Targeting lactylation in a novel metabolic-epigenetic-inflammatory axis mitigates inflammation and promotes healing following corneal injury

PURPOSE: To investigate the potential roles of histone lactylation, N6-methyladenosine (m6A) modification, and the Janus kinase-signal transducer and activator of transcription 3 (JAK-STAT3) pathway in regulating the wound healing following corneal alkali burns. METHODS: An in vivo corneal alkali burn model was established using C57BL/6J mice. Corneal inflammation, fibrosis, histone lactylation, pan-lactylation, m6A modification, and JAK-STAT3 pathway activation were assessed at indicated time points post-injury using slit-lamp examination, hematoxylin and eosin (H&E) staining, RT‒qPCR, and western blotting. In vitro, the effects of histone lactylation and m6A modification on JAK-STAT3 signaling and inflammation were investigated in interleukin-1β (IL-1β)-stimulated keratocytes. The impact of histone lactylation on corneal wound repair was evaluated by employing the glycolysis inhibitors 2-deoxy-D-glucose (2-DG) and rotenone, which modulates glycolysis via mitochondrial complex I inhibition. RESULTS: Analysis of the GSE191228 dataset revealed that JAK-STAT pathway plays an important role in corneal wound healing. Gene Set Enrichment Analysis (GSEA) of proteomic data further demonstrated substantial upregulation of the IL-6-JAK-STAT pathway in alkali-burned corneas. Additionally, the analysis revealed a positive correlation between both hypoxia and glycolysis pathways and the corneal alkali burns condition. In the mouse model, corneal alkali burns activated JAK-STAT3 pathway, elevated methyltransferase-like 3 (METTL3) expression, and increased global m6A modification. Furthermore, histone H3 lysine 18 lactylation (H3K18la) levels were significantly increased, accompanied by characteristic changes in pan-lactylation expression patterns. At day 7 post-alkali burn, treatment with 2-DG alleviated corneal inflammation and fibrosis while decreasing the levels of H3K18la, METTL3, and pSTAT3. In vitro, knockdown of LDHa significantly attenuated IL-1β-stimulated upregulation of H3K18la, METTL3 and pSTAT3 in keratocytes. Consistently, treatment with 2-DG or rotenone decreased or increased the expression of H3K18la, METTL3, and pSTAT3, as well as inflammatory factor IL-6, respectively. Moreover, reducing METTL3 expression via siRNA-METTL3 transfection decreased pSTAT3 activation. In TGF-β-induced fibrotic response of keratocytes, 2DG suppressed the TGF-β-mediated elevation of H3K18la, METTL3 and αSMA. Additionally, LPS similarly elevated H3K18la levels in keratocytes. CONCLUSIONS: Hypoxia and glycolysis following corneal alkali burn initiate lactylation, subsequently activating m6A modification and the JAK-STAT3 signaling pathway. This lactylation-induced cascade critically regulates corneal wound healing and represents a promising therapeutic target.