Slit2-Robo1信号通路调控肝纤维化的作用与机制研究

Recent studies suggest that the activation of Slit2-Robo1 signaling pathway is involved in inflammation and the formation of a variety of tumors including hepatocellular carcinoma (HCC). However, it is still poorly understood that whether Slit2-Robo1 signaling pathway mediates the pathogenesis of liver fibrosis-the precursor of HCC. It has been demonstrated that sustained activation of TGF-β1/Smad3 pathway plays a pivotal role in the pathogenesis of liver fibrosis. In addition, upregulation of TGF-β1 is dependent on the activation of NF-κB signaling. We have previously shown that Slit2-Robo1 signaling pathway was activated in the fibrogenic livers of carbon tetrachloride (CCl4)-treated Slit2 transgenic mice. Moreover, the activation of Slit2-Robo1 signaling pathway further stimulated TGF-β1/Smad3 pathway, accelerating liver fibrosis. Here, we hypothesizes that Slit2-Robo1 signaling pathway mediates the pathogenesis of liver fibrosis through activating NF-κB signaling and subsequently stimulating TGF-β1/Smad3 pathway, eventually accelerating liver fibrosis. To demonstrate the hypothesis, our project will investigate the action and mechanism of Slit2-Robo1 regulating the pathogenesis of liver fibrosis, which is beneficial to offerring novel anti-fibrogenic approaches and drug targets for the treatment of liver fibrosis.

近年研究表明：Slit2-Robo1信号通路的激活参与了肝癌等多种肿瘤的形成及炎症反应。然而Slit2-Robo1信号通路能否调节肝癌重要前期病变- - 肝纤维化的病理进程尚不明确。TGF-β1/Smad3通路的持续激活在促进肝纤维化病程中起着关键作用，TGF-β1的上调依赖于NF-κB信号的活化。我们前期发现：在CCl4诱导的Slit2转基因鼠肝纤维化组织中，Slit2-Robo1信号通路处于激活状态，并进一步激活TGF-β1/Smad3通路，促进小鼠肝纤维化进程。据此推测，Slit2-Robo1信号通路可通过活化NF-κB信号进而激活TGF-β1/Smad3通路，促进肝纤维化进程。为证实该假说，本课题拟在基因工程小鼠及临床肝纤维化组织标本等不同水平深入研究Slit2-Robo1信号通路在肝纤维化中的作用和机制，为肝纤维化疾病的防治提供新思路和药物作用的新靶点。

肝纤维化（Liver Fibrosis）是由多种损伤因子持续刺激肝脏，促使肝肌成纤维细胞分泌大量细胞外基质（Extracellular Matrix, ECM）的病理过程。而肝肌成纤维细胞主要由肝星状细胞（Hepatic Stellate Cells, HSCs）转化而来。肝星状细胞（HSCs）的活化与肝纤维化的形成密切相关，但具体机制尚不明确。临床样本研究发现：与正常对照相比，肝硬化患者的肝脏组织和血清Slit2和Robo1表达量显著升高，提示Slit2-Robo1信号可能与临床肝纤维化的发生发展密切相关。鉴于临床数据的提示，我们在Slit2-Tg小鼠和Robo1/2+/-小鼠肝纤维化模型上，观察到Slit2过表达加重了肝纤维化病程，而Robo1敲除可明显改善CCl4诱导的肝损伤和纤维化程度。随后我们提取了小鼠原代肝HSCs，证实Slit2-Robo1信号通过调节Smad2/3和PI3K/AKT的磷酸化，进而介导HSCs的活化，促进肝纤维化发生发展。同时我们采用R5对CCl4诱导的C57小鼠进行治疗，证实R5对改善CCl4诱导的小鼠肝纤维化具有显著的治疗效果，其机制可能是通过抑制HSCs的活化来实现。通过细胞定位研究，我们发现Slit2和Robo1主要在Kupffer细胞和HSCs表达，而在肝细胞中没有表达；并且Kupffer细胞表达的Slit2和Robo1明显多于HSCs中的Slit2和Robo1，提示Kupffer细胞中的Slit2-Robo1信号可能参与了肝纤维化过程。最后我们明确了Slit2-Robo1信号介导CCl4诱导的小鼠肝Kupffer细胞中TGF-β1的表达。Kupffer胞内Slit2-Robo1信号对HSCs细胞的活化可能发挥重要调节作用。本项目的完成揭示了Slit2-Robo1信号在小鼠肝纤维化形成中的重要作用及其分子机制，为肝纤维化的防治提供一种新思路和药物作用新靶点。