Intrinsic Regeneration Mechanisms of Murine Lacrimal Gland Epithelial Cells for Therapeutic Applications in Aqueous- Deficient Dry Eye Disease

Purpose Aqueous-deficient dry eye disease (ADDE) results from lacrimal gland dysfunction leading to tear film instability and chronic ocular surface inflammation. Despite the gland’s known regenerative potential, the intrinsic molecular mechanisms enabling epithelial recovery remain poorly understood. This study aimed to characterize intrinsic regeneration processes of murine lacrimal gland epithelial cells (mLGECs) and identify pathways with therapeutic potential. Methods Primary mLGECs were isolated from neonatal C57BL/6J mice and characterized by RT-PCR, immunocytochemistry, and β-hexosaminidase secretion assays. Acute injury was induced by brief exposure to 20% ethanol. Cell viability, apoptosis, and transcriptional responses were analyzed during early recovery phases. Pathway enrichment was conducted using Gene Ontology and KEGG annotations. Regeneration-associated growth factors were validated by quantitative PCR, ELISA, and functional assays assessing cell viability and wound closure in the presence or absence of the MAPK inhibitor SCH772984. Results Cultured mLGECs preserved epithelial and lacrimal-specific markers and maintained regulated secretory activity. RNA-seq two hours post-injury revealed robust activation of MAPK signaling, accompanied by strong upregulation of regenerative ligands including Hbegf, Areg, Ngf, and Gdnf. Transient protein induction of HBEGF and AREG was confirmed. Functionally, HBEGF, AREG, NGF, and GDNF markedly enhanced epithelial survival, ATP recovery, and wound closure, while MAPK inhibition abolished regeneration, demonstrating pathway dependence. Conclusion These results identify MAPK-dependent mechanisms as central to the intrinsic regenerative response of lacrimal gland epithelial cells following acute injury. HBEGF and AREG emerged as key mediators driving epithelial survival and proliferation. The findings provide a mechanistic basis for developing defined growth factor–based therapies as standardized, safe, and effective alternatives to blood-derived treatments for patients with severe ADDE.