Modulation of mitochondrial respiration to treat colitis

炎症性肠病（IBD）中粘膜愈合的重要性来自临床数据， 证明它是病情缓解的预测指标不幸的是，许多IBD患者仍然难治性或失去治疗。 对治疗的反应性导致持续性粘膜溃疡。前瞻性儿科研究表明， 线粒体基因表达降低预示溃疡性结肠炎（UC）的不良结局。一致认为， 线粒体呼吸链基因表达的增加已被证明预示着有利的克罗恩病 疾病（CD）结果。基于这些结果和我们自己的数据，我们假设慢性疾病的一个关键驱动因素 IBD中的溃疡是肠上皮细胞（IEC）不能增加线粒体呼吸。研究中 在提交本申请之前，我们发现线粒体缺陷导致溃疡愈合不良， 溃疡边缘出现隐窝分裂（进展报告）。这些发现使我们开发了一种新的金（Au）- 金诺芬是基于目前安全有效的风湿性关节炎金疗法的基础药物。这个新 药物AuPhos定位于线粒体，在那里它增加线粒体呼吸和线粒体反应性。 氧物种（mtROS）的产生。已知低水平的“内源性”mtROS的诱导促进 细胞中健康的适应性反应，包括抗氧化酶活性（背景）。初步 研究中，我们发现口服AuPhos 1）增强DSS结肠炎的粘膜修复，2）增加IEC线粒体 氧消耗率（OCR）和氧化磷酸化（OXPHOS），3）mtROS和H2 O2的产生， IEC以及4）磷酸肌醇-3激酶（PI 3 K）和核因子红细胞2相关因子（Nrf 2）信号传导 5）诱导线粒体生物发生[过氧化物酶体增殖物激活]的蛋白质的表达提高 受体-γ共激活因子1 α（PGC 1 α）和线粒体转录因子A（TFAM）。拟议的研究 目标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、电子传递链（ETC）活性的变化， 诱导适应性反应[抗氧化剂过氧化氢酶、MnSOD（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（4 HNE，蛋白质羰基化， 硝基酪氨酸IHC）。目标2将建立在这些研究的基础上，通过评估AuPhos在TNF刺激的小细胞中的作用， 肠（SB）和结肠类器官，来自退伍军人（正常和IBD）。3D类器官的研究将允许 AuPhos诱导的隐窝出芽和IEC基因表达的机制测试。目标2的结果将指导 目的3A中研究AuPhos对IEC基因表达和隐窝分裂的治疗作用的研究， DSS结肠炎溃疡愈合。在Aim 3B中，用AuPhos孵育的人活检组织的scRNAseq分析将允许 我们研究离散IEC簇中线粒体基因表达的诱导。拟议的研究是推动 通过一个新的假设，即恢复IEC线粒体呼吸是粘膜愈合的关键步骤。在一起， 这些对VA赞助的新药（AuPhos）进行的“临床前”研究有可能使这种药物 治疗退伍军人炎症性肠病

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.