The influence of strain on human keratocyte redox homeostasis and behavior

Aims/Purpose: To study the influence of strain on the expression of specific proteins in human keratocytes and its consequential effects on keratocyte intracellular redox homeostasis and behavior.

Methods: Strain was applied to human keratocytes using the Flexcell® Tension Systems. Proteomics and western blot were used to identify proteins regulated in response to strain. Immunofluorescence (IF) staining and live-cell imaging were employed to monitor reactive oxygen species (ROS) production and mitochondrial membrane potential (ΔΨM). Expression of oxidative stress-related protein, and its influence on nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) nuclear translocation and keratocyte proliferation and migration, were assessed by western blot, IF staining and bromodeoxyuridine (BrdU) assay. Mouse injury models and keratocytes from keratoconus patients were used to assess how strain (intraocular pressure, IOP) influences downstream protein in vivo.

Results: The expression of protein Cytochrome P450 1B1 (CYP1B1) and Aldehyde Dehydrogenase 3 Family Member A1 (ALDH3A1) was significantly upregulated in strained keratocytes. Increased CYP1B1 effectively suppressed H₂O₂-induced ROS production and mitigated the dissipation of ΔΨM. ALDH3A1 expression was negatively related to ROS accumulation. When the expression of ALDH3A1 was knocked down, NF-κB nuclear translocation was promoted, ultimately resulting in increased keratocyte proliferation and migration. Mice and keratoconus patients with increased corneal strain also showed elevated ALDH3A1.

Conclusions: Corneal strain significantly upregulates the expression of CYP1B1 and ALDH3A1. Increased CYP1B1 helps maintain keratocyte intracellular redox homeostasis and subsequently regulates keratocyte proliferation and migration via ALDH3A1.