Toll-like receptor-4 siRNA protects mice from acute liver injury induced by lipopolysaccharide and D-galactosamine / 中华传染病杂志

ABSTRACT

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(50 μg 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 nflammatory protein(MIP)-2 mRNA levels in liver tissues were determined by reverse transcriptasepolymerase 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.ResultsPretreatment 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).ConclusionIt suggest that inhibition of TLR-4 expression by TLR-4 siRNA may provide potential application value for preventing liver injury.