Modulation of mitochondrial respiration to treat colitis

黏膜愈合在炎症性肠病(IBD)中的重要性源于临床数据 证明它是病情缓解的预测指标。不幸的是，许多IBD患者仍然难以治愈或失去治疗。 对导致持续性粘膜溃疡的治疗的反应性。未来的儿科研究表明 线粒体基因表达降低预示着溃疡性结肠炎(UC)的不良预后。始终如一， 线粒体呼吸链基因的高表达被证明是有利的克罗恩病的预测因素 疾病(CD)结局。根据这些结果和我们自己的数据，我们假设慢性病的一个关键驱动因素 IBD的溃烂是肠上皮细胞(IEC)不能增加线粒体呼吸。在研究中 在提交这篇论文之前，我们发现线粒体缺陷导致溃疡愈合不佳，这是由于 溃疡边缘发生隐窝分裂(进展报告)。这些发现使我们开发了一种新型的金(Au)- 基于目前治疗类风湿关节炎的安全有效的黄金疗法(Auranofin)的药物。这是一项新的 药物AuPhos定位于线粒体，在那里它增加了线粒体的呼吸和线粒体的反应 氧物种(MtROS)产生。已知诱导低水平的内源性mtRO可以促进 细胞中健康的适应性反应，包括抗氧化酶活性(背景)。在预赛中 研究发现，口服AuPhos 1)促进DSS结肠炎的粘膜修复，2)增加IEC线粒体 耗氧率(OCR)和氧化磷酸化(OXPHOS)，3)线粒体ROS和H_2O_2产生 IEC以及4)磷脂酰肌醇-3激酶(PI3K)和核因子红系相关因子(Nrf2)信号转导 5)提高了诱导线粒体生物发生的蛋白质的表达[过氧化物酶增殖物激活 受体-γ共激活因子1α(α)和转录因子A，线粒体(TFAM)。建议进行的研究 目的1在基线条件下询问线粒体功能，以评估AuPhos诱导的 线粒体质量、活性(MtROS)和OCR、ATP周转率、电子传输链(ETC)活性的变化； 诱导适应性反应[抗氧化剂过氧化氢酶、超氧化物歧化酶和谷胱甘肽 (还原/氧化)]以及评估组织氧化应激的变化(4HNE，蛋白质羰化， 硝基酪氨酸(IHC)。Aim 2将在这些研究的基础上，评估AuPhos对肿瘤坏死因子刺激的小鼠的影响 退伍军人(正常和IBD)的肠道(SB)和结肠类器官。对3D有机化合物的研究将允许 AuPhos诱导的隐窝萌发和IEC基因表达的机制测试。目标2的结果将指示 Aim 3A研究AuPhos对IEC基因表达和隐窝分裂的治疗作用 治疗DSS结肠炎的溃疡。在AIM 3B中，对与AuPhos孵育的人类活组织进行scRNAseq分析将允许 US检测离散IEC集群中线粒体基因表达的诱导。拟议的研究是由 一种新的假说认为，IEC线粒体呼吸的恢复是粘膜愈合的关键步骤。一起， 这些用退伍军人管理局赞助的新药(AuPhos)进行的“临床前”研究有可能使这种药物过渡 退伍军人IBD的治疗。

The importance of mucosal healing in inflammatory bowel disease (IBD) derives from clinical data that demonstrate it to be a predictor of remission. Unfortunately, many IBD patients remain refractory or lose responsiveness to therapies leading to persistent mucosal ulceration. Prospective pediatric studies show that reduced mitochondrial gene expression predict unfavorable outcomes in ulcerative colitis (UC). Consistently, increased expression of mitochondrial respiratory chain genes has been shown to predict favorable Crohn’s disease (CD) outcomes. Based on these results and our own data, we hypothesize that a critical driver of chronic ulceration in IBD is the failure of intestinal epithelial cells (IEC) to increase mitochondrial respiration. In studies leading up to this submission we found that mitochondrial deficiency caused poor ulcer healing due to inadequate crypt fissioning occurring at ulcer edges (Progress Report). These findings led us to develop a novel gold (Au)- based drug based on the current safe and effective gold therapy for Rheumatoid arthritis (Auranofin). This new drug, AuPhos, localizes to mitochondria where it increases mitochondrial respiration and mitochondrial reactive oxygen species (mtROS) production. Induction of low levels of “endogenous” mtROS are known to promote healthy adaptive responses in cells including anti-oxidant enzyme activities (Background). In preliminary studies, we found that oral AuPhos 1) enhances mucosal repair in DSS colitis, 2) increases IEC mitochondrial oxygen consumption rate (OCR) and oxidative phosphorylation (OXPHOS), 3) mtROS and H2O2 production in IEC as well as 4) phosphoinositides-3 kinase (PI3K) and nuclear factor erythroid 2-related factor (Nrf2) signaling with 5) improved expression of proteins that induce mitochondrial biogenesis [peroxisome proliferator activated receptor-γ coactivator 1 alpha (PGC1α) and transcription factor A, mitochondrial (TFAM). Studies proposed in Aim 1 interrogate mitochondrial function under baseline conditions to allow assessment of AuPhos-induced changes in mitochondrial mass, activity (mtROS) and OCR, ATP turnover, electron transport chain (ETC) activity, induction of adaptive responses [anti-oxidant catalase, MnSOD (superoxide dismutase) and glutathione (reduced/oxidized)] as well as assess changes in tissue oxidant stress (4HNE, protein carbonylation, nitrotyrosine IHC). Aim 2 will build upon these studies by evaluating AuPhos effects in TNF-stimulated small bowel (SB) and colonic organoids from veterans (normal and IBD). Studies in 3D organoids will allow for mechanistic testing of AuPhos-induced crypt budding and IEC gene expression. Results of Aim 2 will instruct studies in Aim 3A that study therapeutic effects of AuPhos on IEC gene expression and crypt fissioning that heal ulcers in DSS colitis. In Aim 3B, scRNAseq analysis of human biopsies incubated with AuPhos will allow us to examine induction of mitochondrial gene expression in discrete IEC clusters. Proposed studies are driven by a novel hypothesis that restoration of IEC mitochondrial respiration is a key step in mucosal healing. Together, these “preclinical” studies with a VA-sponsored new drug (AuPhos) have the potential of transitioning this agent to the therapy of IBD in veterans.