重组血栓调节蛋白N端结构域和可溶性晚期糖化终末产物受体对急性肝衰竭模型鼠的保护作用

Protective effect of recombinant TM-N and recombinant soluble RAGE in a mouse model of acute hepatic failure

目的 探索重组血栓调节蛋白N端结构域（rTM-N）和可溶性晚期糖化终末产物受体（rsRAGE）对急性肝衰竭模型小鼠的保护作用. 方法 原核表达、纯化得到rTM-N和rsRAGE,腹腔注射rTM-N或rsRAGE至急性肝衰竭模型小鼠,用不同方法检测肝组织高迁移族蛋白（HMG） B1及核因子-κ B的表达水平、血清中肿瘤坏死因子α和白细胞介素1 β的含量.对数据进行单因素方差分析. 结果 成功表达并纯化rTM-N和rsRAGE,相对分子质量约为1600和31600.各实验组小鼠肝组织坏死情况明显好转,免疫组织化学检测显示,HMGB1表达水平在实验1组和实验2组明显低于对照组；免疫荧光检测核因子-κB的表达水平,在实验1组和实验2组也明显低于对照组.RT-PCR检测HMGB1 mRNA显示,实验1组峰值（114.99±6.53）和实验2组峰值（98.44±13.69）明显低于对照组峰值（199.30±11.51）,差异有统计学意义（P＜0.05）.血清肿瘤坏死因子α在实验1组12h的峰值为（355.42±53.55）pg/ml、实验2组12h的峰值为（451.15±79.66）pg/ml,也显著低于对照组6h的峰值（634.23±35.18）pg/ml,差异有统计学意义（F=109.31,P＜0.05）.血清白细胞介素1 β在实验l组的峰值（457.32±34.99） pg/ml和实验2组的峰值（530.66±38.83）pg/ml显著低于对照组在6h达到的峰值（688.91±48.27） pg/ml,差异有统计学意义（F=65.48,P＜0.05）.结论 rsRAGE和rTM-N均可显著减少急性肝衰竭模型小鼠肝组织HMGB1的表达水平,显示对急性肝衰竭鼠较强的保护作用.

Objective To evaluate the roles of N-terminal lectin-like domain of thrombomodulin （TM-N） and receptor for advanced glycation end products （RAGE） in acute hepatic failure using a mouse model system.Methods Acute hepatic failure was induced in Kunming mice by intraperitonenl injection of D-galactosamine （D-Galn at 600 mg/kg） and lipopolysaccharide （LPS at 5 μg/kg） and mice were divided into groups for injection with saline,recombinant （r）TM-N protein,or recombinant soluble （rs）RAGE protein.Unmanipulated model mice served as the negative controls.Effects on liver expression of high mobility group box-1 （HMGB1） were detected by immunohistochemistry and real time RT-PCR.Effects on serum levels of tumor necrosis factor-alpha （TNFα） and interleukin-1 beta （IL）-1b were quantified by ELISA.Results Treatment with rTM-N and rsRAGE both alleviated the acute liver damage induced by D-Galn/LPS exposure,and decreased the hepatic expression of HMGB1 as well as the serum levels of TNFα and IL-1b.Conclusion Intraperitoneal delivery of rTM-N and rsRAGE can alleviate acute liver damage by modulating the expression of necrosis-and inflammatión-related factors.