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; NRF2; ROS-NLRP3-IL-1β.

Graphical abstract

Fig. 1

Calcitriol protected human corneal epithelial cells against hyperosmotic stress-induced cytotoxicity and subsequent NLRP3 activation. (a,b) Cell viability of iHCECs or priHCECs treated with hyperosmotic medium (350, 400 or 450 mOsM) or co-treated with calcitriol (10⁻⁶ M) for 24 h. (c) Calcitriol (10⁻⁶ M) inhibited NLRP3 related gene expression in iHCECs that exposed to 450mOsM hyperosmotic stress. (d,e) Calcitriol treatment (10⁻⁶ M) suppressed IL-1β production by iHCECs or priHCECs in response to 450 mOsM hyperosmotic stress. (f) The synergetic effects of NLRP3 antagonist glyburide (100 μM) or the caspase-1 specific inhibitor YVAD (100 μM) with a physiologic concentration of calcitriol (10⁻⁹ M) on suppressing IL-1β production by iHCECs in response to 450mOsM hyperosmotic stress. Data are shown as mean ± SD from four separate experiments. * P < 0.05, * * P < 0.01, * P < 0.001, as compared with 450 mOsM.

Fig. 2

Calcitriol alleviated hyperosmotic stress-induced cytotoxicity and IL-1β secretion through VDR activation. (a) VDR gene expression and (b) protein expression in negative control (nc) siRNA treated- and VDR siRNA treated group. (c) The suppressive effect of calcitriol treatment (10⁻⁶ M) on IL-1β production by iHCECs in response to hyperosmotic stress was almost abolished by VDR siRNA. (d) The effect of VDR silencing on cell survival rate in response to hyperosmotic stress, examined by a CCK-8 assay. Data are shown as mean ± SD from four separate experiments. * P < 0.05, **P < 0.01, *P < 0.001.

Fig. 3

Calcitriol attenuated hyperosmotic stress-induced oxidative stress in iHCECs. (a, b) ROS generation detected by CM-H₂DCFDA probe in cells treated with calcitriol (10⁻⁶ M) or antioxidant NAC (30 mM) for 24 h. (c, d) Immunofluorescent staining of 8-OHdG positive cells under hyperosmotic stress or co-treated with calcitriol (10⁻⁶ M). Images were taken at 200 × magnification. *P < 0.05, **P < 0.01; (mean ± SD, n = 4).

Fig. 4

Calcitriol induced the activation of Nrf2-antioxidant signaling in iHCECs under hyperosmotic stress. (a) The cytoplasm and nucleus levels of NRF2 protein in iHCECs under hyperosmotic stress or co-treated with calcitriol (10⁻⁶ M). (b) NRF2 gene expression in negative control (nc) siRNA treated- and NRF2 siRNA treated group. (c) The suppressive effect of calcitriol treatment on IL-1β production was significantly impeded by NRF2 silencing. (d-f) Enzymatic activities of SOD, catalase, and glutathione reductase (GR) were significantly increased by calcitriol treatment. *P < 0.05; (mean ± SD, n = 4).

Fig. 1

Calcitriol inhibited JNK activation induced by hyperosmotic stress in iHCECs.