暴发性肝衰竭中Toll样受体2表达的实验研究

The experimental study on the expression of toll-like receptor 2 in fulminant hepatic failure

目的 分析D-氨基半乳糖(D-Gal)/脂多糖(LPS)诱导的暴发性肝衰竭模型中肝组织Toll样受体2(TLR2)的表达变化及与细胞因子白细胞介素-18(IL-18)、肿瘤坏死因子α(TNF-α)、γ干扰素(IFN-γ)表达的关系,探讨TLR2在启动炎性应答而致肝损伤中的作用。 方法 BALB/C小鼠腹内联合注射D-Gal 900 mg/kg与LPS 10μg/kg后观察其存活率,并检测不同时间点血清转氨酶和血浆IL-18、TNF-α和IFN-γ含量。用半定量逆转录-聚合酶链反应和Tanon Gis2.0软件分析各时间点肝组织中TLR2 mRNA表达,并与血浆IL-18、TNF-α、IFN-γ含量进行相关分析;免疫组织化学观察肝组织TLR2蛋白的表达。 结果 给药后4 h,血清转氨酶明显升高(与0 h比较,P<0.05);10 h小鼠死亡率达80％。血浆IL-1 8、TNF一α和IFN-γ含量逐步上升,IL-18在1 h即显著升高,之后持续高表达;TNF-α在2 h、5 h有两个分泌高峰;IFN-γ在2 h前增加不明显(F=2.5 7,P=0.1 3),但3 h及以后则显著升高(与0 h比较,P<0.01)。正常小鼠肝组织少量表达TLR2 mRNA,给药后1 h表达即显著增强(与0 h比较,P<0.05);免疫组织化学也显示TLR2蛋白有类似的变化,尤其肝窦内皮细胞、库普弗细胞表达更为显著;且部分肝细胞凋亡、坏死后,残存肝组织仍有较高TLR2表达。相关分析表明,肝组织TLR2

Objective In order to explore the role of toll-like receptors 2 (TLR2) in initiating inflammatory response, the expression of TLR2 of the liver and IL-18, TNF- α and IFN- γ of plasma in fulminant hepatic failure was analysed. Methods D-galactosamine (D-Gal, 900 mg/kg) and lipopolysaccharide (LPS, 10μg/kg) were administered intraperitoneally into the BALB/C mice. To evaluate the hepatic injury, serum transaminase (ALT and AST) and plasma IL-18, TNF- α and IFN-γ were determined and the mortality was observed at various time points following the intraperitoneal injection. The level of TLR2 mRNA was measured by semiquantitative RT-PCR. The protein expression of TLR2 in the liver was detected by immunohistochemistry. The data was analyzed by SAS software. Results After 4 hours of intraperitoneal injection of D-Gal/LPS, the serum transaminase and plasma IL-18, TNF- α and IFN- γ levels were elevated. The treated mice began to die at 7 hours. The mortality reached up to 80% at 10 h. TLR2 mRNA was expressed at a low level in liver tissues of normal mice, while it was significantly increased and maintained at a higher level following intraperitoneal injection with D-Gal/LPS (compared to zero hour time point, P < 0.05). The expression of TLR2 protein was similar to that of the TLR2 mRNA, and the expression of TLR2 mRNA was positively correlated with the concentration of plasma IL-18, TNF- α and IFN- γ (r = 0.36, P = 0.02; r = 0.48, P = 0.003; r = 0.72, P<0.001) at different time points. Conclusions Our results showed that TLR2 was involved in initiating and inducing the expression of proinflammation cytokines in this model of fulminant hepatic failure. The results suggest that adjusting the expression of TLR2 might be a new strategy in preventing the development of infectious diseases.