Circadian rhythms and the control of NRF2-based antioxidant signalling as a therapeutic target in dermal tissue repair and pathological wound healing

病理性伤口愈合与衰老和许多慢性疾病有关。它是发达世界的主要医疗负担之一，是全世界死亡和疾病的主要原因之一。伤口愈合的各个阶段通常以可预测和及时的方式进行；如果他们不这样做，愈合可能不适当地进展到慢性伤口，如糖尿病溃疡或纤维化疤痕，如硬皮病。慢性伤口和纤维化病理的患病率达到约8000万，如果我们包括与外科手术相关的纤维化，这一数字将增加到1亿。目前对慢性伤口和纤维化瘢痕的治疗效果有限，因此迫切需要更好地了解促进愈合同时防止瘢痕形成的生物学机制，以实现更好的治疗。世界各地的科学家以及我们自己的实验室最近进行的研究发现，生物钟在最佳伤口愈合和抑制损伤后的纤维化瘢痕方面发挥着至关重要的作用。生物钟是我们体内的计时机制，它在生理和行为上产生24小时节律，比如睡眠/觉醒周期、体温和激素水平。它们几乎存在于我们身体的所有组织和细胞中。轮班工作和老龄化对他们的干扰是慢性病的一个重要风险因素。我们使用啮齿动物模型的新试点数据表明，皮肤伤口愈合速度在白天/晚上有明显的变化，因此夜间受伤的皮肤愈合速度比白天受伤的皮肤愈合速度更快。这种夜间更快的愈合与参与基质生产和组织的基因水平的增加有关，基质是愈合伤口的一个非常重要的组成部分。我们还发现，我们细胞中的抗氧化保护在白天和晚上是不同的，并且在损伤后的夜间愈合中起着重要作用。更重要的是，如果在一天中的适当时间使用，能够提高我们细胞中抗氧化水平的小药物分子会有更高的效率。这些观察结果为解决病理性伤口修复机制提供了令人兴奋的新途径。在这个新项目中，我们将回答一个主要问题，即存在于参与伤口愈合的关键细胞（称为成纤维细胞）中的生物钟是否对我们在试点研究中观察到的修复能力的日常变化至关重要。我们将利用两种转基因啮齿动物模型：1)我们可以看到（通过荧光标记）在成纤维细胞中特异性激活的基质基因（称为胶原蛋白）；2)另一种是我们在成纤维细胞中特异性地删除了时钟基因。利用这些独特的啮齿动物模型，我们将能够监测伤口内时钟基因活动和基质组织的时间变化。这项研究将揭示关键伤口愈合细胞内强大的时钟对于愈合率和最佳伤口修复组织的时间变化的关键重要性。此外，我们将利用先进的分子生物学工具来发现生物钟用来调节在愈合中重要的基质基因（如胶原蛋白）的遗传机制。使用高科技生化方法，我们将进一步发现生物钟是否能帮助我们的细胞“告诉时间”何时产生适量的抗氧化剂，以对抗受伤后危险自由基的增加。最后，我们将测试基于抗氧化剂的时间疗法（根据一个人的生物钟进行治疗）是否对慢性伤口或疤痕状况（如糖尿病和硬皮病）有有益的愈合效果。为了验证这一点，我们将使用糖尿病啮齿动物模型以及硬皮病患者的皮肤组织活检和细胞。这些新的研究将为未来的研究提供关键的证据，以确定生物钟作为慢性伤口和疤痕管理的新治疗靶点。

Pathological wound healing is associated with ageing and many chronic diseases. It is one of the major medical burdens in the developed world, contributing towards leading causes of death and disease worldwide. The stages of wound healing normally progress in a predictable, timely manner; if they do not, healing may progress inappropriately to either a chronic wound such as diabetic ulcer or fibrotic scarring such as scleroderma. The prevalence of chronic wounds and fibrotic pathologies arrives at the number of ~80 million, which increases to >100 million if we include fibrosis associated with surgical procedures. Current treatments for chronic wounds and fibrotic scarring have limited success, so better understanding of biological mechanisms that promote healing whilst preventing scarring is urgent to achieve better therapies.Research carried out by scientists around the world as well as our own laboratory has recently discovered a crucial role for biological clocks in optimal wound healing and in curbing fibrotic scarring following injury. Biological clocks are timing mechanisms in our body that generate 24h rhythms in physiology and behavior, such as sleep/wake cycles, body temperature and hormone levels. They exist in almost all tissues and cells in our body. Their disruption due to shift work and ageing is a strong risk factor for chronic diseases. Our new pilot data using rodent models shows that there is a robust day/night variation in skin wound healing rates so that skin heals faster when injured during the night then when injured during the day. This faster healing at night is associated with increased levels of genes involved in production and organisation of matrix, a very important component for closing the wound. We have also discovered that antioxidant protection in our cells varies between day and night, and has an important role in faster night-time healing following injury. Even more, small, drug-like molecules capable of boosting antioxidant levels in our cells have a greater efficiency when used at the right time of day. These observations suggest exciting new ways to tackle pathological wound repair mechanisms.In this new project, we will answer the main question as to whether the biological clock present in key cells involved in wound healing (called fibroblasts) is critical for daily variation in repair capacity that we observed in our pilot studies. We will make use of two genetically modified rodent models 1) one in which we can visualise (by fluorescent tags) a matrix gene (called collagen) specifically activated in fibroblast cells and 2) the other in which we have genetically deleted the clock gene specifically in fibroblast cells. Using these unique rodent models, we will be able to monitor temporal changes in clock gene activity and matrix organisation within wounds. This research will uncover critical importance of robust clocks within key wound healing cells for time-of-day variation in healing rate and organisation of optimal wound repair. Furthermore, we will take advantage of advanced molecular biology tools to find out which genetic mechanisms biological clocks use to regulate matrix genes important in healing such as collagen. Using high-tech biochemical approaches, we will further find out whether biological clocks help our cells 'tell the time' when to produce the right amounts of antioxidants to fight off rises in dangerous free radicals following injury. Finally, we will test whether antioxidant-based chronotherapy (giving treatments according to one's body clock) has beneficial healing effects in chronic wound or scarring conditions such as diabetes and scleroderma. To test this, we will use rodent models of diabetes as well as skin tissue biopsies and cells from patients with Scleroderma. These new studies will provide crucial evidence to support future studies pinning down biological clocks as a new therapeutic target for the management of chronic wounds and scarring.