Toll样受体-4小干扰RNA预处理防治小鼠急性肝损伤

Toll-like receptor-4 siRNA protects mice from acute liver injury induced by lipopolysaccharide and D-galactosamine

目的观察Toll样受体（TLR）-4小干扰RNA（siRNA）对D-氨基半乳糖盐酸盐／脂多糖（I）IGalN／LPS）诱导的肝损伤小鼠的保护作用。方法150只雄性C57BL／6小鼠均分为PBS预处理组、阴性对照质粒预处理组、TS4预处理组、TS6预处理组和TS7预处理组。腹腔内联合注射LPS（10ng／g）及D-GalN（1mg／g）诱导小鼠急性肝损伤。在D-GalN／LPS联合注射前24及48h通过尾静脉高压水注射法导入siRNA质粒50mg／L。末端脱氧核苷酸转移酶介导的dUTP缺口标记术（TUNEL）检测细胞凋亡水平，免疫组织化学法检测肝组织中TLR-4表达，RT-PCR检测TLR-4、TNF-α及巨噬细胞炎性蛋白（MIP）-2 mRNA水平，ELISA检测小鼠血清中MIP-2及TNF-α水平，标准自动分析仪检测血清中ALT及AST水平，苏木精-伊红染色观察肝脏组织学变化，Fisher确切概率法比较各组间小鼠存活率。结果TLR-4 siRNA可减轻肝细胞坏死、减轻炎性反应，并亩丁显著降低血清转氨酶水平。TS4预处理组（0.065±0．015）比PBS预处理组（0．346±0．062）的TUNEL标记指数（LI）明显降低（t=9．796，P〈0.05）。TLR-4 siRNA预处理下调TLR-4 mRNA及蛋白表达水平，显著降低TNF-α及MIP-2 mRNA表达及细胞因子水平，显著降低D-GalN／LPS联合诱导的急性肝损伤C57BL／6小鼠的死亡率和肝损伤。结论TLR-4 siRNA抑制TLR-4表达在防治肝损伤方面可能具有潜在应用价值。

Objective To observe the protective effects of Toll-like receptor （TLR）-4 siRNA against acute liver injury in mice induced by lipopolysaccharide（LPS） and D-galactosamine（D-GalN）. Methods One hundred and fifty C57BL/6 male mice were divided into 5 groups： phosphate buffered solution （PBS） pretreatment group, negative control plasmid pretreatment group, TS4 pretreatment group, TS6 pretreatment group and TS7 pretreatment group. Acute liver injury was induced in mice by intraperitoneal coinjection of LPS （10 ng/g） and D-GalN （1 mg/g）. In vivo delivery of siRNA was performed via the tail vein by hydrodynamic injections （50μg siRNA dissolved in 1 mL PBS） 24 h and 48 h before coinjection of LPS and D-GalN. Expression of TLR-4 in liver tissues was measured by immunohistochemistry. The changes of TLR-4, tumor necrosis factor （TNF）-α and macrophage inflammatory protein （MIP）-2 mRNA levels in liver tissues were determined by reverse transcriptase-polymerase chain reaction （RT-PCR） analysis. MIP-2 and TNF-α concentrations in the sera of mice were determined by enzyme-linked immunosorbent assay （ELISA）. Levels of alanine transaminase （ALT） and aspartate transaminase （AST） in serum were measured by standard autoanalyzer techniques. Liver pathological changes were observed by haematoxylin-eosin staining, while cell apoptosis levels in liver were determined by terminal deoxynucleotidyl-mediated-dUTP nick end labeling （TUNEL） assay. The difference of survival rates in 5 groups was analyzed by Fisher＇s exact probability test. Results Pretreatment with TLR-4 siRNA down-regulated the TLR-4 mRNA and protein expressions, and significantly decreased the mortality and liver injury caused by coinjection of LPS and D-GalN in C57BL/6 mice. TLR-4 siRNA significantly down-regulated the TNF-α and MIP-2 mRNA expression and cytokine levels as determined by RT-PCR and ELISA, respectively. TLR-4 siRNA abrogated hepatocyte necrosis and inflammatory infiltration and also remarkably reduced serum concentrations of transaminases. The percentage of TUNEL-positive hepatocytes was significantly reduced in TLR-4 siRNA pretreatment group （TS4 pretreatment group： 0.065±0.015 vs PBS pretreatment group： 0.346±0.062, P〈 0.05）. Conclusion It suggest that inhibition of TLR-4 expression by TLR-4 siRNA may provide potential application value for preventing liver injury.