（χ2、H혹F치분별위23.836、70.290、8.803、38.599화75.076，P치균＜0.05），NEC모형조균고우기타4조（P치균＜0.05）。쌍기간균간예조적NEC발생비례여3개대조조비교，차이균무통계학의의（P치균＞0.05）；장도손상평분고우쌍기간균대조조급모유위양대조조（P치균＜0.01），단여인공위양대조조비교，차이무통계학의의（P＞0.05）；TLR4화NF-κB p65단백표체량균저우인공위양대조조화쌍기간균대조조（P치균＜0.05），단여모유위양대조조비교，차이균무통계학의의（P치균＞0.05）。쌍기간균간예조TLR2단백표체량여3개대조조비교，차이균무통계학의의（P치균＞0.05）。결론쌍기간균가능통과억제치병균혹통과TLR2부반궤조절등방식，감소장점막세포TLR2급TLR4적단백표체，병진일보억제NF-κB통로，사염증반응득이완해，발휘대NEC적방치작용。
Objective To discuss the possible molecular mechanisms involved in the protective effects of Biifdobacterium on intestinal tissue of necrotizing enterocolitis (NEC) newborn rats. Methods Seventy-five newborn Sprague-Dawley rats (born within 2 h) were randomly divided into five groups, each group with 15 rats. Group A was the NEC model group, and the rats were fed lipopolysaccharide (LPS) and formula. Group B was the Biifdobacterium treatment group, and the rats were fed LPS and formula and Biifdobacterium micro-capsule. Group C was the artificial feeding control group, and the rats were fed formula. Group D was the Biifdobacterium control group, and the rats were fed formula and Biifdobacterium micro-capsule. Group E was the breastfeeding control group, and the rats were fed rat breast milk by mothers. LPS 30 mg/kg was administered by gavage once per day for 3 days. Bifidobacterium micro-capsules were given as 1×1010 colony forming units/ml by gavage with formula once per day. After fed for 72 h and fasted for 12 h, the five groups of rats were killed by decapitation. Morphological changes in the terminal ileum tissue were observed under a light microscope and intestinal injury was scored. The expression of Toll-like receptor (TLR) 2, TLR4, and nuclear transcription factor (NF)-κB p65 was detected by immunohistochemical methods. Kruskal-Wallis test, analysis of variance, corrected Chi-square test and Fisher's exact test were used for statistics. Results The morbidity of NEC in group A to E was 11/15, 4/15, 3/15, 2/15 and 0/15, respectively;the intestinal injury score in group A to E was 3.37±0.27, 1.53±0.44, 1.75±0.37, 0.92±0.39 and 0.30±0.18, respectively; the expression level of TLR2 in group A to E was 0.35±0.05, 0.30±0.03, 0.32±0.04, 0.30±0.02 and 0.29±0.03, respectively;the expression level of TLR4 in group A to E was 0.48±0.05, 0.34±0.03, 0.36±0.03, 0.37±0.04 and 0.35±0.02, respectively;the expression level of NF-κB p65 in group A to E was 0.43±0.03, 0.29±0.03, 0.35±0.02, 0.32±0.02 and 0.30±0.02, respectively. The differences in NEC morbidity, intestinal injury score, and the expression levels of TLR4, TLR2 and NF-κB p65 among the five groups were all statistically significant (χ2, H or F=23.863, 70.290, 8.803, 38.599 and 75.076, respectively, all P<0.05). The values in the NEC model group were all significantly higher than those in the other four groups (all P<0.05). The morbidity of NEC in the Biifdobacterium treatment group compared with the three control groups was not significantly different (all P > 0.05). The intestinal injury score in the Bifidobacterium treatment group was significantly higher than that in the Bifidobacterium control group and the breastfeeding control group (both P < 0.01), but was not significantly different to that in the artificial feeding control group (P > 0.05). The expression levels of TLR4 and NF-κB p65 in the Biifdobacterium treatment group were significantly lower than those in the artificial feeding control group and the Biifdobacterium control group (all P < 0.05), and were not significantly different to those in the breastfeeding control group (P>0.05). The expression level of TLR2 in the Biifdobacterium treatment group compared with the three control groups was not significantly different (all P > 0.05). Conclusions Biifdobacterium may inhibit pathogenic bacteria or regulate the negative feedback of TLR2 to reduce the expression of TLR2 and TLR4 in intestinal mucosa cells, inhibit the NF-κB pathway, attenuate the inflammatory reaction, and play a role in the prevention and control of NEC.
