Toll样受体4在实验性急性坏死型胰腺炎形成中的介导作用

Impact of lipoplysacchride receptor-toll like receptor 4 in animal model of acute necrotizing pancreatitis

目的 应用抗内毒素受体 Toll样受体 4 (TLR4 )抗体体内注射观察其对小鼠实验性急性坏死型胰腺炎 (ANP)的影响 ,探讨内毒素信号通路 ,尤其是Toll样受体 4在ANP发生中的介导作用。方法 1 0只BALB/c小鼠随机分为ANP组和ANPTLR4抗体处理组。ANP模型制备采用腹腔内注射雨蛙肽和脂多糖 (LPS)。TLR4抗体处理组于LPS注射前 1 5min静脉注射TLR4单克隆抗体 ,剂量为 2 0 μg。 1 2h后处死小鼠并收取标本。测定血清淀粉酶和乳酸脱氢酶 (LDH)水平 ;胰腺组织切片行HE染色并作病理评分 ;RT PCR检测胰腺组织炎性介质TNF α、IL 1 β和ICAM 1mRNA表达变化 ;免疫组化和westernblot检测胰原组织核因子 -κΒ (NF κΒ)p6 5亚单位蛋白表达的改变 ;酶组织化学方法检测中性粒细胞特异性髓过氧化物酶(MPO)活性的变化。结果 与ANP模型组相比 ,TLR4抗体处理组血清淀粉酶活性 [1 2 6 1 2± 5 70 6U/Lvs2 341 2± 892 2U/L ,P <0 0 5 ]和LDH活性 [1 92 6± 4 0 0U/Lvs2 5 74± 2 77U/L ,P <0 0 5 ],较ANP模型组显著升高 ;病理评分结果显示 ,TLR4抗体处理组胰腺组织坏死和炎症反应程度明显加重 ;胰腺组织炎性介质TNF α、IL 1 β和ICAM 1mRAN表达呈显著上调 ;胰原组织NF κBp6 5亚单位的蛋白表达也呈上调趋势 ;

Objective To explore the role of lipopolysacchride receptor toll like receptor 4(TLR4) in the pathogenesis during the course of acute necrotizing pancreatitis(ANP) by appiying anti TLR4 antibody to the animal model of ANP in mice.Methods Ten BALB/c mice were randomly divided into two groups,ANP group and ANP treated with anti TLR4 antibody group.ANP model was induced by administration of cerulein(50μg/kg·body weight),challenged by lipopolysaccharide(LPS)(5mg/kg) intraperioneal injection.Treatment with anti TLR4 antibody was started 15 minutes before LPS injection.At 12hr after the first injection of cerulein,serum levels of amylase and lactate dehyrogenase(LDH)were measured.The sevrity of pancreatitis was evaluated by histological scoring system.Intrapancreatic TNF α、IL 1β and ICAM 1 mRNA expressions were studied by RT PCR.The activation of nuclear factor κB(NF κB)in the pancreas was investigated by the methods of immunohistochemistry and western blot.The activity of PMN myeloperoxidase(MPO) was determined by zymohistochemistry.Results Compared with ANP group,a marked elevation of serum amylase[12 612±5 706 U/L vs 23 412±8 922U/L,P<0 05] and LDH[1 926±400 U/L vs 2 574±277 U/L,P<0 05])was observed in anti TLR4 group.Histologically,treatment with anti TLR4 group increased the severity of pancreatic injury including inflammatory cell infiltration and necrosis.Intrapancreatic TNF α、IL 1β and ICAM 1 mRNA levels were increased.The activity of MPO was increased significantly in anti TLR4 antibody group.Immunohistochemistry and western blotting showed up regulation of NF κB.Conclusion When anti TLR4 antibody was applied to the ANP model,the severity of pancreatitis was aggravated.It might be deduced that this monoclonal antibody shares the same antigen epitope with LPS,therefore it mimics the effect of LPS and is capable of transducing endotoxic signal which in turn induce the activation of NF κB,up regulation of proinflammatory mediartors and activation of PMN.This suggests that endotoxin signal transduction pathway promotes the pathogenesis of ANP. [