Eye field specification requires precise spatiotemporal signaling. Among these signals, the morphogen Noggin blocks both BMP and Activin signaling pathways to generate retinal tissue from pluripotent frog animal cap cells. We previously found that Pax6 and Tbx3 are sufficient to replace Noggin in generating retinal tissue, but it was unclear which signaling pathway affected tbx3 expression. We had also found that Noggin, while activating all other eye field transcription factors (EFTFs), repressed tbx3 expression in animal cap cells, and that Otx2, a transcription factor essential for anterior neural plate induction, is expressed prior to tbx3 and can overcome Noggin-mediated repression to induce tbx3 in vitro. Since we have also found that Tbx3 is required for eye formation, we sought to determine how it is regulated. Here, we show that in vivo tbx3 expression is affected by both BMP and Activin signaling, and that the BMP/Activin chemical inhibitor LDN-193189 preferentially expands tbx3 expression in the eye field while repressing the cement gland, the anterior-most structure in the frog embryo. We further show that otx2, tbx3 and rax expression are all expanded in the eye field following LDN-193189 treatment. To determine how tbx3 is regulated in the eye, we identify evolutionarily conserved regions of the tbx3 gene that drive GFP expression in the eyes of transgenic tadpoles. We observed direct binding by myc-tagged Otx2 to activate tbx3 and one region that myc-tagged Otx2 directly binds. Together, these findings close the regulatory loop, demonstrating that tbx3 expression and early eye formation are directly regulated by Otx2-mediated activation.
Corneal fibrosis is a dysregulated wound-healing response in which epithelial and basement membrane failure, limbal dysfunction, unresolved inflammation, persistent myofibroblasts, vascular amplification, and extracellular matrix disorganisation impair corneal transparency and vision. Although transforming growth factor-β is central, persistence reflects interconnected epithelial, stromal, immune, vascular, and mechanobiological networks. This review frames fibrosis as a stage-dependent continuum from early fibrotic commitment through active stromal remodelling to established structural scar and evaluates interventions according to their most plausible biological window. Anti-inflammatory and pathway-directed treatments are considered primarily as preventive or active-stage interventions, whereas limbal restoration, mesenchymal stromal cell- and extracellular vesicle-based approaches, amniotic membrane-derived interventions, bioengineered constructs, and keratoplasty address surface stabilisation, regeneration, or structural replacement. Current evidence supports prevention and modulation of early or active fibrosis more strongly than pharmacological reversal of mature scar and remains dominated by preclinical studies and small clinical datasets. Translation will require stage- and phenotype-matched models, objective structural and optical endpoints, biologically informed patient stratification, and clear separation of prevention, suppression of active remodelling, regenerative stabilisation, and true scar reversal.
Dry eye disease (DED) is a multifactorial ocular surface disorder for which effective therapeutic options remain limited. This study aimed to investigate the therapeutic effects of naringin (NAR) in DED and elucidate its underlying mechanisms. DED was induced by topical administration of 0.075% BAC twice daily for 7 days. During days 8–14, BAC administration was continued while mice received NAR eye drops Corneal epithelial integrity and tear secretion were evaluated using corneal fluorescein sodium staining and the phenol red cotton thread test, respectively. In parallel, an in vitro hyperosmotic stress model was constructed by exposing human corneal epithelial cells to hyperosmotic sodium chloride medium (90 mM). Inflammation, oxidative stress, apoptosis, and autophagy-related phenotypes and mechanisms were analyzed using real-time quantitative PCR, Western blot, flow cytometry, and immunofluorescence. NAR eye drops significantly alleviated ocular surface damage in DED mice, enhanced tear secretion, reduced corneal inflammatory factor expression, and increased conjunctival goblet cell density. Furthermore, NAR attenuated oxidative stress and apoptosis through the promotion of autophagic flux. Mechanistic investigations revealed that NAR downregulated the PI3K/AKT/mTOR signaling pathway, thereby enhancing autophagic flux, and that its anti-inflammatory and antioxidant effects are closely associated with the inhibition of this pathway. Collectively, these findings suggest that NAR represents a promising therapeutic candidate for DED, with its beneficial effects mediated through enhancing autophagic flux via the PI3K/AKT/mTOR pathway, leading to the alleviation of oxidative stress and ocular surface inflammation.
Purpose To investigate whether didymin confers neuroprotection against retinal ischemia–reperfusion (IR) injury by modulating microglial pyroptosis and to elucidate the underlying molecular mechanisms. Methods A mouse retinal IR injury model was used to evaluate the neuroprotective effects of didymin and its regulation of pyroptosis and inflammation in vivo. LPS/ATP-stimulated BV-2 microglia were used to further evaluate the inhibitory effect of didymin on microglial pyroptosis in vitro. Retinal structure and function were assessed via histology and visual behavioral tests. The regulation of pyroptosis and inflammation was detected by quantitative real-time PCR (qPCR), western blotting, and immunofluorescence. Network pharmacology, molecular docking, and cellular thermal shift assay (CETSA) were employed to identify and validate EGFR as a target of didymin. Pharmacological inhibition of EGFR was employed in vivo to verify its functional involvement. Results Didymin treatment significantly alleviated IR-induced retinal damage and preserved visual function. Mechanistically, didymin primarily targeted microglia, effectively suppressing NLRP3/GSDMD-dependent pyroptosis and neuroinflammation. Didymin was found to bind and activate EGFR. Crucially, pharmacological inhibition of EGFR with AG1478 abolished the anti-pyroptotic, anti-inflammatory, and neuroprotective effects of didymin, confirming that didymin acts by activating EGFR signaling. Conclusions Our study demonstrates that didymin exerts potent neuroprotective effects in IR-induced retinal damage by activating EGFR signaling, thereby inhibiting microglial pyroptosis and neuroinflammation via the NLRP3/GSDMD pathway. Didymin may represent a potential immunomodulatory and neuroprotective candidate for ischemia-associated retinal neurodegenerative conditions.
