C21 ameliorates microglial ferroptosis and neuronal apoptosis after ischemic brain injury by activating the METTL3/USP18/FASN axis.

BACKGROUND: Microglial ferroptosis is a key risk factor promoting neuroinflammation after ischemic brain injury (IBI). Although Compound 21 (C21) has neuroprotective and anti-inflammatory properties, its role in modulating microglial ferroptosis in IBI remains unexplored. METHODS: IBI was induced in C57BL/6 mice via transient middle cerebral artery occlusion (tMCAO). Parallelly, an in vitro oxygen-glucose deprivation (OGD) model was established using BV2 microglial cells to mimic ischemic conditions. Gene and protein expression levels were quantified using quantitative real-time polymerase chain reaction (qRT-PCR), western blot, and immunofluorescence staining. Cell viability and apoptosis were assessed using cell counting kit-8 (CCK-8) assay and flow cytometry, respectively. The interactions between methyltransferase-like 3 (METTL3), ubiquitin-specific peptidase 18 (USP18), and fatty acid synthase (FASN) were analyzed by RNA immunoprecipitation (RIP) and co-immunoprecipitation (Co-IP) assays. The ubiquitination of FASN was assessed by immunoprecipitation (IP) assay. The N6-methyladenosine (m6A) modification of USP18 mRNA was analyzed by m6A RNA immunoprecipitation (Me-RIP) and dual-luciferase reporter assays. RESULTS: C21 treatment alleviated neuroinflammation and neuronal apoptosis in IBI models by inhibiting microglial ferroptosis. Mechanistically, C21 upregulated USP18 via m6A-dependent mRNA stabilization through increasing METTL3 expression. In addition, C21 stabilized FASN protein through promoting USP18-mediated deubiquitination. Functional experiments further demonstrated that C21 ameliorated microglial ferroptosis and neuronal apoptosis after IBI by activating the METTL3/USP18/FASN axis. CONCLUSION: C21 ameliorated neuroinflammation and neuronal apoptosis following IBI by inhibiting microglial ferroptosis through activating the METTL3/USP18/FASN axis. Our findings provide mechanistic insights into the protective role of C21 in IBI and establish a preclinical foundation for further evaluation of its therapeutic potential.