m6A demethylase ALKBH5 reduces ferroptosis in diabetic retinopathy through the m6A-YTHDF1-ACSL4 axis

Diabetic retinopathy (DR) represents the main ocular complication of diabetes. Targeting ferroptosis is a promising treatment of choice for various diabetic complications. N6-methyladenosine (m6A) demethylase alkylation repair homolog protein 5 (ALKBH5) functions as a pivotal regulator of ferroptosis, and we investigated its role and molecular mechanisms in ferroptosis in DR.

A DR mouse model was developed by streptozotocin (STZ) intraperitoneal injection. High glucose (HG)-induced human retinal pigment epithelial cells (ARPE-19) were used as a DR model in vitro. ALKBH5, YTH N6-methyladenosine RNA binding protein 1 (YTHDF1) and acyl-CoA synthetase long-chain family member 4 (ACSL4) expression levels were examined by RT-qPCR and Western blot. The biological functions of ALKBH5 in vitro and in vivo were investigated by gain-of-function and loss-of-function analyses. ALKBH5's downstream regulatory mechanisms were detected by bioinformatics analysis, RNA pull-down, MeRIP-qPCR and actinomycin D assay.

ALKBH5 was under-expressed while YTHDF1 and ACSL4 were up-regulated in the retinal tissues of STZ-induced DR mice and HG-stimulated ARPE-19 cells. Ectopically expressed ALKBH5 or YTHDF1 knockdown partially reversed the increased ferroptosis in vitro and in vivo, evidenced by decreased levels of Fe²⁺, malondialdehyde and reactive oxygen species yet increased glutathione level. ALKBH5 mediated m6A modification of ACSL4 mRNA and disrupted its stability in a YTHDF1-dependent manner. Importantly, in vivo data demonstrated that overexpression of ALKBH5 or YTHDF1 knockdown repressed ferroptosis and alleviated DR by down-regulating ACSL4.

These findings suggest that ALKBH5 may delay DR progression by reducing ferroptosis through the m6A-YTHDF1-ACSL4 axis, offering therapeutic paradigms for the treatment of DR.