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

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. 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. 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. 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.