肠三叶因子对新生鼠坏死性小肠结肠炎模型iNOS及TNF-α、NO的影响及意义

Effect of intestinal trefoil factor on expression of iNOS, TNF-α and NO in neonatal rat model of necroti-zing enterocolitis

目的探讨肠三叶因子(intestinal trefoil factor,ITF)对坏死性小肠结肠炎(necrotizingenterocolitis,NEC)新生大鼠的肠黏膜组织中iNOS、TNF-α、NO的含量的影响,及ITF对NEC是否具有保护作用.方法建立NEC模型,对新生1日龄Wistar大鼠予100%二氧化碳,5 min后再予以100%氧气,5 min后放回母鼠身边喂养,第4天处死,取肠组织待检.新生鼠40只随机分为五组,每组8只,A组为NEC模型后予以腹腔注射ITF 0.5 mg;B组为NEC模型后予以皮下注射ITF 0.2mg C、D组为NEC模型后予以腹腔注射生理盐水分别为0.5 ml和0.2 ml;E组为正常对照组.取近回盲部1～2cm肠道组织固定包埋、切片、HE染色做病理学检查及免疫组化观察iNOS的表达,其他肠道组织制备组织匀浆取上清液检测TNF-α、NO的含量.结果A、B及E组iNOS呈弱阳性表达,C、D组iNOS呈强阳性表达;A、B组组织匀浆中TNF-α的含量各为[(30.515±2.731)和(32.229±4.978)pg/mg·pro]均较C、D组[(39.957±8.283)、(39.960±8.374)pg/mg·pro]明显下降(P＜0.01),与E组[(33.523±6.752)pg/mg·pro]差异无统计学意义(P＞0.05);A、B组组织匀浆中NO的含量各为[(0.37±0.07)和(0.54±0.08)μmol/mg tissue]较C、D组[(0.76±0.01)和(0.82±0.04)μmol/mg tissue]明显下降(P＜0.01),与E组[(0.41±0.02)μmol/mg tissue]差异无统计学意义(P＞0.05);A、B组间及C、D组间TNF-α、NO含量差异无统计学意义(P＞0.05);病理切片显示C、D组HE染色切片见肠壁损伤轻重不一,可见全肠黏膜绒毛坏死,病理评分的中位积分为3分;A、B组肠上皮细胞少量脱落,顶端绒毛坏死,病理评分的中位积分为1分.结论通过腹腔和皮下注射ITF可以减轻NEC后的肠道炎症反应,ITF有可能为治疗NEC提供新的方法.

Objective To study the effects of intestinal trefoil factor（ITF） on inducible nitric oxide synthase（iNOS）,tumor necrosis factor-α（TNF-α） and NO density in neonatal rat model of necrotizing enterocolitis（NEC）. Methods Forty neonatal rats were randomly divided into five groups： Group A, NEC＋ITF 0.5 rag; Group B, NEC＋ITF 0.2 mg; Group C, NEC＋N. S 0.5 ml; Group D, NEC＋N. S 0.2 ml; Group E, normal control. NEC model was established as following method： one-day old Wistar rats were put into 100% carbon dioxide for 5 rain and then 100% oxygen for 5 min before returned to their mothers. This was done once daily for 3 days. On the 4th day, all rats were sacrificed and the intestinal tissue located at the boundary of ileum and cecum was obtained for histological examination by HE staining and iNOS, TNF-α and NO level by immunohistopathology. Resuits iNOS was found negative in group A, B and E, but positive in group C and D. The density of TNF-α were significantly decreased in group A[（30. 515±2. 731） pg/mg·pro] and B[（32. 229±4. 978） pg/mg·pro] than that in group CF（39. 957±8. 283） pg/mg·pro] and DE（39. 960±8. 374） pg/mg· pro, P〈0.01] and no difference with that in group E[（33. 523±6. 752） pg/mg·pro, P〉 0.05）]. The density of NO were significantly decreased in group A[（0. 37±0. 07） μmol/mg tissue] and B[（0. 54±0. 08） μmol/mg tissue] than that in group C[（0. 76±0.01） μmol/mg tissue] and D [（0.82±0.04）μmol/mg tissue （P〈0.01）], and no difference with that in group E[（0.41±0.02）μmol/mg tissue（P〉0.05）]. The TNF-α and NO expression showed no difference among group A and B, group C and D. The pathological lesions indicate that severe intestinal tissue necrosis in group C and D, and mild in group A and B. Conclusions Intestinal inflammation could be ameliorated after ITF injection hypodermically or intraperitoneally. ITF may provide a brand-new way for the management of NEC in neonatal rats.