肠三叶因子在内毒素致幼鼠肠损伤中的作用及意义

Protective effects of recombinant intestinal trefoil factor against intestinal injuries induced by endotoxin in young rats

目的探讨内毒素(LPS)致幼鼠肠损伤中二胺氧化酶(DAO)及肿瘤坏死因子(TNF-α)的变化及基因重组肠三叶因子(rITF)的保护作用,为临床治疗提供实验依据。方法Wistar幼鼠10日龄96只分为3组,A组:生理盐水对照组;B组:LPS组;C组:LPS+rITF组,每组32只。以生理盐水(1mL/kg),EcoliO55:B5(1mL/kg)、rITF(0.1mL/只、配制浓度5g/L)腹腔注射后2,6,24,72h断头处死动物,取动静脉混合血,测血DAO活性。留取肠组织,免疫组化法检测TNF-α蛋白含量,PCR法检测TNF-αmRNA表达。同时作电镜观测肠组织超微结构变化。结果B组血浆DAO活性自LPS作用后2h即开始升高,至6h达到最高值,较A组差异有显著性(1.519±0.13U/Lvs1.081±0.04U/L,P<0.01),72h仍持续高值;C组血浆DAO活性较B组明显下降(P<0.01或P<0.05);与A组6h比较差异有显著性(P<0.01),其余各时间点差异无显著性(P>0.05);B组TNF-α积分光密度含量明显高于A组,以LPS作用后6h达高峰(37247.64±3387.59vs6191.02±482.32,P<0.01),C组TNF-α含量较B组明显降低,但仍高于A组(P<0.01);TNF-αmRNA在A组各时间点有微弱的表达,在B组各时间点表达明显增强,较A组差异显著(P<0.01);而C组各时间点表达明显减少,较B组差异显著(P<0.01或P<0.05)。电镜下肠组织超微结构:A组正常,B组变化明显,C组变化较B组减轻。结论ITF可降低血浆DAO活性,抑制肠组织TNF-αmRNA及蛋白表达,减轻LPS所致幼鼠肠损伤,发挥保护作用。

Objective This study aimed to investigate the protective effects of recombinant intestinal trefoil factor （rlTF） against intestinal injuries and the possible mechanism by examining the changes of diamine oxidase （DAO） and TNF-α and the intestinal ultrastructural changes in lipopolyseccharide （LPS） induced intestinal injuries. Methods Ninety-six ten-day-old Wistar rats were randomly injected with either normal saline （ 1 mL/kg, Control group ） , LPS （1 mL/kg） or LPS （1 mL/kg） ＋ rlTF （0.1 mL） intraperioneally. At 2, 6, 24 and 72 hrs after administration plasma DAO activity was determined using absorption spectrometry; and the intestinal protein and mRNA expression of TNF-α were measured using immunohistochemistry and RT-PCR methods. The intestinal ultrastructural changes were observed by electron microscopy. Results The plasma DAO activity in the LPS group began to increase at 2 hrs, peaked at 6 hrs and remained at significantly higher levels until 72 hrs after administration compared with the Control group （P 〈 0.01 ）. The plasma DAO activity in the LPS ＋ rlTF group decreased noticeably compared with the LPS group at all time points（P 〈 0. 01 or 0. 05 ）. A significant difference in the plasma DAO activity was only observed at 6 hrs after administration between the LPS ＋ rlTF and the Control group. The expression of TNF-α protein in the LPS group significantly increased at each time point, peaking at 6 hrs after LPS administration, with the IODT of TNF-α of 37 247.64 ± 3387.59 vs 6 191.02 ± 482.32 （P 〈0. O1 ） compared with the Control group, rlTF treatment decreased the expression of TNF-α protein although it remained significantly higher than in the Control group （ P 〈 0.01 ）. The TNF-α mRNA was weakly expressed in the Control group but strikingly increased after LPS injection （P 〈0.01 ）. Compared with the LPS group, the TNF-α mRNA expression in the LPS ＋ rlTF group decreased at all time points（P 〈0.01 or 0.05）. Vacuole changes of mitochodrium,cell nucleus condense, break and depletion of part of microvilli, and widen.and disrupted tight junction were observed in the LPS group. The ultrastructural changes of intestinal tissues were improved in the LPS ＋ rlTF group. Conclusions rlTF can decrease the plasma DAO activity and inhibit the expression of TNF-α, resulting in a protective effect against intestinal injuries induced by LPS in young rats.