Abstract
Purpose :
Corneal fibrosis is a common sequela of traumatic or chemical injury, corneal infections, and photorefractive surgery that can result in vision loss. There are limited treatment options to prevent or resolve corneal fibrosis, therefore we sought to evaluate the anti-fibrotic effect of isoliquiritigenin (ISL), a molecule with demonstrated anti-fibrotic properties, in human corneal fibroblasts (HCFs) in vitro stimulated with the pro-fibrotic cytokine, TGF- β1.
Methods :
HCFs were isolated from corneal donor tissue (n = 3 donors) and cell viability assays (MTT) were performed. HCFs were treated with 25 μM, 50 μM, or vehicle control in the presence or absence of TGF- β1 (10 ng/mL). After treatment, total RNA and protein were isolated for qRT-PCR and western blotting to quantify alpha-smooth muscle actin (αSMA) expression, which is upregulated during a pro-fibrotic response, normalized to GAPDH. Immunofluorescence was performed to label cellular αSMA and F-actin. An RNA sequencing analysis was performed and a select group of genes was chosen for qRT-PCR validation (n = 4 donors). Data were analyzed using an ANOVA.
Results :
Cell viability was significantly reduced at ISL concentrations ≥ 121.5 μM. A dose-dependent inhibition of αSMA mRNA expression was identified with ISL (25 μM, 2.5-fold reduction, P = 0.0025; 50 μM, 5-fold reduction, P = 0.0001) compared with vehicle control in the presence of TGF- β1. Additionally, αSMA protein expression was decreased by ISL (25 μM, 2-fold reduction, P = 0.1847; 50 μM, 3-fold reduction, P = 0.0352). Immunofluorescence revealed reduced αSMA and F-actin staining, confirming the expression results and the downregulation of the anti-fibrotic response. The RNAseq analysis revealed a reduction in fibrosis-associated extracellular matrix (ECM) gene expression. In a larger cohort, qRT-PCR confirmed the downregulation of fibronectin 1 (P = 0.0247), collagen type 1 α1 (P = 0.0018), and connective tissue growth factor (P = 0.0036).
Conclusions :
Treatment with ISL inhibits αSMA expression in HCFs stimulated with TGF- β1 at both the mRNA and protein level, and the polymerization of F-actin, all features of an anti-fibrotic response. RNA sequencing and validation suggest that ISL prevents the upregulation of ECM genes associated with fibrosis. Future studies will be focused on the anti-fibrotic mechanism(s) of ISL in HCFs and determining the anti-fibrotic effect in vivo.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.