Fig 1: SIRT7 attenuates ferroptosis by regulating the FOXO4-GPX4 signaling pathway. A Schematic diagram of pharmacological inhibition or activation of ferroptosis intervention in SIRT7-KO mice. B Representative H&E-stained lung tissue sections in mice described in (A) (n = 3, up: scale bar = 2 mm; down: scale bar = 500 μm). C Levels of pro-inflammatory factors (IL-6, TNF-α and IL-1β) in BALF from mice across different groups as described in (A) (n = 6). One-way ANOVA followed by Tukey's post-hoc test for multiple comparisons. D Lung injury score across different groups as described in (A) (n = 3). One-way ANOVA followed by Tukey's post-hoc test for multiple comparisons. E W/D weight ratio across different groups as described in (A) (n = 6). One-way ANOVA followed by Tukey's post-hoc test for multiple comparisons. F-G Levels of total protein and total cells in BALF from mice across different groups as described in (A) (n = 6). One-way ANOVA followed by Tukey's post-hoc test for multiple comparisons. H Schematic diagram of pharmacological inhibition or activation of ferroptosis in SIRT7-KO or SIRT7-OE MLE-12 cells. I-J Levels of pro-inflammatory factors (IL-6, TNF-α and IL-1β) in MLE-12 cells across different groups as described in (H) (n = 6). One-way ANOVA followed by Tukey's post-hoc test for multiple comparisons. K JASPAR database prediction of three putative FOXO4 binding sites in the GPX4 promoter. L ChIP-qPCR showed FOXO4 binding to three putative sites in the GPX4 promoter (n = 3). Student's t-test. M Dual-luciferase reporter analysis of FOXO4-mediated transcriptional activation of GPX4 in MLE-12 cells (n = 3). One-way ANOVA followed by Tukey's post-hoc test for multiple comparisons. N–O Western blotting analysis of GPX4 protein expression in MLE-12 cells after FOXO4-OE or KD (n = 3). Student's t-test. P FOXO4-OE rescues GPX4 expression in SIRT7-KO MLE12 cells, with the desuccinylation-mimicking mutant K139R exerting a stronger effect than WT (n = 3). One-way ANOVA followed by Tukey's post-hoc test for multiple comparisons. LPS was applied at a concentration of 10 μg/mL. Data are presented as mean ± SD. Each experiment was conducted with three or six independent biological replicates. *p < 0.05, **p < 0.01, and ***p < 0.001; ns, not significant.
Fig 2: SIRT7 interacts with FOXO4. A The schematic diagram of IP-MS. B Molecular docking analysis of the interaction between SIRT7 and FOXO4. C Venn diagram illustrating the overlap of proteins identified by mass spectrometry from the IgG-enriched and SIRT7 immunoprecipitation samples. D FOXO4 is identified as a highly abundant protein in anti-SIRT7 immunoprecipitates by MS. E Silver staining of proteins from anti-SIRT7 immunoprecipitates. F Co-IP analysis of the endogenous FOXO4-SIRT7 interaction in MLE-12 cell lysates. G Co-IP analysis of the interaction between HA-tagged SIRT7 and Flag-tagged FOXO4 in co-transfected HEK293T cells. H Confocal microscopy analysis of SIRT7 (green) and FOXO4 (red) subcellular localization in MLE-12 cells following PBS or LPS treatment (upper and middle panels); and of HA-tagged SIRT7 (green) and Flag-tagged FOXO4 (red) in transfected HEK293T cells (lower panel). scale bar = 10 μm. LPS was applied at a concentration of 10 μg/mL for 24 h. I Co-IP analysis of endogenous total FOXO4 and succinylated FOXO4 in MLE-12 cells using the indicated antibodies. J Co-IP analysis of endogenous total FOXO4 and succinylated FOXO4 in HEK293T cells using the indicated antibodies. K In vitro succinylation assay of Flag-FOXO4 immunoprecipitated from HEK293T cells. The immunoprecipitates were incubated with 2 mM succinyl-CoA for 30 min and analyzed for FOXO4 succinylation. The data shown are representative of at least three independent biological experiments.
