Chicken erythrocytes are nucleated blood cells that provide a useful model for assessing oxidative stress-related membrane injury, but the conventional hemolysis assays do not resolve single-cell nanoscale mechanical alterations. Here, we established an atomic force microscopy (AFM)-based workflow to characterize structural and nanomechanical alterations associated with 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH) exposure and curcumin-associated protection in chicken erythrocytes. Hemolysis assays were used to select a moderate injury condition and a protective curcumin dose. Confocal microscopy was used to assess filamentous actin (F-actin) organization and nuclear morphology, and short-term storage conditions were optimized to preserve fragile damaged cells before AFM measurements. The AAPH-induced concentration-dependent hemolysis, cytoskeletal disruption, and nuclear shrinkage altered erythrocyte morphology, characterized by decreased cellular dimensions and increased cell height. These treatments also affected nuclear mechanical properties, as indicated by increased indentation depth of the nuclear region and decreased apparent Young’s modulus and adhesion force at the nuclear region. Storage in Alsever’s solution at 4 °C best preserved the damaged erythrocytes for AFM analysis. Curcumin pretreatment reduced hemolysis and partially preserved cytoskeletal and nuclear morphology. AFM further showed that curcumin attenuated the nanoscale topographical and mechanical changes induced by AAPH. These results link biochemical injury, subcellular structural remodeling and single-cell nanomechanical phenotypes, and provide a quantitative AFM workflow for evaluating oxidative damage-associated structural and mechanical alterations and curcumin-associated preservation in fragile blood cells.

Red blood cells (RBCs) are the most abundant cells in peripheral blood and perform critical functions including oxygen and carbon dioxide transport, acid base buffering, regulation of hemorheology, and modulation of immune signaling. Due to their high content of hemoglobin and labile iron, prolonged exposure to high oxygen tension, membrane enrichment with polyunsaturated fatty acids, and the absence of both nucleus and mitochondria, mature RBCs have limited capacity for damage repair and protein re-synthesis, making them highly susceptible to attack by reactive oxygen species (ROS) and reactive nitrogen species (RNS). Hydrogen peroxide (H₂O₂) and 2,2′-azobis(2-methylpropionamidine) dihydrochloride (AAPH) are the two most commonly used inducers in the in vitro models of RBC oxidative injury: H₂O₂ primarily generates hydroxyl radicals via hemoglobin/ferrous ion-dependent Fenton reactions, simulating acute oxidative stress. AAPH releases peroxyl radicals upon thermal decomposition, mimicking persistent lipid peroxidation in cell membranes. Curcumin, a representative polyphenolic compound derived from turmeric, exerts multiple effects including free radical scavenging, metal ion chelation, membrane stabilization, anti-inflammatory activity, and regulation of redox homeostasis. This review systematically summarizes the pathological basis of RBC oxidative damage and the protective effects of curcumin on membrane systems, antioxidant defenses, morphology, and function, based on the core evidence chain “H₂O₂/AAPH—RBCs—curcumin”, integrating recent experimental findings on H₂O₂, AAPH, blood storage-induced injury, and curcumin formulations. It emphasizes that mature RBCs lack nuclei and mitochondria, and therefore mechanisms such as Nrf2/ARE signaling, HO-1 induction, mitochondrial apoptosis, caspase cascades, and inflammasome activation should not be directly equated with transcriptional regulatory pathways within mature RBCs, but rather interpreted as indirect evidence originating from nucleated cells, erythroid progenitors, or the blood microenvironment. The article further proposes that future research should focus on standardized RBC models, physiologically relevant dosages, nanodelivery systems, and translational applications in blood storage, to facilitate the transition of curcumin’s in vitro antioxidant evidence into clinical transfusion medicine and precision nutritional interventions.