脂多糖结合蛋白在实验性急性坏死型胰腺炎发生中的作用

The role of lipopolysacchride-binding protein in the pathogenesis of animal model of acute necrotizing pancreatitis

目的 探讨抗脂多糖 (LPS)结合蛋白 (LBP)抗体对小鼠实验性急性坏死型胰腺炎 (ANP)的影响 ,了解LBP在ANP发生中的作用。方法 6 0只BALB/c小鼠分为 4组 :ANP组 (n =18)、ANPLBP抗体处理组 (n =18)、LBP抗体单独处理组 (n =18)和正常对照组 (n =6 )。于第 9、12、2 4h分批处死小鼠并收取标本。测定血清淀粉酶和乳酸脱氢酶 (LDH)活性 ;对胰腺组织HE染色进行病理评分 ;RT PCR检测胰腺组织炎性介质肿瘤坏死因子α(TNF α)、白细胞介素 1β(IL 1β)、细胞黏附分子(ICAM 1)和E 选择素mRNA表达变化 ;应用免疫组化和Western印迹检测核因子 κB(NF κB)蛋白表达的改变 ;酶组织化学法检测中性粒细胞髓过氧化物酶 (MPO)的活性改变。结果 与ANP组相比 ,ANPLBP抗体处理组血清淀粉酶活性于 9h和 12h显著升高 ,LDH活性于 2 4h显著升高。病理学评分结果显示 ,ANPLBP抗体处理组胰腺组织水肿程度于 9h、12h加重 ,而炎性细胞浸润和坏死则于 2 4h明显加重。反映中性粒细胞活化程度的胰腺组织MPO活性于 12h和 2 4h明显升高 ,胰腺组织内炎性介质TNF α、IL 1β、ICAM 1和E 选择素mRNA的表达呈不同程度的上调。免疫组化和Western印迹显示NF κB的表达明显上调。而LBP抗体单独处理组血清生化指标和病理学评分结果与正常对照?

Objective To explore the pathogenic role of lipopolysacchride binding protein (LBP) in the pathogenesis of acute necrotizing pancreatitis (ANP) by applying anti LBP antibody to the animal model of ANP in mice. Methods Sixty BALB/c mice were randomly divided into four groups, including ANP group( n =18), ANP treated with anti LBP antibody group ( n =18), anti LBP antibody group ( n =18) and normal control ( n =6). ANP model was induced by seven times administration of cerulein (50 μg/kg·body weight), challenged by lipopolysaccharide (LPS) (5 mg/kg)intravenous injection. Treatment with anti LBP antibody was started 15 minutes before LPS injection in ANP treated with anti LBP antibody group. Anti LBP antibody group only received intravenous injection of anti LBP antibody, normal saline was administrated intraperitoneally instead of cerulein and LPS. At 9 h,12 h and 24 h after the first injection of cerulein (or saline), the serum levels of amylase and lactate dehydrogenase (LDH) were measured. The severity of pancreatitis was evaluated by histological scoring system. Intrapancreatic TNF α 、IL 1β、ICAM 1 and E selectin mRNA expressions were studied by semi quantitative 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 the ANP group, a marked elevation of serum amylase was observed 9 h and 12 h after cerulein administration and a marked elevation of serum LDH was observed 24 h after cerulein administration in the ANP treated with anti LBP antibody group. Histologically, treatment with anti LBP group increased the severity of pancreatic injury including edema at 9 h and 12 h after, and inflammatory cell infiltration and necrosis 24 h after. Intrapancreatic TNF α, IL 1β, ICAM 1 and E selectin mRNA levels were increased. The activity of MPO was increased significantly 12 h and 24 h after in the anti LBP antibody group. Immunohistochemistry and Western blotting showed up regulation of NF κB. However, there was no significant difference in serum parameters and pathologic scoring results between LBP antibody group and normal control. Conclusion LBP plays a protective role in the pathogenesis of ANP, and this action may be mediated by inhibiting of NF κB activation and down regulation of proinflammatory mediators.