TLR信号通路蛋白TAK1调控乙肝病毒复制的机制研究

The chronic liver diseases caused by hepatitis B virus (HBV）infection remains a major health burden. Recent studies have shown that Toll like receptor (TLR)-mediated innate immune responses play an important role in the control of HBV replication and disease progression. However, the molecular pathway of intracellular antiviral mechanism remains unclear. From our previous studies, the ligands of TLR2 and TLR4 could inhibit HBV replication in hepatocytes through IFN-independent pathways. By use of siRNAs screening which targeting TLR2/4 pathway proteins, knock down of transforming growth factor-β-activated kinase 1 (TAK1) resulted in significant upregulation of HBV replication. In contrast, overexpression of TAK1 and its dowstreaming proteins IKKα/β inhibited HBV replication and gene expression. Hence, we speculated that TAK1 and its downstream pathway contributed to control of HBV replication and gene expression in hepatocytes. The current project supposed to verify TAK1 antiviral effect on HBV replication in hepatoma cell lines in vitro and in hydrodynamic tail vein injection mouse model in vivo. Further to explore the possible mechanisms by luciferase reporter assay, signaling pathway inhibitors, immunoprecipitation technology, and so on. This study will be helpful to identify the novel molecular mechanism of TLR-mediated anti-HBV response and provide experimental and theoretical evidences for clinical application of TLR ligands in HBV therapy.

乙型肝炎病毒（HBV）感染所致慢性肝病危害严重，Toll样受体（TLR）介导的天然免疫应答在控制HBV复制和感染进展中发挥着重要作用，其胞内抗病毒分子机制尚不明确。本课题组前期研究发现TLR2/4配体可激活肝细胞内非干扰素依赖信号通路抑制HBV复制，进一步利用小干扰RNA沉默TLR2/4通路蛋白筛选发现，降低转化生长因子激酶1（TAK1）表达后显著上调HBV复制，过表达TAK1及其下游分子IKKα/β可以抑制HBV复制和基因表达。由此，我们推测，TAK1及其下游通路参与控制胞内HBV复制及基因表达。本课题拟在肝癌细胞系体外模型和高压尾静脉注射的小鼠模型中确证TAK1调控HBV复制的效应，利用荧光素酶报告基因实验、信号通路抑制剂、免疫共沉淀等方法探讨其可能的作用机制。本研究有助于阐述TLR介导的天然免疫应答调控HBV复制的胞内分子机制,为TLR配体用于乙肝治疗提供实验和理论依据。

肝细胞内乙肝病毒（HBV）的复制受到宿主免疫系统及其相关的胞内信号通路的限制。转化生长因子β激酶1（TAK1）是Toll样受体和炎症因子通路的一个重要接头蛋白。我们研究发现沉默或者抑制肝癌细胞系内源性的TAK1的表达或者功能可以导致HBV复制、转录、抗原表达的上调。反之，过表达TAK1显著抑制HBV复制，而激酶活性缺失的TAK1则无效应。通过siRNAs或者抑制剂筛选TAK1相关的信号通路，我们发现MAPK-JNK通路参与TAK1介导的HBV抑制。更进一步地，沉默TAK1或者抑制JNK通路可以诱导与HBV核心启动子结合的法尼醇受体α(FXRα)的表达。最后，在pAAV-HBV1.2质粒高压尾静脉注射的慢性HBV感染小鼠模型中，TAK1过表达可以显著抑制肝内和血清中HBV DNA和抗原的表达。总结来说，我们的研究发现TAK1可以通过MAPK-JNK通路在转录水平抑制HBV复制和基因表达，是肝细胞控制HBV感染的内源性限制因子。