Defining the oxidative stress-related mechanisms by which activation of the transcription factor Nrf2 arrests and resolves liver fibrosis

肝脏的反复伤口愈合会导致纤维化，导致疤痕形成，如果严重，还会导致肝硬化。这是一种严重的疾病，可能会导致肝功能衰竭和肝癌。肝硬变是由多种原因引起的，包括病毒、酒精、自身免疫、代谢状况和肥胖相关的肝损伤。肝硬变的人数急剧增加，这对我们的国民健康保险制度构成了巨大的挑战。最令人担忧的是，除了移植，目前还没有有效的治疗肝纤维化的方法。最近我们发现，通过激活细胞内抗氧化系统的主要调节因子Nrf2，我们能够阻止和/或逆转小鼠已建立的和正在进行的肝纤维化。本项目的目的是确定Nrf2的激活如何改变肝瘢痕组织的形成和去除。这种疤痕是由肝脏中称为肝星状细胞(HSCs)的特定细胞形成的，并被招募到肝脏的称为巨噬细胞的免疫细胞清除。肝纤维化的一个重要特征是，它的启动、持续和发展涉及肝脏内不同类型细胞之间的合作，包括免疫细胞，这是由各种信号分子的产生所控制的，这些分子允许不同类型的细胞之间的通信。一类这样的分子是氧化剂，称为活性氧簇(ROS)，它会触发促纤维化和促炎的信号级联反应。由于肝纤维化的发展涉及肝干细胞产生ROS，从而触发氧化还原信号，导致其分化为可增殖的胶原分泌的肌成纤维细胞，我们设想HSC中Nrf2的激活通过诱导抗氧化剂基因来阻止肝脏纤维瘢痕组织的合成，从而使ROS失活并阻止氧化还原信号。ROS还模拟招募到肝脏的巨噬细胞的炎症反应，所以它们不是允许它们杀死HSCs和消化细胞外疤痕物质，而是造成更多的损害，我们设想在这些免疫细胞中激活Nrf2可以抑制炎症，并通过诱导抗氧化剂基因来抑制ROS并阻止氧化还原信号，从而使细胞化解纤维化，从而促进纤维化的解决。为了评估这些假说，我们将使用转基因小鼠来探索持续肝纤维化小鼠HSC中Nrf2的药理和遗传激活是否通过钝化氧化还原信号来阻止肝脏中纤维瘢痕蛋白的合成，并将确定Nrf2调控的哪些基因有助于抑制纤维化形成。以类似的方式，我们将使用转基因小鼠来探索Nrf2在患有肝纤维化但肝损伤已经停止的小鼠的免疫细胞中的药物和遗传激活，是否通过钝化氧化还原信号来加速纤维瘢痕的去除，并将确定Nrf2调控的哪些基因有助于肝纤维化的解决。为了检验结果的临床意义，我们将检测从肝硬变患者分离的HSC中Nrf2的丰度，并评估Nrf2的激活是否诱导靶基因，抑制ROS水平和抑制纤维蛋白的合成。此外，我们还将从肝硬变患者的血液中提取巨噬细胞，看看Nrf2的激活是否会诱导Nrf2靶基因，抑制ROS水平，并促进抗炎和支持分解的细胞类型。最后，我们将评估肝病患者肝脏中Nrf2测量的价值，以预测临床结果，并阐明导致肝纤维化的分子过程。因此，我们将从肝病患者的肝脏切片中检测Nrf2的水平，并评估在严重的肝硬变病例中Nrf2是否下调及其靶基因的表达，并探索已知的Nrf2负调控蛋白是否上调。

Repetitive wound healing in the liver results in fibrosis leading to scarring and, if severe, to cirrhosis. It is a serious condition that can lead to liver failure, and liver cancer. Liver cirrhosis arises due to a variety of causes including viruses, alcohol, auto-immunity, metabolic conditions and obesity-related liver damage. There has been a huge increase in cirrhosis, which poses an enormous challenge to our NHS. Most alarmingly, there is currently no effective therapy for liver fibrosis, except transplantation. Recently we discovered that by activating a major regulator of intracellular antioxidant systems, called Nrf2, we were able to arrest and/or reverse established and ongoing liver fibrosis in the mouse. The purpose of this project is to determine how activation of Nrf2 changes the formation and removal of liver scar tissue. The scarring is laid down by specific cells in the liver called hepatic stellate cells (HSCs), and is removed by immune cells recruited into the liver called macrophages. An important feature of liver fibrosis is that its initiation, perpetuation and progression involve cooperation between different cell types within the liver, including the immune cells, which is governed by production of various signalling molecules that allow communication between the different cell types. One such class of molecule are oxidants, called reactive oxygen species (ROS), that trigger pro-fibrotic and pro-inflammatory signalling cascades. As the development of liver fibrosis involves production by HSCs of ROS, which trigger redox signalling that causes their differentiation into collagen-secreting myofibroblasts that proliferate, we envisage that activation of Nrf2 in HSCs blocks synthesis of fibrous scar tissue in the liver by inducing antioxidant genes that inactivate ROS and block redox signalling. ROS also simulate inflammatory responses in macrophages recruited into the liver, so rather than allowing them to kill HSCs and digest extracellular scar material, they cause more damage, we envisage that activation of Nrf2 in these immune cells dampens inflammation and promotes resolution of fibrosis by inducing antioxidant genes that inactivate ROS and block redox signalling, allowing the cell to resolve the fibrosis. To evaluate these hypotheses, we will use transgenic mice to explore whether pharmacological and genetic activation of Nrf2 in HSCs of mice with ongoing liver fibrosis arrests synthesis of fibrous scar protein in the liver by blunting redox signalling and will identify which of the genes regulated by Nrf2 contributes to inhibition of fibrogenesis. In a similar manner, we will use transgenic mice to explore whether pharmacological and genetic activation of Nrf2 in immune cells of mice that have liver fibrosis but in which liver injury has ceased, accelerates removal of the fibrous scar by blunting redox signalling, and will identify which of the genes regulated by Nrf2 contribute to resolution of fibrosis. To examine the clinical significance of results we will examine the abundance of Nrf2 in HSCs isolated from the livers of patients with cirrhosis and evaluate whether activation of Nrf2 induces target genes, suppresses ROS levels and inhibits the synthesis of fibrous protein. Also, we will take macrophages from the blood of patients with cirrhosis and see if activation of Nrf2 induces Nrf2-target genes, suppresses ROS levels and promotes an anti-inflammatory and pro-resolving cell type. Lastly, we will evaluate the value of Nrf2 measurements in the liver of patients with liver disease to predict clinical outcome and shed light on the molecular processes that lead to liver fibrosis. Thus, we will measure Nrf2 levels in archived liver sections from patients with liver disease and assess whether Nrf2 is downregulated and expression of its target genes suppressed in severe cases of liver cirrhosis, and explore whether proteins that are known to be negative regulators of Nrf2 are upregulated.