An Experimental Study On The Effects Of TLR4 SiRNA For Preventing Acute Liver Injury In Mice

Liver failure induced by various pathogenic factors (such as hepatitis virus, alcohol, drugs and hepatotoxicants, etc.) is a very common clinical condition that affects millions of people every year. Despite a combination of all available treatment, the mortality of hepatic failure is higher than 60%. Bioartificial liver support and hepatocyte transplantation are promising treatment options, but currently orthotopic liver transplantation remains the only treatment modality that has an impact on survival. Because of limitations of donor organs, high expense and immunologic rejection, it is urgent to find a more effective therapeutic measurement for liver failure.Intestinal endotoxemia plays a crucial role in the pathogenesis of liver failure. A correlation between endotoxemia and liver injury was demonstrated in animal models and patients suffering from liver failure. Intestinal endotoxemia commonly results in excessive inflammatory responses, with serious hepatic necrosis, further severe hepatitis and even induces acute liver failure.Endotoxins consist of lipopolysaccharide(LPS) and lipoprotein complexes. LPS is the major active component of endotoxins and performs important pathophysiological functions. TLR4 (Toll-like receptor 4) has been identified as a critical receptor for LPS signaling. Sensitivity to LPS is likely to be controlled through modulation of TLR4 expression. However, there is little evidence in vivo to support this assumption. Therefore, we considered that it would be more informative to study the therapeutic efficacy of TLR4 gene silencing in vivo.RNAi is a powerful tool to silence gene expression post-transcriptionally. In the current study, we designed and constructed TLR4 siRNA expression vectors based on pSilencer 4.1-CMV neo siRNA expression vector. We then tested the silence potency of these TLR4 siRNA expression vectors through transfection into RAW264.7 cells. Then we evaluated the therapeutic efficacy of TLR4 gene silencing with siRNA in vivo in a murine model of liver injury induced by D-GalN/LPS. The experimental results are as follows:1.The recombinant vectors were successfully constructed as confirmed by enzyme digest and sequencing. The in vitro experiment indicated that TLR4 siRNA markedly down-regulated the TLR4 mRNA and protein levels on RAW264.7 cells. Treatment with TLR4 siRNA significantly decreased the rise amplitude of tumor necrosis factor-α(TNF-α) and macrophage inflammatory protein 2 (MIP-2) caused by LPS. TLR4 siRNA treatment also down-regulated the phosphorylation levels of p38-MAPK and ERK 1/2 activiated by LPS.2. Pretreatment with TLR4 siRNA 48h and 24h before D-GalN/LPS challenge markedly inhibited elevation of serum ALT and AST levels. The mRNA and cytokine levels of TNF-αand MIP-2 were also been attenuated. TLR4 siRNA significantly reduced the percentage of TUNEL-positive hepatocytes. Pretreatment with TLR4 siRNA significantly decreased the mortality and liver injury caused by coinjections of D-GalN and LPS in C57BL/6 mice. ConclusionCollectively, our findings suggest that inhibition of TLR4 expression by TLR4 siRNA may be therapeutically beneficial in controlling the overall responses of immune cells to LPS and preventing liver injury.