PPP4C-CALML5 axis alleviates corneal injury and corneal pain in dry eye disease

Corneal nerve damage in dry eye disease (DED) disrupts the lacrimal functional unit, leading to pain, reduced tear secretion, and impaired wound healing. Current treatments remain inadequate for addressing DED-induced corneal injury and neuropathic pain. We established a DED mouse model through lacrimal gland excision and performed comprehensive analyses including Schirmer tests, behavioral assessments, histopathology, and molecular biology techniques (qRT-PCR, Western blot, immunofluorescence). Viral vector-mediated gene overexpression, knockdown, and comprehensive rescue experiments were employed to investigate CALML5 and PPP4C functions in DED. CALML5 expression was significantly downregulated in DED corneas. CALML5 overexpression alleviated corneal damage by increasing neuronal marker expression (β III tubulin), reducing pain markers (SP, P2 × 3 and PKC), and suppressing neuroinflammation (reduced Iba-1 and GFAP). Moreover, CALML5 overexpression improved tear secretion, reduced corneal fluorescence staining, and mitigated pain behaviors. PPP4C showed similar downregulation in DED and interacted with CALML5. PPP4C overexpression recapitulated the therapeutic effects of CALML5 overexpression while upregulating CALML5 expression. Furthermore, comprehensive rescue experiments demonstrated that CALML5 knockdown abolished the therapeutic benefits of PPP4C overexpression on DED mouse tear secretion, corneal integrity, pain behaviors, nerve density, pain marker expression, and neuroinflammation, suggesting CALML5 as a potential downstream effector. Our study identified CALML5 as a key regulator in DED-induced corneal injury and ocular pain-related behaviors and demonstrated that PPP4C may exert protective effects through CALML5-dependent mechanisms. These findings provide potential therapeutic targets for DED treatment.