METTL3-mediated m⁶A modification of Smad3 affects ferroptosis in septic acute lung injury by mediating GRSF1/GPX4

Ferroptosis is recognized as a critical driver in the pathogenesis of septic acute lung injury (ALI). Although both METTL3-mediated m⁶A modification and the GRSF1/GPX4 axis are known to regulate ferroptosis, the potential interplay between these pathways in the context of septic ALI remains unexplored.

Using a murine model of cecal ligation and puncture (CLP) and lipopolysaccharide (LPS)-treated MLE-12 cells, we identified a novel METTL3-m⁶A-Smad3-GRSF1/GPX4 signaling axis engaged in ferroptosis and the subsequent ALI pathogenesis. Inhibition of METTL3 alleviated lung injury in CLP mice, which was associated with reduced m⁶A modification levels and suppression of ferroptosis. In vitro, METTL3 knockdown upregulated the GRSF1/GPX4 axis, thereby ameliorating LPS-induced mitochondrial dysfunction and ferroptosis in lung epithelial cells. Through integrated RNA sequencing, bioinformatic predictions, and mechanistic assays (MeRIP, RIP, ChIP, and dual-luciferase reporter assays), we demonstrated that METTL3 promoted Smad3 expression by enhancing Smad3 mRNA stability, which in turn down-regulated GRSF1 and its downstream target GPX4. The aforementioned protective effects of METTL3 inhibition were effectively reversed by either Smad3 overexpression or GRSF1 knockdown. Consistently, METTL3 inhibition in vivo reduced Smad3 m⁶A modification and expression, while restoring GRSF1 and GPX4 protein levels in the lungs of septic mice.

Our findings reveal a novel METTL3-m⁶A-Smad3-GRSF1/GPX4 regulatory axis that modulates ferroptosis and affects the development of septic ALI. This axis represents a potential therapeutic target for this disease. Considering the complicated ferroptosis regulatory network, more efforts should be made to elucidate the interactions of this axis with other pathways in this network.