脾切除对内毒素诱发肝脏脂多糖结合蛋白和TNFα基因表达的影响

Effect of splenectomy on endotoxin mediated lipopolysaccharide-binding protein and TNF-α gene expression

目的:观察脾切除动物受内毒素攻击后肝脏脂多糖结合蛋白及肿瘤坏死因子基因表达的变化,探讨脂多糖结合蛋白在脾切除后感染中的可能作用。 方法:(?)Wister大鼠112只,随机分为2组:假手术对照组(n=56只):行大网膜切除;脾切除组(n=56只):行脾切除术,每组根据时间点分为内毒素注射前、注射后1/6,0.5,1.5,4,12,24h组;各组均留取肝组织标本,采用改良过氯酸法预处理组织匀浆液、鲎试剂基质显色法检测组织内毒素水平;采用RF-PCR法检测组织LBP,TNF-αmRNA表达水平,ELISA法测定组织TNF-α含量,对肝脏行常规病理检查。 结果:静脉注射内毒素后,脾切除动物肝组织内毒素较有脾动物明显升高(21.5±4.3EU/g vs 10.9±3.3EU/g,P<0.01)。脾切栋大鼠内毒素攻击后肝组织LBP mRNA及TNF-αmRNA表达峰值较对照组明显增高(LBP mRNA:1.45±0.31 vs 1.09±0.17,TNF mRNA:1.39±0.19 vs 0.97±0.16,P<0.05),且峰值持续时间延长,脾切除动物肝组织TNF水平较有脾动物明显增高,相关分析表明,组织TNF-α水平与TNF-αmRNA表达呈显著正相关,TNF-αmRNA表达与LBP mRNA表达显著相关。HE染色光镜下示有脾动物肝细胞空泡及脂肪变性,无脾大鼠可见肝小叶内散在小灶性坏死。 结论:脾切除后机体对内毒素清除延迟,内毒素在组织积聚,组织细胞LBP mRNA?

AIM: To investigate the role of lipopolysaccharide-binding protein (LBP) gene expression in the pathogenesis of endotoxemia-mediated systemic inflammation and organ damages after splenectomy.METHODS:Total 112 male Wistar rats were randomly divided into two groups: control group (n = 56), including omentectomy and mobilization of the spleen; s plenectomy group (n = 56): including 10 minutes, 0.5, 1.5,4, 12 and 24 hours subgroups after endotoxin injection. Tissue specimens from liver were obtained. Endotoxin concentrations in the tissue were measured with chromogenic limulus amebocyte lysate (LAL), which was modified by perchloric acid ( PCA) pretreatment for samples. LBP and TNF-α mRNA expression were semi -quantitated by the reverse transcription polymerase chain reaction (RT-PCR) using (-actin as an internal standard. Enzyme-linked immunosorbent assay ( ELISA ) was performed for tumor necrosis factor-α(TNF-α) measurement. The biopsy were carried out for the histological examination.RESULTS: After endotoxin administration, liver endotoxin concentrations were higher in splenectomized rats than those in controls at 10 minutes(21.5 ± 4.3EU/g vs 10.9 ± 3. 3EU/g, P < 0. 01). Maximal liver LBP mRNA and TNF-α mRNA expression was higher in animals with asplenia than those with eusplenia (LBP mRNA: 1.45 ±0.31 vs 1.09± 0.17 TNF mRNA 1.39±0.19 vs 0.97±0.16, P<0.05). Liver TNF-α levels increased markedly in splenectomy groups following endotoxin administration. There was a positive correlationbetween tissue TNF-α levels and TNF-α mRNA expression. Similar result also obtained between TNF-α mRNA and LBP mRNA expression. H-E staining showed hepatic cell vacudation in control groups and steatosis in model groups.CONCLUSION: Splenectomy can lead to impairment of tissue clearance of endotoxin in liver. Endotoxin accumulated in the local site may markedly up-regulate LBP mRNA expression, and excessive LBP mRNA expression enhances synthesis and release of inflammatory factor. Thus, LBP increases LPS-induced production of inflammatory cytokines.