Single-cell Raman spectroscopy reveals multidimensional spectral phenotypes associated with curcumin protection in high-glucose-stressed cone cells.

High-glucose-induced oxidative stress plays a critical role in retinal cellular dysfunction associated with diabetic retinopathy. We investigated the protective effects of curcumin in high-glucose-exposed 661 W cone cells and evaluated single-cell Raman spectroscopy for label-free characterization of treatment-associated cellular responses. Cells were exposed to 55 mmol/L glucose for 24 h and then treated with 1.5 μmol/L curcumin for 6 h. Cell viability, reactive oxygen species (ROS) accumulation, TUNEL positivity, and Nrf2/HO-1 expression were assessed alongside Raman spectra from individual intact cells. Exposure to high glucose resulted in reduced cell viability, increased ROS accumulation and TUNEL-positive ratio, alongside downregulated levels of Nrf2, HO-1 mRNA and corresponding immunofluorescence signals. Curcumin attenuated ROS accumulation and TUNEL positivity, and increased the mRNA and protein expression of Nrf2 and HO-1. Raman analysis identified high-glucose-associated changes at 408, 525, 633, 683, 1046, and 1536 cm-1 (all adjusted P ≤ 0.00216). Five of these regions shifted toward control values after curcumin treatment (adjusted P values ranging from 6.58 × 10-11 to 0.00128). Literature-based assignments suggested overlapping contributions from carbohydrates, amino acids, coenzymes, and redox-related molecules. Candidate-based pathway association analysis ranked starch and sucrose metabolism highest after FDR correction (FDR = 0.00323). A 20-band CRT achieved 77.9 ± 1.5% accuracy and 77.8 ± 1.5% balanced accuracy in repeated nested cross-validation; corresponding run-held-out values were 70.1% and 70.0%. These findings show that single-cell Raman spectroscopy captures a coordinated, treatment-responsive spectral phenotype that complements conventional measures of oxidative injury and antioxidant responses.