Toll样受体4在对乙酰氨基酚肝损伤过程肝细胞再生中的作用

Previous studies have shown that Toll-like receptor 4(TLR4) is involved in the process of APAP-evoked acute liver injury, and the inflammatory cytokines downstream of TLR4 signal play an important role in this process. Our preliminary study has observed that APAP-induced acute liver injury is accompanied by hepatocyte necroptosis and regeneration. We hypothesize that endogenous ligand HMGB1 activates TLR4 signaling and regulates hepatocyte necroptosis and regeneration during APAP-induced acute liver injury. To explore the role of TLR4 signaling and downstream inflammatory factor on APAP-induced hepatocyte necroptosis and regeneration, we are to measure hepatocyte necroptosis and proliferation in APAP-treated tlr4 mutant mice and wild type mice. The present study is to investigate the role of inflammatory cytokine IL-6 which downstream of macrophage TLR4 signaling in regulating hepatocyte regeneration during APAP-induced acute liver injury. To exlpore whether TLR4-mediated IL-6/STAT3 signaling is involved in hepatocyte regeneration during APAP- evoked acute liver injury, the present study is to investigate the effects of the IL-6 neutralizing antibody and STAT3 inhibitor on hepatocyte proliferation in APAP-treated mice. This study will provide theoretical basis for clinical treatment of APAP-induced liver injury.

既往研究表明Toll样受体4（TLR4）参与对乙酰氨基酚（APAP）急性肝损伤过程；且TLR4信号下游炎性细胞因子在急性肝损伤过程中起重要作用。前期研究发现APAP肝损伤过程伴随有肝细胞程序性坏死和肝细胞再生。课题组假设：APAP急性肝损伤过程释放的内源性配体（HMGB1）通过激活TLR4信号，调节肝细胞程序性坏死和再生。本课题拟对比tlr4基因突变型与野生型小鼠在APAP急性肝损伤过程肝细胞程序性坏死和增殖情况，以阐明TLR4信号及其下游炎症因子在APAP肝损伤不同阶段肝细胞坏死和再生中的作用；重点阐明巨噬细胞TLR4下游炎性细胞因子IL-6在调节肝细胞再生中的作用；通过观察IL-6中和抗体和STAT3抑制剂对APAP急性肝损伤不同阶段肝细胞再生的拮抗效应，进一步探讨肝细胞IL-6/STAT3信号调控APAP急性肝损伤肝细胞再生的分子机理。本课题为临床治疗APAP急性肝损伤提供理论依据。

本项目为探讨TLR4信号在APAP肝损伤过程中肝脏再生中的作用。做了以下研究：一、体外培养小鼠正常肝细胞AML12，APAP（2.5mmol/L）分别作用于该细胞12 h、24 h、48 h。结果提示APAP处理后48 h时可能出现肝细胞损伤后再生现象。因此选择STAT3抑制剂AG490(50μmol/L)作用于该细胞。与正常对照组相比，APAP组p-STAT3蛋白和Ki67的mRNA水平升高；与APAP组相比，AG490+APAP组的细胞活力下降，PCNA、CyclinD1、p-STAT3的蛋白和PCNA、CyclinD1、Ki67的mRNA水平均降低。提示AG490通过抑制STAT3磷酸化从而抑制APAP肝损伤后肝细胞增殖。二、APAP作用于AML12细胞0h、4h、12h、24h、48 h，加入IL-6中和抗体（0.01 μg/ml）作用于该细胞。与APAP组相比，APAP+IL-6Ab组CyclinD1、PCNA、p-STAT3、HGF蛋白和PCNA 、CyclinD1的mRNA水平均降低。提示IL-6中和抗体通过抑制IL-6/STAT3通路，抑制了肝细胞再生。三、健康ICR小鼠分成对照组、APAP组和APAP+TAK-242（TLR4抑制剂）组。APAP给药后不同时点（第0、4、12、24、48、72h）剖杀小鼠。结果显示TLR4抑制剂在APAP诱导的小鼠急性肝损伤早期（12h前）可能通过抑制炎症因子（MCP-1、IL-1β、TNF-α、IL-6）的表达进一步减轻肝细胞损伤。而TLR4抑制剂在APAP诱导的小鼠急性肝损伤中、后期可能通过抑制p-STAT3、Cyclin D1、Ki-67 及PNCA的蛋白或mRNA水平，从而进一步抑制了肝脏再生。以上结果提示TLR4在APAP诱导的急性肝损伤早期可能通过促进炎症因子表达而加重肝损伤；中后期可能通过促进肝脏再生相关蛋白和基因表达而促进肝脏再生。IL-6的早期激活加重了肝损伤；而IL-6/STAT3信号通路的激活在APAP急性肝损伤过程中促进了肝损伤后期的肝再生。