The in vitro effect of human orbital adipose-derived stem cell exosomes on the expression of genes involved in apoptosis, inflammation, and neurogenesis in corneal keratocytes
Description
Background: Pathological insults often disrupt corneal keratocyte homeostasis, leading to viability loss and visual impairment. While orbital adipose stem cell-derived exosomes (OASC-Exos) are recognized for regenerative potential, their specific mechanisms in keratocyte remodeling remain underexplored. This study investigates the effects of OASC-Exos on human corneal keratocytes. Methods: OASC-Exos were isolated and validated via TEM and surface markers (CD9, CD63, CD81). We evaluated keratocyte metabolic viability across a concentration gradient (0.0032-320 µg/mL). Gene expression of apoptotic regulators (BCL-2, BAX), neurotrophic factors (NGF, TRKB, LIF, MAPK), and cytokines (IL-1β, IL-6) was quantified by qRT-PCR, alongside ELISA for IL-1β and IL-6 secreted proteins. Morphometric structural changes were analyzed via high-throughput pipeline and microstructural imaging. Results: OASC-Exos demonstrated high cytocompatibility, with 0.0032 µg/mL supporting optimal viability (106.20 ± 5.67%). Treatment significantly enhanced pro-survival signaling, increasing the BCL-2/BAX ratio by 4.35 ± 0.52 (p = 0.003). Crucially, OASC-Exos activated a robust neurotrophic program, characterized by significant upregulation of NGF (4.56 ± 0.76), TRKB (2.03 ± 0.34), and MAPK (4.12 ± 0.61). While TGF-β (2.01 ± 0.06, p < 0.001) and IL-6 (4.19 ± 0.49, p = 0.003) mRNA increased, secreted IL-6 protein remained stable. IL-1β protein secretion increased significantly, accompanying a marginal, statistically non-significant upward trend in IL-1β mRNA expression. Morphometric analysis revealed significant cellular elongation (p < 0.001) and a transition to a spindle-shaped morphology, with high-resolution imaging confirming the emergence of radiating, neurite-like arborizations. Conclusion: OASC-Exos primed human corneal keratocytes toward an early neurogenic, regenerative phenotype driven by the NGF-TRKB-MAPK signaling axis and bolstered by BCL-2-mediated pro-survival mechanisms. These findings elucidate the molecular and structural pathways through which OASC-Exos promote corneal stromal repair, offering a foundation for therapeutic applications in corneal regenerative medicine.