Fosfenopril Attenuates Inflammatory Response in Diabetic Dry Eye Models by Inhibiting the TLR4/NF-κB/NLRP3 Signaling Pathway

Abstract

Purpose: The purpose of this study was to investigate the involvement of the TLR4/NF-κB/NLRP3 signaling pathway and its underlying mechanism in diabetic dry eye.

Methods: Two models of diabetic dry eye were established in high glucose-induced human corneal epithelial (HCE-T) cells and streptozotocin (STZ)-induced C57BL/6 mice, and the TLR4 inhibitor fosfenopril (FOS) was utilized to suppress the TLR4/NF-κB/NLRP3 signaling pathway. The expression changes in TLR4, NF-κB, NLRP3, and IL-1β, and other factors were detected by Western blot and RT‒qPCR, the wound healing rate was evaluated by cell scratch assay, and the symptoms of diabetic mice were evaluated by corneal sodium fluorescein staining and tear secretion assay.

Results: In the diabetic dry eye model, the transcript levels of TLR4, NF-κB, NLRP3, and IL-1β were raised, and further application of FOS, a TLR4 inhibitor, downregulated the levels of these pathway factors. In addition, FOS was found to be effective in increasing the wound healing rate of high glucose-induced HCE-T cells, increasing tear production, and decreasing corneal fluorescence staining scores in diabetic mice, as measured by cell scratch assay, corneal sodium fluorescein staining assay, and tear production.

Conclusions: The current study found that the TLR4/NF-κB/NLRP3 signaling pathway regulates diabetic dry eye in an in vitro and in vivo model, and that FOS reduces the signs of dry eye in diabetic mice, providing a new treatment option for diabetic dry eye.

Figure 1.

Results of cell morphology and cell activity. Alterations in HCE-T cells stimulated with high glucose. In this study, we observed the morphology and size of cells treated with different glucose concentrations (25, 30, and 35 mM) using inverted microscopy (A). Effects of different concentrations (25, 30, and 35 mM) of glucose on cell survival (B). Each experiment was repeated three times (mean ± SD; *P < 0.05 versus the NC group).

Figure 2.

Changes in TLR4, NLRP3, NF-κB , and IL-1β in high glucose-induced HCE-T cells. HCE-T cells were stimulated with glucose at a final concentration of 25 mM. RT‒qPCR was used to determine the mRNA expression of the TLR4/NF-κB/NLRP3 signaling pathway (A); Western blotting analysis was used to determine the protein expression of the TLR4/NF-κB/NLRP3 signaling pathway (B, C), and the grayscale of the bands was analyzed with β-tubulin as an internal reference gene. Each treatment was tested in triplicate in each experiment, and each experiment was repeated three times (mean ± SD; ns P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001).

Figure 3.

Effects of the TLR4 inhibitor FOS on TLR4, NLRP3, NF-κB , and IL-1β-related factors in high glucose-induced HCE-T cells. High glucose stimulation of HCE-T cells with FOS. RT‒qPCR was used to determine the mRNA expression of the TLR4/NF-κB/NLRP3 signaling pathway (A); Western blotting analysis was used to determine the protein expression of the TLR4/NF-κB/NLRP3 signaling pathway (B, C), and the grayscale of the bands was analyzed with β-tubulin as an internal reference gene. Each treatment was tested in triplicate in each experiment, and each experiment was repeated three times (mean ± SD; ns P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001).

Figure 4.

LPS increases the expression of inflammatory mediators in high glucose-stimulated HCE-T cells. RT‒qPCR was used to determine the mRNA expression of the TLR4/NF-κB/NLRP3 signaling pathway (A); Western blotting analysis was used to determine the protein expression of the TLR4/NF-κB/NLRP3 signaling pathway (B, C), and the grayscale of the bands was analyzed with β-tubulin as an internal reference gene. Each treatment was tested in triplicate in each experiment, and each experiment was repeated three times (mean ± SD; ns P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001).

Figure 5.

Results of cell scratching experiments. HCE-T cells were grown in media for 24 hours. Vertical lines were made on six-well plates using a 1000 µL pipette tip. Then, different media were used for incubation, and the healing of HCE-T cells was observed after 0, 6, 12 hours (A, C, E). ImageJ was used for calculating the repair areas (B, D, F). The identical experiment was repeated three times (mean ± SD; ns P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001).

Figure 6.

Progression of dry eye in diabetic mice. Streptozotocin was injected intraperitoneally to induce type 1 diabetes in mice, and blood glucose was measured at 10 days and 4 weeks after the last injection (B) (n = 5). Corneal fluorescein sodium staining (A and C) (n = 5) and tear secretion (D) (n = 5) were compared between STZ-induced diabetic mice at 4 weeks, 6 weeks, and 8 weeks (ns P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001).

Figure 7.

TLR4 inhibitor FOS reduces TLR4, NLRP3, NF-κB, and IL-1β protein expression in the corneas of diabetic mice. The expression of the TLR4/NF-κB/NLRP3 signaling pathway was determined by Western blotting analysis (A, B), analyzing the grayscale of the bands with β-tubulin as an internal reference gene (C–F). Each treatment was tested in triplicate in each experiment, and each experiment was repeated three times (mean ± SD; ns P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001).

Figure 8.

Effect of the TLR4 inhibitor FOS on dry eye in diabetic mice. Corneal sodium fluorescein staining (A, B) (n = 5) and tear secretion assessment (C) (n = 5) were performed in diabetic mice with topical administration of 1 mL FOS for 1 week, and PBS was used as a mediator control (ns P > 0.05, **P < 0.01, ***P < 0.001).