YTHDF1 promotes myelin phagocytosis through m6A-dependent regulation of Galectin-3 to enhance macrophage glycolysis in painful diabetic neuropathy.

Painful diabetic neuropathy (PDN) represents a prevalent complication of diabetes, impacting sensory, motor, and autonomic nerves, with its pathogenesis remaining unclear, thereby hindering effective treatment. This study investigates the mechanisms underlying PDN and aims to identify potential molecular treatment targets. Male C57BL/6 J wild-type mice were employed to establish a PDN model, receiving intrathecal administration of shRNA targeting Galectin-3 (sh-Gal-3), shRNA targeting YTHDF1 (sh-YTHDF1), a YTHDF1 overexpression vector, or the m6A inhibitor 3-deazaadenosine (3-DAA), either individually or in combination. Macrophages underwent gene knockdown or overexpression and/or treatment with the glycolysis inhibitor 2-deoxy-d-glucose (2-DG) or 3-DAA. Diabetes was confirmed by monitoring blood glucose levels. Pain behavior was evaluated using mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) assessments. Expression levels of Gal-3 and YTHDF1 were analyzed via Real-time PCR and Western blot, while myelin phagocytosis was evaluated through immunofluorescence and/or transmission electron microscopy. Glycolysis was assessed by measuring glucose uptake, lactate production, extracellular acidification rate (ECAR), and oxygen consumption rate (OCR). The RNA pull-down assay facilitated the detection of YTHDF1 binding to Gal-3 mRNA, and the half-life of Gal-3 mRNA was measured following transcription blockade using actinomycin D. Additionally, meRIP-qPCR assessed the m6A modification on Gal-3 mRNA. In vivo analyses revealed upregulation of Gal-3, which colocalized with IBA1. Silencing Gal-3 alleviated mechanical allodynia and diminished myelin phagocytosis. In vitro, Gal-3 silencing inhibited glycolysis, while Gal-3 overexpression enhanced myelin phagocytosis, an effect reversed by 2-DG treatment. Furthermore, high glucose stimulation elevated YTHDF1 expression, subsequently increasing Gal-3 levels; this induction was abrogated by YTHDF1 knockdown. Mechanistically, YTHDF1 enhanced Gal-3 mRNA stability through an m6A-dependent mechanism, promoting glycolysis and myelin phagocytosis. Consistently, YTHDF1 overexpression exacerbated PDN symptoms and myelin phagocytosis in vivo, which were mitigated by YTHDF1 knockdown or 3-DAA administration. YTHDF1 enhances Gal-3 mRNA stability and expression via an m6A-dependent mechanism, thereby facilitating glycolysis and myelin phagocytosis in PDN.