Chapter 4 - Experimental Results - data.

Aging and lifestyle-related diseases, such as cardiovascular diseases, diabetes, cancer, and neurodegenerative disorders, are major global health challenges. These conditions are often linked to redox imbalances, where cells fail to regulate reactive redox species (RRS), leading to oxidative stress and cellular damage. Although antioxidants are known to neutralize harmful RRS, their clinical efficacy remains inconsistent. One reason for this inconsistency is the inadequacy of current in vitro models to accurately mimic in vivo redox conditions. This study addresses the gap in understanding the heterogeneity of redox responses in cells by using metabolically primed human dermal fibroblasts (NHDF), a model relevant for precision mitochondrial medicine. We investigated how metabolic priming, which enhances mitochondrial bioenergetics, influences redox responses to oxidative stress induced by hydrogen peroxide (H2O2) and tert-butyl hydroperoxide (tBHP). Specifically, we explored the impact of cell population density and cell cycle distribution on redox dynamics. Our findings indicate that NHDF cells cultured in oxidative phosphorylation-promoting medium (OXm) exhibit significantly larger variability in oxidative stress responses. This variability suggests that enhanced mitochondrial bioenergetics necessitates a constant regulation of the cellular redox machinery, potentially leading to heterogeneous responses. Additionally, cells grown in OXm showed increased mitochondrial polarization and a lower percentage of cells in the G2/M phase, contributing to the observed heterogeneity. Key factors influencing this variability included cell population density at the time of oxidant exposure and fluctuations in cell cycle distribution. Our results highlight the necessity of employing multiple oxidants in metabolic priming models to achieve a comprehensive understanding of oxidative stress responses and redox regulation mechanisms. Furthermore, the study emphasizes the need to refine in vitro models to better reflect in vivo conditions, which is crucial for the development of effective redox-based therapeutic strategies.

衰老及与生活方式相关的疾病，如心血管疾病、糖尿病、癌症及神经退行性疾病，是全球主要的健康挑战。此类病症通常与氧化还原失衡相关：细胞无法调控活性氧化还原物种（reactive redox species, RRS），进而引发氧化应激与细胞损伤。尽管已知抗氧化剂可中和有害的活性氧化还原物种，但其临床疗效仍存在不一致性。导致这种不一致的原因之一，是当前的体外模型难以精准模拟体内的氧化还原环境。本研究针对细胞氧化还原应答异质性的认知空白，采用代谢预激活的人真皮成纤维细胞（metabolically primed human dermal fibroblasts, NHDF）——一种适用于精准线粒体医学的模型——展开研究。我们探究了可增强线粒体生物能的代谢预激活，如何影响细胞对过氧化氢（hydrogen peroxide, H₂O₂）与叔丁基过氧化氢（tert-butyl hydroperoxide, tBHP）诱导的氧化应激的应答。具体而言，我们分析了细胞群密度与细胞周期分布对氧化还原动力学的影响。研究结果显示，在促进氧化磷酸化的培养基（oxidative phosphorylation-promoting medium, OXm）中培养的NHDF细胞，其氧化应激应答的异质性显著更高。这种异质性表明，增强的线粒体生物能需要持续调控细胞氧化还原机制，进而可能导致应答的异质性。此外，在OXm中培养的细胞，其线粒体极化水平更高，且处于G2/M期的细胞比例更低，这一特征也加剧了观测到的异质性。影响该异质性的关键因素包括氧化剂暴露时的细胞群密度，以及细胞周期分布的波动。我们的研究结果表明，在代谢预激活模型中需采用多种氧化剂，才能全面理解氧化应激应答与氧化还原调控机制。此外，本研究强调，需要优化体外模型以更好地反映体内环境，这对于开发基于氧化还原的有效治疗策略至关重要。