TLR4 siRNA预处理防治小鼠急性肝损伤

Effect of TLR4 siRNA for Preventing Acute Liver Injury in Mice

目的急性肝功能衰竭是重要的临床综合征。近年来,对急性肝功能衰竭的治疗进展甚微,如何从根本上解决这一难题依然任重道远。方法本研究基于pSilencer 4.1-CMV neo siRNA表达载体,设计并构建了TLR4 siRNA表达载体。经体外细胞转染实验,筛选了3条抑制效果较好的表达载体,进一步行体内动物实验。进行酶切及测序鉴定,体外评估其对转染RAW264.7细胞TLR4 mRNA、蛋白表达水平的抑制作用,进一步评价了TLR4 siRNA对TNF-α、MIP-2水平的影响,及对LPS诱导的丝裂原活化蛋白激酶(MAPK)信号通路的作用;之后再采用3条抑制效率较高的TLR4 siRNA进行体内试验,采用尾静脉高压水注射法转染C57BL/6小鼠,静默TLR4的表达,再采用D-Ga1N/LPS联合诱导小鼠急性肝损伤,观察TLR4 siRNA对D-GalN/LPS诱导的肝损伤小鼠是否有保护作用,观察TNF-α、MIP-2水平的变化,观察肝细胞凋亡水平变化。结果 D-GalN/LPS联合给药前48 h及24 h TLR4 siRNA预处理可明显降低ALT及AST的升高幅度,TNF-α及MIP-2的mRNA及细胞因子水平亦被抑制,TUNEL阳性肝细胞百分率下降。TLR4siRNA预处理可明显减轻D-GalN/LPS对C57BL/6小鼠的肝损害作用,降低小鼠的死亡率。通过TLR4 siRNA阻断TLR4表达可能有助于控制LPS炎性反应,防治肝损伤。结论通过本研究可望进一步阐明LPS/TLR4在急性肝衰竭发病机制中的作用,为今后从RNA角度治疗肝功能衰竭提供理论和实验依据。

Objective Acute liver failure is an important clinical syndrome. In recent years, advances in the treatment of acute liver failure is minimal, how to solve this fundamental problem remains to be done. Methods In the current study, we designed and constructed TLR4 siRNA expression vectors based on pSilencer 4.1-CMV neo siRNA expression vector. After in vitro transfection experiments, screen three better expression vectors to further in vivo animal experiments. By enzyme digestion and sequencing, in vitro evaluate the inhibition of TLR4 mRNA and protein expression levels of the transfected RAW264.7 cells, further evaluate the effects of TLR4 siRNA on TNF- α, MIP-2 level, and on the role of LPS-induced mitogen-activated protein kinase （MAPK） signaling pathway, After then, in vivo test using three TLR4 siRNA with high inhibition efficiency. And transfect C57BL/6 mice by intravenous injection of high pressure water, then using D-GalN/LPS joint induce acute liver injury in mice. Observe TLR4 siRNA on liver injury in mice induced by D-GalN/LPS whether there is protective effect, changes in TNF-α, MIP-2 levels and changes in the level of liver cell apoptosis. Results Pretreatment with TLR4 siRNA 48 h and 24 h before D-GalN/LPS challenge markedly inhibited elevation of serum ALT and AST levels. The mRNA and cytokine levels of TNF-α and 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. Blocking TLR4 expression by TLR4 siRNA may help control inflammatory response, preventing and treating liver injury. Conclusion Through this study it is expected to further clarify the role of LPS/TLR4 in the pathogenesis of acute liver failure, providing theoretical and experimental evidence for the future treatment of liver failure from the perspective of RNA.