Validation of neutrophil elastase as a target to improve muscle regeneration in Duchenne muscular dystrophy

在Duchenne肌营养不良症(DMD)中，肌肉细胞极其脆弱，在使用肌肉时很容易分解。被诊断为DMD的儿童预期寿命较短，肌肉质量和力量会逐渐丧失，这将导致他们在12岁时被轮椅束缚，并经常在20岁之前需要外部呼吸支持。DMD是无法治愈的。虽然我们的身体中有干细胞可以再生受损的肌肉，但在DMD中，这些细胞会过早耗尽，它们缺乏功能会导致进行性肌肉丧失。目前以使用皮质类固醇为基础的护理标准在减缓疾病进展方面略有效果，但代价是严重的不良反应。我们研究了小鼠的肌肉营养不良症，我们发现营养不良小鼠的肌肉中一种由白细胞产生的名为弹性蛋白酶的蛋白质比未受影响的小鼠的肌肉增加。弹性蛋白酶是一种蛋白酶，这意味着它是一种能分解其他蛋白质的蛋白质。然后我们继续了解这种增加意味着什么，我们发现弹性蛋白酶损伤肌肉干细胞，当受损时，肌肉干细胞负责再生肌肉组织。因此，我们推测，如果我们能够阻断营养不良肌肉中弹性蛋白酶的活性，我们可能能够保存肌肉干细胞的功能，并促进肌肉再生，以应对疾病对肌肉细胞的持续损害。为了验证这一假设，我们进行了一项初步实验，我们抑制了弹性蛋白酶的活性，并确实观察到营养不良小鼠肌肉病理的改善。然而，这些实验中使用的弹性蛋白酶抑制剂不能开给患者，因为它不是合成的化合物，而是从细菌中提纯的化合物。我们现在的目标是在小鼠身上测试可以开给患者的药物，这些药物已经开发出来，可以抑制多种疾病的弹性蛋白酶活性，如囊性纤维化、支气管扩张和慢性阻塞性肺疾病。我们建议开展的项目将分两个阶段进行。首先，我们将在DMD患者的肌肉干细胞培养上测试几种这样的弹性酶抑制剂，以找到一种更有效地抑制细胞中弹性酶影响的药物。其次，我们将用这种药物治疗患有肌肉营养不良的小鼠，并研究它们是否有所改善。在治疗过程中，我们将测量他们的握力，以及他们自愿在轮子上跑步的量，看看他们的肌肉功能是否有所改善。在治疗结束时，我们将扑杀小鼠，收集它们的肌肉，制作薄片并对它们进行染色，看看治疗是否促进了再生，这将表现为肌肉质量的增加和肌肉内疤痕组织的减少。此外，我们将通过量化炎症的特定标记物，如肌肉中炎症细胞的存在和促炎分子的水平，来确定肌肉中的炎症是否已经减少。最后，我们将量化血液中某些分子的水平，这些分子是肌肉损伤或炎症的指标。通过这个项目，我们的目标是开发一种基于弹性酶抑制的DMD治疗方法，通过促进肌肉再生来帮助维持肌肉质量和力量。

In Duchenne muscular dystrophy (DMD), muscle cells are extremely fragile and break down easily as the muscles are used. Children diagnosed with DMD have a shorter life expectancy and will undergo progressive loss of muscle mass and strength that will lead them to be wheelchair bound by the age of 12 and often require external respiratory support by the age of 20. There is no cure for DMD. Although our body contains stem cells that can regenerate damaged muscle, in DMD these cells become prematurely exhausted and their lack of function leads to progressive muscle loss. Current standard of care based on the use of corticosteroids is mildly effective at slowing down disease progression but at the cost of significant adverse effects. We have studied muscular dystrophy in mice and we have discovered that a protein produced by white blood cells, called elastase, is increased in the muscles of dystrophic mice as compared to the muscles of non-affected mice. Elastase is a protease, which means is a protein that cleaves other proteins. We then went on to understand what this increase means and we found that elastase damages muscle stem cells, which are responsible for regenerating the muscle tissue when is damaged. Thus, we speculated that if we could block the activity of elastase in dystrophic muscle, we might be able to preserve muscle stem cell function and promote muscle regeneration in response to the continuous damage to muscle cells caused by the disease. To test this hypothesis we have carried out a preliminary experiment where we have inhibited elastase activity and indeed observed an improvement in muscle pathology in dystrophic mice. However the elastase inhibitor used in these experiments cannot be prescribed to patients because is not a synthetic compound but one purified from bacteria. We now aim to test in mice drugs that can be prescribed to patients and that have been developed to inhibit elastase activity in a variety of diseases such as cystic fibrosis, bronchiectasis and chronic obstructive pulmonary disease. The project we propose to carry out will develop in two phases. Firstly, we will test a few of these elastase inhibitors on muscle stem cell cultures that have been derived from patients with DMD to find the one drug that is more potent at inhibiting the effects of elastase in cells. Secondly, we will treat mice that have muscular dystrophy with this drug and study whether they improve. During the course of the treatment we will measure their grip strength and also how much they voluntarily run on a wheel to see if their muscle function has improved. At the end of the treatment we will cull the mice, collect their muscles, make thin sections and stain them to see whether the treatment has improved regeneration, which will be shown as an increase in muscle mass and a decrease in scar tissue within the muscle. Moreover, we will find out whether inflammation has decreased in the muscles, by quantifying specific markers of inflammation such as the presence of inflammatory cells and the levels of pro-inflammatory molecules in the muscle. Lastly, we will quantify the levels in the blood of certain molecules that are indicators of either muscle damage or inflammation.With this project we aim to develop a novel treatment for DMD based on elastase inhibition that would help maintain muscle mass and strength by promoting muscle regeneration.