Calcitriol inhibits ROS-NLRP3-IL-1β signaling axis via activation of Nrf2-antioxidant signaling in hyperosmotic stress stimulated human corneal epithelial cells

Abstract

Purpose: The activation of ROS-NLRP3-IL-1β signaling axis induced by hyperosmotic stress (HS) has been recognized as a key priming stage of epithelial inflammation in dry eye pathogenesis. The current study aims to investigate whether calcitriol, the active metabolite of vitamin D3, could protect cells against HS-induced inflammation through modulating this critical step. Methods: Human corneal epithelial cells (iHCECs) were cultured in hyperosmotic medium (450 mOsM) with various concentrations of calcitriol. Small interfering RNA (siRNA) was used to knock down the expression of vitamin D receptor (VDR) in iHCECs. NLRP3 activation and IL-1β generation were detected by RT-qPCR or ELISA, respectively. Oxidative stress markers including ROS and 8-OHdG were examined by fluorometric analysis. The nuclear translocation of NRF2 was assessed by western blotting. Results: Calcitriol could protect cells against HS-induced injury through inhibiting ROS-NLRP3-IL-1β signaling axis. Calcitriol remarkably suppressed the expression of NLRP3 inflammasome related genes and the production of IL-1β in cells that were exposed to HS. It could also significantly attenuate HS-induced oxidative stress, shown as the reduced intracellular ROS generation and 8-OHdG staining cells after calcitriol treatment. Calcitriol induced the translocation of NRF2 to the nucleus, and thereby triggered the expression of several antioxidant enzymes. Conclusion: The current study indicated that calcitriol could inhibit the priming stage of HS-induced cellular inflammation, highlighting its potential capacity to prevent and mitigate dry eye related corneal inflammation at an earlier stage. Keywords: Calcitriol, Dry eye, Inflammasomes, ROS-NLRP3-IL-1β, NRF2