Wnt信号通路对肝星状细胞收缩与门脉高压的调控机制

Hepatic stellate cells (HSCs) critically regulate liver microcirculation. Their abnormal contraction is one of major contributors to cirrhosis portal hypertension (PH). Recent evidence indicates that Wnt pathway plays a pivotal role in the pathogenesis of chronic liver disease and HSC activation. We previously elucidated the molecular mechanisms underlying HSC involvement in liver pathological angiogenesis, and our prior data suggested that Wnt signaling affected cell contraction-related molecules in HSCs and their biological behaviors. Based on these observations, the current program will deeply investigate the role of Wnt pathway in HSC contraction and cirrhosis PH. Firstly, we will establish two kinds of cirrhosis portal hypertension models with different etiologies in rats and isolate the primary HSCs for culture to establish the essential relevance of Wnt pathway to PH. Then normal rat primary HSC and human LX-2 cells will be used to explore the molecular mechanisms of Wnt pathway modulation of HSC contraction and determine whether Wnt regulation is dependent on β-catenin. Finally, we will use in vivo and in vitro models to investigate how Wnt pathway regulates glycolysis that are closely related to HSC contraction. These investigations will from two aspects of signal transduction and energy metabolism to systematically reveal the key role of Wnt pathway in regulation of HSC contraction and PH pathology, and provide potential molecular targets for intervention of cirrhosis PH.

肝星状细胞（HSC）对肝脏微循环有重要调节作用，HSC收缩是导致肝硬化门脉高压（PH）的主要原因之一。新近发现Wnt通路在慢性肝病及HSC活化过程中起重要作用。申请者前期揭示了HSC参与肝脏病理性血管生成的分子机制，且预实验显示Wnt通路可激活HSC收缩相关蛋白并影响其生物学行为。在此基础上，本项目拟深入研究Wnt通路在HSC收缩及肝硬化PH中的作用。首先建立大鼠两种PH模型并分离原代HSC，体内外考察Wnt通路与PH及HSC收缩的相关性；然后以大鼠原代HSC和人LX-2细胞探究Wnt通路调控HSC收缩的分子机制，并明确是否依赖于β-catenin；再以体内外模型研究Wnt通路调控HSC糖酵解继而影响HSC收缩及门脉高压的分子机制。以此从信号转导与能量代谢的角度揭示Wnt通路是HSC收缩和PH病理的关键调控节点，为有效干预PH提供潜在分子靶标。

肝硬化门脉高压是严重危害人类生命健康的常见疾病。肝脏特异性血管周细胞肝星状细胞（HSC）能够与肝窦毛细血管紧密接触，影响肝脏血管的结构与功能。近年来发现HSC的收缩特性是调节肝脏微循环与门静脉血流的关键环节，决定了门静脉阻力及门静脉血流动力学的改变，因而是门脉高压发生发展的核心因素。鉴于Wnt信号通路在慢性肝病及HSC活化过程中的重要作用，本项目提出Wnt信号通路是调控HSC收缩与门脉高压病理过程关键分子途径的科学假说。为此，我们检测了临床肝纤维化、肝硬化病人的肝组织样本，在体内实验中建立了经典的CCl4和胆管结扎诱导大鼠或小鼠的肝硬化模型，在体外实验中培养HSC进行细胞形态学与功能学实验，发现：（1）在人、大鼠和小鼠肝脏中Wnt/β-catenin信号通路的活化与HSC活化及肝硬化门脉高压的形成过程密切相关；（2）Wnt/β-catenin信号通路通过TCF4依赖性的Sufu转录抑制促进Gli1的核转位，进而诱导了HSC收缩；（3）Wnt/β-catenin信号通路还可通过上调LDH-A的转录与表达，进而增强了HSC的有氧糖酵解能量代谢以及细胞收缩过程；（4）Gli1介导的Hedgehog信号通路通过转录激活HSP90促进HIF-1α的胞内集聚，从而正向调控HSC的周细胞功能；天然产物Ligustrazine通过阻断Hedgehog/Gli信号通路抑制了HSC周细胞功能。通过本项目研究，我们建立了较为完备的以HSC为靶细胞的肝脏病理性血管重构机制与药物干预机制的研究技术体系；通过将分子病理机制与药物调控机制研究相结合，从信号转导与能量代谢两个方面，揭示了Wnt信号通路及其与Hedgehog/Gli1信号通路之间的串话调控HSC收缩及肝硬化门脉高压的新机制，为药物干预肝硬化门脉高压及肝脏病理性血管重构提供了潜在的分子靶标，具有显著的理论与转化意义。