Alkaptonuria (AKU) is a rare metabolic disease correlated with the deficiency of homogentisate 1,2-dioxygenase and leading to an accumulation of the metabolite homogentisic acid (HGA) which can be subjected to oxidation and polymerization reactions. These events are considered a trigger for the induction of oxidative stress in AKU but, despite the large description of an altered redox status, the underlying pathogenetic processes are still unstudied.

In the present study, we investigated the molecular mechanisms responsible for the oxidative damage present in an osteoblast-based cellular model of AKU. Bone, in fact, is largely affected in AKU patients: severe osteoclastic resorption, osteoporosis, even for pediatric cases, and an altered rate of remodeling biomarkers have been reported.

In our AKU osteoblast cell model, we found a clear altered redox homeostasis, determined by elevated hydrogen peroxide (H₂O₂) levels and 4HNE protein adducts formation. These findings were correlated with increased NADPH oxidase (NOX) activity and altered mitochondrial respiration. In addition, we observed a decreased activity of superoxide dismutase (SOD) and reduced levels of thioredoxin (TRX) that parallel the decreased Nrf2-DNA binding.

Overall, our results reveal that HGA is able to alter the cellular redox homeostasis by modulating the endogenous ROS production via NOX activation and mitochondrial dysfunctions and impair the cellular response mechanism. These findings can be useful for understanding the pathophysiology of AKU, not yet well studied in bones, but which is an important source of comorbidities that affect the life quality of the patients.

碱尿症（AKU）是一种罕见的代谢疾病，与高纯酸1,2-双加氧酶的缺乏有关，导致代谢产物高纯酸（HGA）的积聚，可使其发生氧化和聚合反应。这些事件被认为是诱发AKU中氧化应激的触发因素，但是，尽管大量描述了氧化还原状态的改变，但仍未研究潜在的致病过程。

在本研究中，我们调查了在AKU的成骨细胞细胞模型中造成氧化损伤的分子机制。实际上，骨骼在AKU患者中受到的影响很大：严重的破骨细胞吸收，骨质疏松症，甚至对于小儿病例，也已经报道了重塑生物标志物的速率改变。

在我们的AKU成骨细胞模型中，我们发现氧化还原稳态明显改变，这取决于过氧化氢（H ₂ O ₂）水平升高和4HNE蛋白加合物形成。这些发现与NADPH氧化酶（NOX）活性增加和线粒体呼吸改变有关。此外，我们观察到超氧化物歧化酶（SOD）的活性降低和硫氧还蛋白（TRX）的水平降低，与Nrf2-DNA结合的降低平行。

总体而言，我们的结果表明，HGA能够通过NOX激活和线粒体功能障碍调节内源性ROS产生，从而改变细胞的氧化还原稳态，并损害细胞的应答机制。这些发现对于理解AKU的病理生理学可能是有用的，AKU的病理生理学尚未在骨骼中进行很好的研究，但它是影响患者生活质量的合并症的重要来源。