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

肝脏中重复的伤口愈合会导致纤维化，导致疤痕，如果严重的话，会导致肝硬化。这是一种严重的疾病，可导致肝功能衰竭和肝癌。肝硬化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。肝硬化患者大幅增加，这对我们的NHS构成了巨大挑战。最令人担忧的是，除了肝移植，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/或逆转小鼠已建立和正在进行的肝纤维化。这个项目的目的是确定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一种叫做肝星状细胞（hsc）的特殊细胞形成的，然后被吸收到肝脏中的免疫细胞——巨噬细胞——清除。肝纤维化的一个重要特征是，它的开始、延续和进展涉及肝脏内不同细胞类型之间的合作，包括免疫细胞，这是由各种信号分子的产生所控制的，这些信号分子允许不同细胞类型之间的通信。其中一类分子是氧化剂，被称为活性氧（ROS），它会引发促纤维化和促炎症信号级联反应。由于肝纤维化的发展涉及造血干细胞产生ROS， ROS触发氧化还原信号，导致其分化为分泌胶原的肌成纤维细胞增殖，我们设想造血干细胞中Nrf2的激活通过诱导抗氧化基因灭活ROS和阻断氧化还原信号来阻止肝脏纤维瘢痕组织的合成。活性氧还能模拟进入肝脏的巨噬细胞的炎症反应，因此，它们不是让它们杀死造血干细胞和消化细胞外疤痕物质，而是造成更多的损伤。我们设想，激活这些免疫细胞中的Nrf2可以抑制炎症，并通过诱导抗氧化基因抑制活性氧和阻断氧化还原信号，促进纤维化的消退。为了评估这些假设，我们将使用转基因小鼠来探索持续肝纤维化小鼠hsc中Nrf2的药理学和遗传学激活是否通过钝化氧化还原信号来阻止肝脏中纤维疤痕蛋白的合成，并将确定Nrf2调节的哪些基因有助于抑制纤维发生。以类似的方式，我们将使用转基因小鼠来探索Nrf2在肝纤维化但肝损伤停止的小鼠免疫细胞中的药理和遗传激活是否通过钝化氧化还原信号来加速纤维疤痕的清除，并将确定Nrf2调节的哪些基因有助于纤维化的消退。为了检验结果的临床意义，我们将检测从肝硬化患者肝脏分离的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，我们将从肝硬化患者的血液中提取巨噬细胞，观察Nrf2的激活是否会诱导Nrf2靶基因，抑制ROS水平，促进抗炎和促溶解细胞类型。最后，我们将评估Nrf2在肝病患者肝脏中的测量值，以预测临床结果，并阐明导致肝纤维化的分子过程。因此，我们将测量肝脏疾病患者存档肝脏切片中的Nrf2水平，评估Nrf2在严重肝硬化病例中是否下调，其靶基因的表达是否受到抑制，并探索已知的Nrf2负调节蛋白是否上调。

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.