Purpose: To investigate the pathogenesis of age-related cataract, we aim to examine the expression and distribution of Calb1 in cultured human lens epithelial cells (HLECs) subjected to calcium overload in vitro, as well as in the lenses of selenium-induced cataract SD rats in vivo.

Methods: The human lens epithelial cell line (SRA01/04) was treated with ionomycin at concentrations of 0.5 μmol/L, 1.0 μmol/L, 1.5 μmol/L, and 2.0 μmol/L for 1 hour to induce intracellular calcium overload in order to determine the optimal treatment concentration. Cell morphology changes were observed under an inverted microscope, cell viability was assessed using CCK-8 assay, intracellular calcium concentration was measured using a fluorescence microplate reader, and the distribution of calcium ions was visualized through confocal microscopy in HLECs labeled with Fluo-4/AM fluorescent probe at time points of 6 h, 12 h, 24 h, and 48 h post-treatment. Immunohistochemistry and Western Blot analysis were used to locate and quantify CALB1 protein expression levels while qRT-PCR was used to detect Calb1 mRNA expression levels in cultured HLECs as well as in the lenses of selenium cataract SD rats. Statistical comparisons were performed using one-way ANOVA followed by independent sample t-test.

Results: A treatment concentration of 1.0 μM ionomycin was determined as optimal. Between 6h and 12h, cell viability reached its lowest point while intracellular Ca2+ concentration peaked. Subsequently, cell viability gradually increased from 24h to 48h, while intracellular Ca2+ concentration remained stable and low. Upon calcium overload, the number of Calb1 spots decreased at 6h before increasing at 12h and becoming concentrated near the nucleus without a distinct pattern by 24h. At 48h, both fluorescence intensity and range expanded compared to that observed at 24 h. In selenium-induced cataractous lenses of SD rats, CALB1 protein gradually increased from 2 w to 8 w with prominent localization within the internal nucleus region at 12 w. qRT-PCR revealed that Calb1 mRNA expression significantly increased after calcium overload with peak expression occurring at 48h (p<0.01). Similarly, in selenium cataract SD rats, Calb1 mRNA expression was significantly higher compared to control groups from 4w to 12w (p<0.01), peaking at 8w before slightly decreasing by 12w. Western Blot demonstrated that following calcium overload there was no change in relative CALB1 protein expression within each time point for control groups; however, it significantly increased after calcium overload with peak expression occurring at 48 h (p<0.01) for experimental groups while lens CALB1 protein decreased significantly 2-week post-selenium induction but then increased 4-week post induction before being highly expressed during 8w and 12w.

Conclusion: The CALB1 protein is mainly found in the cytoplasm of HLECs and the perinuclear region of lens fibers. After calcium overload, Calb1 may play a crucial role in reducing and stabilizing intracellular Ca2+ levels, protecting the lens from oxidative damage and promoting transparency.