Protective Effects of Coixol Against Nε-Carboxymethyllysine-Induced Injury in IMR-32 Neuronal Cells: Modulation of Endoplasmic Reticulum Stress and Amyloidogenic Pathways.

The accumulation of Nε-carboxymethyllysine (CML), a major advanced glycation end product (AGE), has been implicated in neuronal dysfunction by promoting oxidative stress, endoplasmic reticulum (ER) stress, and dysregulation of amyloid-β (Aβ) metabolism. This study evaluated the neuroprotective properties of coixol, a naturally occurring polyphenolic compound derived from the outer layers of Coix lacryma-jobi L. var. ma-yuen, in a CML-induced injury model using IMR-32 human neuronal-like cells. Cells were pretreated with coixol (1 μmol/L), N-acetyl-L-cysteine (NALC, 1 mmol/L), or 4-phenylbutyric acid (4-PBA, 200 μmol/L) for 1 h prior to CML (100 μmol/L) exposure for 24 h. Cell viability was determined by colorimetric analysis of 3-(4,5-dimethyl-2-yl)-2,5-diphenyltetrazolium bromide, while intracellular reactive oxygen species (ROS) generation was quantified using a fluorescence-based oxidative stress probe. Activities of key antioxidant enzymes and caspase-3 were determined using commercial assay kits. The expression of Aβ isoforms, amyloidogenic enzymes, ER stress markers, and apoptosis-related signaling proteins was quantified through validated immunoassays. Coixol pretreatment significantly enhanced cell viability by attenuating ROS accumulation and restoring antioxidant enzyme activities. Concurrently, coixol suppressed ER stress signaling via downregulation of the protein kinase R-like ER kinase/C/EBP homologous protein axis and modulated apoptosis by increasing B-cell lymphoma (Bcl)-2, reducing Bcl-2-associated X protein expression, and inhibiting caspase-3 activation and DNA fragmentation. Furthermore, coixol regulated Aβ metabolism by inhibiting the expression of β-site amyloid precursor protein-cleaving enzyme 1 and presenilin 1, while restoring insulin-degrading enzyme and neprilysin levels, leading to reduced accumulation of Aβ40 and Aβ42. Compared to NALC and 4-PBA, coixol demonstrated comparable or superior modulation across multiple pathological pathways. These findings highlight coixol's potential as a neuroprotective candidate in AGE-associated neurodegenerative conditions.