Fig 3: SIRT7 acts as a negative regulator of LPS-induced ferroptosis in vitro. A Western blotting analysis of ferroptosis-related markers (PTGS2, xCT, GPX4, and FTH1) in SIRT7-KO MLE-12 cells (n = 3). One-way ANOVA followed by Tukey's post-hoc test for multiple comparisons. B–C Blind quantification of positively stained cells with ferroptosis-typical mitochondrial morphology across designated experimental groups in MLE-12 cells. Representative TEM images are shown (n = 3, up: scale bar = 1 μm; down: scale bar = 500 nm). Two-way ANOVA followed by Tukey's post-hoc test for multiple comparisons. D Assessment of cellular ROS levels in MLE-12 cells following SIRT7-KO using the DCFH-DA fluorescent probe (n = 6, scale bar = 50 μm). Representative images are shown. Two-way ANOVA followed by Tukey's post-hoc test for multiple comparisons. E CCK-8 assay to assess the impact of SIRT7-KO on MLE-12 cell viability (n = 6). Two-way ANOVA followed by Tukey's post-hoc test for multiple comparisons. F-J The effects of SIRT7-KO on intracellular Fe2+, MDA, 4-HNE, SOD, and GSH levels in MLE-12 cells (n = 6). Two-way ANOVA followed by Tukey's post-hoc test for multiple comparisons. K Western blotting analysis of ferroptosis-related markers (PTGS2, xCT, GPX4, and FTH1) in SIRT7-OE (SIRT7) MLE-12 cells (n = 3). One-way ANOVA followed by Tukey's post-hoc test for multiple comparisons. L-M Blind quantification of positively stained cells with ferroptosis-typical mitochondrial morphology across designated experimental groups in MLE-12 cells. Representative TEM images are shown (n = 3, up: scale bar = 1 μm; down: scale bar = 500 nm). Two-way ANOVA followed by Tukey's post-hoc test for multiple comparisons. N Assessment of cellular ROS levels in MLE-12 cells following SIRT7-OE using the DCFH-DA fluorescent probe (n = 6, scale bar = 50 μm). Representative images are shown. Two-way ANOVA followed by Tukey's post-hoc test for multiple comparisons. O CCK-8 assay to assess the impact of SIRT7-OE on MLE-12 cell viability (n = 6). Two-way ANOVA followed by Tukey's post-hoc test for multiple comparisons. P-T The effects of SIRT7-OE on intracellular Fe2+, MDA, 4-HNE, SOD, and GSH levels in MLE-12 cells (n = 6). Two-way ANOVA followed by Tukey's post-hoc test for multiple comparisons. LPS was applied at a concentration of 10 μg/mL. Data are presented as mean ± SD. Each experiment was conducted with three or six independent biological replicates. *p < 0.05, **p < 0.01, and ***p < 0.001; ns, not significant.
Fig 4: FOXO4 desuccinylation at K139 protects against LPS-challenged ALI in SIRT7-KO mice. A Schematic illustration of AAV6-mediated intervention in SIRT7-KO mice. Intratracheal LPS instillation to induce ALI following a 3-week indicated AAV6 vector delivery period. B–C Representative H&E-stained lung tissue sections and quantitative lung injury scores in SIRT7-KO mice across different groups described in (A) (n = 6, up: scale bar = 2 mm; down: scale bar = 500 μm). One-way ANOVA followed by Tukey's post-hoc test for multiple comparisons. D-I Lung W/D weight ratio and BALF parameters, including total cells, total protein, and pro-inflammatory cytokines (IL-6, TNF-α and IL-1β) levels in SIRT7-KO mice across different groups described in (A) (n = 6). One-way ANOVA followed by Tukey's post-hoc test for multiple comparisons. J Succinylated FOXO4 protein levels across different groups described in (A) (n = 5). One-way ANOVA followed by Tukey's post-hoc test for multiple comparisons. K–O Levels of 4-HNE Fe2+, MDA, SOD, and GSH across different groups described in (A) (n = 6). One-way ANOVA followed by Tukey's post-hoc test for multiple comparisons. P Representative DHE staining of lung tissue sections showing ROS levels across different groups described in (A) (n = 3). One-way ANOVA followed by Tukey's post-hoc test for multiple comparisons. Q Blind quantification of positively stained cells with ferroptosis-typical mitochondrial morphology across different groups described in (A) in mouse lung tissue. Representative TEM images are shown (n = 3, up: scale bar = 5 μm; down: scale bar = 2 μm). One-way ANOVA followed by Tukey's post-hoc test for multiple comparisons. Data are presented as mean ± SD. Each experiment was conducted with three, five, or six independent biological replicates. *p < 0.05, **p < 0.01, and ***p < 0.001.
Fig 5: Graphic overview. Schematic illustration of a mechanistic pathway wherein SIRT7 desuccinylated FOXO4 to suppress ferroptosis and mitigate LPS-induced ALI.
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