Abstract: Objective  To investigate whether the ferroptosis inhibitor ferrostatin-1 (Fer-1) attenuates cerebral edema 24 h after middle-cerebral-artery occlusion-reperfusion (MCAO/R) in mice and to explore the underlying mechanisms focusing on nuclear factor kappa B (NF-κB), matrix metalloproteinase-9 (MMP-9) and aquaporin-4 (AQP4) polarity. Methods  Adult male C57BL/6J mice were randomly allocated to Sham, MCAO/R + vehicle, and MCAO/R + Fer-1 groups (n=10－13 per group). Sixty-minute filament occlusion was followed by reperfusion. Fer-1 (0.8 mg·kg-1) or equal-volume vehicle was administered intravenously at the onset of reperfusion. Twenty-four hours later, brain water content was quantified by the dry-wet weight method; Western blotting was employed to detect total NF-κB, MMP-9 and AQP4 levels as well as the AQP4-M23/M1 isoform ratio; and immunofluorescence co-labelling of AQP4 with CD31 was performed to assess perivascular AQP4 polarization. Image J was used for densitometric analysis of Western blotting bands and quantitative assessment of fluorescence intensity. Results  ① Brain water content was markedly elevated in the MCAO/R group compared with Sham (P<0.01), and this increase was significantly attenuated by Fer-1 (P<0.05). ②The protein expression levels of NF-κB, MMP-9 and total AQP4 were up-regulated in the ischemic hemisphere after MCAO/R (P<0.05 versus Sham); Fer-1 treatment substantially decreased NF-κB and MMP-9 expression (P<0.01) and moderately down-regulated total AQP4 (P<0.05). ③The AQP4-M23/M1 ratio, an index of AQP4 polarization, was significantly decreased after MCAO/R (P<0.01 versus Sham) and was restored by Fer-1 (P<0.05).④ In the ischemic hemisphere, the peak-to-background fluorescence ratio of perivascular AQP4 was lower in MCAO/R mice than that in Sham mice (P<0.05); Fer-1 administration reversed this deficit (P<0.05). Conclusion  Fer-1 attenuates early cerebral edema following MCAO/R by suppressing NF-κB and MMP-9 signaling, down-regulating total AQP4 abundance and preserving perivascular AQP4 polarization. These findings highlight ferroptosis inhibition as a potential therapeutic strategy for ischemia－reperfusion-induced brain edema.  

Key words: ferrostatin-1, cerebral ischemia, brain edema, nuclear factor kappa B（NF-κB）, matrix metalloproteinase-9（MMP-9）, aquaporin-4（AQP4）polarization