Astragaloside IV alleviates neuronal ferroptosis in ischemic stroke by regulating FTO-m6A-ACSL4 axis

Abstract

Abstract Background Ischemic stroke (IS) is a detrimental neurological disease with limited treatments options. Astragaloside IV (As-IV) was a promising bioactive constituent in the treatment of IS. However, the functional mechanism remains unclear. Methods IS cell and mice models were established by oxygen glucose deprivation /re-oxygenation (OGD/R) and middle cerebral artery occlusion (MCAO). RT-qPCR, Western blotting or Immunofluorescence staining were used to measure the gene expression. The N6-methyladenosine (m6A) levels were measured by MeRIP-qPCR and dot blot assays. CCK-8 and TUNEL staining were used to evaluate cell viability and apoptosis. TTC staining analyzed the infract area of brain tissues. The interplays of YTHDF3/ACSL4 and ATF3/FTO were analyzed by RNA-pull down, RIP, ChIP and dual-luciferase reporter assay. Results As-IV treatment promoted HT-22 and Neuro-2 cell viability and upregulated FTO levels in vitro and in vivo, as well as inhibited the levels of MDA, LDH, Fe2 + and ACSL4, while promoted the expression of GSH, SCL7A11 and GPX4. Knockdown of FTO, or overexpression of ACSL4 increased the infract size of brain tissues, neuron damage and the levels of MDA, LDH and Fe2+, while As-IV treatment reversed these changes. FTO regulated the m6A levels of ACSL4. YTHDF3 bound to ACSL4, and modulated its levels through m6A modification. ATF3 bound to FTO and positively regulated its levels. Knockdown of FTO or ATF3 increased the apoptosis of OGD/R cells, and promoted MDA, LDH and Fe2 + levels, while inhibited GSH expression. Knockdown of ACSL4, overexpression of FTO or treatment with As-IV reversed these effects. Conclusion As-IV promoted the transcription of FTO by upregulating ATF3, resulting in the decreased m6A levels of ACSL4, thus improving neuronal injury in IS by inhibiting ferroptosis.