Protective role of aldehyde dehydrogenase 2 in retinal pigment epithelium cells with CoCl₂-induced hypoxic injury: an in-vitro study.

BACKGROUND: Retinal pigment epithelial (RPE) cells are vulnerable to hypoxia-related and oxidative stress. Cobalt chloride (CoCl₂) is widely used as a chemical hypoxia mimetic; however, an appropriate concentration window for producing measurable RPE injury without the more extensive damage associated with higher concentrations remains incompletely defined. Aldehyde dehydrogenase 2 (ALDH2) contributes to reactive-aldehyde detoxification and cellular stress defense. METHODS: ARPE-19 cells were exposed to 0, 10, 100, 500, 1000, or 2000 µM CoCl₂. Scratch-wound recovery and representative morphology were evaluated for up to 72 h, and relative CCK-8 metabolic activity was quantified at 24, 48, and 72 h. The 500 µM condition was selected for molecular and pharmacological experiments based on the integrated dose-response profile. Culture-supernatant 4-HNE and VEGF-A were measured by ELISA, whereas HIF-1α, ALDH2, and BAX were assessed by Western blotting. Alda-1 and Daidzin were used as an ALDH2 activator/chemical chaperone and an enzymatic inhibitory comparator, respectively. RESULTS: CoCl₂ caused concentration- and time-associated impairment of scratch-wound recovery. Wound closure was largely preserved at 0-100 µM, whereas 500-2000 µM produced wound-closure failure. The CCK-8 profile showed that 500 µM retained a nonmaximal metabolic signal compared with the substantially greater late reductions observed at 1000 and 2000 µM. Thus, 500 µM represented the lowest tested concentration producing significant functional injury while avoiding the more extensive injury phenotype observed at higher concentrations. Under 500 µM CoCl₂, HIF-1α increased early, ALDH2 protein abundance decreased, and culture-supernatant 4-HNE and VEGF-A increased at the later time point. Alda-1 improved wound recovery and restored ALDH2 protein abundance, whereas Daidzin aggravated the functional injury phenotype and shifted extracellular stress-marker readouts in an unfavorable direction. CONCLUSIONS: CoCl₂ produced a concentration-resolved, hypoxia-related and aldehyde-associated RPE injury phenotype. Alda-1 improved wound recovery and increased ALDH2 protein abundance, whereas Daidzin aggravated the functional injury phenotype. These findings support ALDH2-associated aldehyde-detoxifying defense as a modulatory component of CoCl₂-induced RPE stress.