Evaluated NSUN3 in reticulocytes from HbH-CS disease that reflects cellular stress in erythroblasts.

Hemoglobin H Disease-Constant Spring (HbH-CS) represents a severe variant of α-thalassemia characterized by a fundamental pathological mechanism involving inadequate synthesis of α-globin chains. This deficiency results in the formation of unstable Hemoglobin H (HbH) due to the aggregation of free β-globin chains, which subsequently induces an imbalance in oxidative stress within erythrocytes. This imbalance leads to an abnormal accumulation of reactive oxygen species (ROS), which in turn promotes lipid peroxidation, culminating in the production of malondialdehyde (MDA) and a significant depletion of glutathione (GSH). Concurrently, Nrf2 is translocated to the nucleus, where it activates the antioxidant response element (ARE) to mitigate cellular stress. Here, we report that NSUN3 (which, together with ALKBH1, maintains mitochondrial function through m5C→f5C modification) is abnormally overexpressed in reticulocytes from patients with HbH-CS, and an in vitro cellular model of NSUN3 overexpression/silencing was constructed using K562 cells, which have the potential for erythroid lineage differentiation and retain an intact cluster of bead protein genes. Functional assays indicated that the overexpression of NSUN3 significantly intensified the accumulation of intracellular ROS and MDA, led to a reduction in GSH levels, and diminished the overall cellular antioxidant capacity (T-AOC). This may be due to ROS accumulation resulting from inhibition of mitochondrial respiratory chain complex I, II, and IV synthesis through aberrant m5C→f5C modification. In addition, NSUN3 overexpression further exacerbates oxidative stress by inhibiting the phosphorylation of Nrf2 hindering its translocation into the nucleus and weakening the cellular antioxidant system. Moreover, we also observed that NSUN3 overexpression exacerbated intracellular DNA damage and inhibited cellular value-added activity, and silencing NSUN3 showed the opposite result. Our research offers initial insights into the molecular mechanisms through which NSUN3 modulates oxidative stress in erythrocytes via its role in epigenetic modifications. These findings contribute to a deeper understanding of the clinical management of patients with Hb H-CS.