Fig 2: AQP4 overexpression reduces transferrin content and neuronal ferroptosis in the brain parenchyma of SAH mice. (a) Representative images of FITC-dextran (40 kD) after post-SAH intravenous injections at 24 hours after SAH (scale bar = 50 µm). (b, c) Western blot analysis showed the levels of transferrin in the cortex after AAV overexpression of AQP4. n = 6 per group. Data are the mean ± SD (ANOVA F(2, 15) = 8.3P = 0.0036; **P = 0.0053, post hoc Tukey's test). (d, e) Representative fluorescence micrographs and quantification of PI-positive and neuron-positive cells in the cortex after AAV overexpression of AQP4, n = 6 per group. Fluorescence colors: NeuN: green; PI: red; DAPI: blue. Scale bar = 20 µm. The quantification of PI-positive and NeuN-positive neurons is expressed as positive cells per square millimeter. Data are the mean ± SD (ANOVA F(3, 20) = 157.2P < 0.0001; ***P < 0.001, post hoc Tukey's test). (f) Electron microscopic images of the SAH+vector group and SAH+AAV-AQP4 group. (g) AAV-AQP4 treatment reduced the mean percentage area of cytoplasm covered by mitochondria. Data are the mean ± SEM (as this is the convention in the electron microscopy field) (*P = 0.0186, t-test).

Fig 3: Hypoxia-activated microglia release pro-inflammatory cytokines and induce astrocyte swelling. Primary microglia were exposed to 1% O2 for 24 h. (A and B) Protein levels of NLRP3, p65, p-p65, IL-1β, p-IL-1β, and ASC were determined by western blotting (A) and quantified by strip grayscale (B, *P < 0.05 and ***P < 0.001 by Student's t-test). (C) Secretion of TNF-α by microglia to the supernatant of culture medium was measured by ELISA (**P < 0.01 and ***P < 0.001 by Student's t-test). (D–G) mRNA levels of Nlrp3, Tnfa, Il1b, and Il18 were determined by real-time PCR (*P < 0.05, **P < 0.01, and ***P < 0.001 by one-way ANOVA). (H) Cells were fixed and stained with NLRP3 and ASC antibodies to display NLRP3 inflammasomes (white arrows). (I–L) Microglia culture medium was harvested and diluted with the same volume of fresh medium and then incubated with astrocytes under hypoxia. The protein level of AQP4 in astrocytes was measured by western blotting (I) and quantified by strip grayscale (J, *P < 0.05 by Student's t-test). Astrocytes were stained with Calcein green AM (K) and the astrocyte swelling status induced by microglia medium was quantified by the dye intensity (L, ***P < 0.001 by Student's t-test). n.s. indicates no significance.

Fig 4: MTA1/HIF1A/AQP4 promotes the apoptosis of colon epithelial cells.A TUNEL staining of the cell apoptosis of colon tissue in DSS-induced experimental colitis. B The cell viability rate detected by CCK-8 assay in LPS-treated cells with MTA1 silencing. C The apoptotic rate detected by flow cytometry in LPS-treated cells with MTA1 silencing. D The cell viability rate detected by CCK-8 assay after AQP4 was overexpressed in LPS-treated cells with MTA1 silencing. E The apoptotic rate detected by flow cytometry after AQP4 was overexpressed in LPS-treated cells with MTA1 silencing. *p < 0.05; **p < 0.01; ***p < 0.001. The measurement data were expressed as mean ± standard deviation. Data between two groups were compared by the unpaired student’s t test, and data among multiple groups were compared by one-way ANOVA, followed by Tukey’s post hoc tests. The experiment was conducted three times independently.

Fig 5: AQP4 overexpression improves SAH-induced AQP4 depolarization. (a, g) Immunofluorescence staining analysis of perivascular AQP4 expression changes in mouse temporal cortex large vessels 24 h after SAH. n = 6 per group. CD31: red, AQP4: green; DAPI: blue. Scale bar = 20 µm. Data are the mean ± standard deviation (SD). (b, c, h, i) Representative immunofluorescence images of blood vessels in the cortex of (b) sham, (c) SAH, (h) SAH+vector, and (i) SAH+AAV-AQP4 group mice stained for AQP4 and CD31, illustrating placement of the 40 µm axis perpendicular to blood vessels for quantification of expression across vessel cross-sections, illustrating AQP4 expression surrounding blood vessels in the cortex. Ten cross-sections of one blood vessel from one mouse were selected. (d) Viral vector pattern diagram. (e) Immunofluorescence intensity statistics for AQP4. Data are the mean ± standard deviation (SD) (ANOVA F(3, 20) = 88.80P < 0.0001; ***P < 0.001, post hoc Tukey's test). (f) Quantification of AQP4 polarization. n = 6 per group (ANOVA F(3, 20) = 56.75P < 0.0001; ***P < 0.001; post hoc Tukey's test).