AIM: 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.
METHODS: 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.
RESULTS: 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 Fe, 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. 2+
CONCLUSION: 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.
