Role of Programmed Cell Death Pathways in Mediating Epithelial Wound Healing and Its Defects in Diabetic Corneas

Purpose: Diabetic keratopathy (DK), a sight-threatening corneal disease, is characterized by delayed epithelial wound healing and ulceration. This study investigated the role of programmed cell death (PCD) pathways-caspase-mediated apoptosis and RIPK1/RIPK3-mediated necroptosis-in corneal wound healing in normal (NL) and diabetes mellitus (DM) B6 mice. Methods: Corneal epithelial debridement wounds were created in normoglycemic, streptozotocin (STZ)-induced, and db/db diabetic mice. Wound closure was quantified by digitizing the residual wound area. PCD pathway activity was assessed by RT-qPCR, immunohistochemistry (IHC), and whole-mount confocal microscopy. Pharmacologic inhibitors were applied to determine the role of specific PCD modulators in corneal wound healing. Results: In healing DM corneas, wounding-induced Casp8 upregulation was blunted, whereas RIPK3 upregulation was enhanced. At the tissue level, cleaved caspase-3 (c-Casp3, active) was more abundant in NL corneas, whereas phosphorylated RIPK3 (pRIPK3) predominated in DM corneas. The pRIPK3 was detected as early as 9 hours post-wounding in DM corneas, whereas c-Casp3 was most abundant at 72 hours post-wounding-a time point when PCD activity was no longer detectable in NL corneas. Pharmacologic inhibition of RIPK3 accelerated wound closure and suppressed inflammation in both NL and DM corneas, with greater benefit in DM corneas. Notably, extracellular HMGB1, a key alarmin/DAMP released during necroptosis, was strongly localized to basal cells. Blocking HMGB1 with glycyrrhizin, a cell-impermeable inhibitor, improved wound healing outcomes in DM corneas. Conclusions: Hyperglycemia skews PCD from apoptosis toward necroptosis. Targeting necroptosis or its downstream effectors, such as HMGB1, may represent a promising therapeutic approach for DK.