Effects of ophthalmic surface anesthetic alcaine on the proliferation and apoptosis of human corneal endothelial cells through HIF-1α regulation

Abstract

The corneal endothelium is a monolayer, which mediates solute and water flux across the posterior corneal surface. Alcaine's main component proparacaine is paramount in human corneal endothelium (HCE) cell regulation. This study explored the mechanism of alcaine in regulating HCE cells. HCE cell morphology under gradient concentrations was observed by an optical microscope. Cell proliferation and viability were detected by MTT assay to determine the half inhibitory concentration (IC 50). Cell apoptosis rate, HIF-1α mRNA expression, and HIF-1α, p/t-JNK and Caspase-3 protein levels were detected by flow cytometry, RT-qPCR, and Western blot. After treatment with alcaine at 0.625-5 g/L concentration range for 24 h, HCE cells showed cytoplasmic vacuolation, cell shrinkage, separation from culture matrix, and eventual death. Alcaine treated-HCE cell proliferation was decreased in a dose-dependent manner. The IC 50 of alcaine was 1.26 g/L. After alcaine treatment, HCE cell apoptosis rate was promoted and HIF-1α levels in HCE cells were stimulated. Knockdown of HIF-1α partially annulled the effects of alcaine on inhibiting HCE cell proliferation and facilitating apoptosis. Alcaine might activate the JNK/caspase-3 pathway by increasing HIF-1α. The inhibition of the JNK/caspase-3 pathway partially abrogated the effects of alcaine on inhibiting HCE cell proliferation and promoting apoptosis. Alcaine might affect HCE cell proliferation and apoptosis by upregulating HIF-1α and activating the JNK/caspase-3 pathway.

Keywords: Alcaine; Apoptosis; Caspase-3; HIF-1α; IC 50; JNK; Proliferation.