Fig 2: Mrp14 inhibitor (ABR-215757) attenuated depressive-like behavior and TLR4/NF-κB signaling pathway activation induced by CUMS. ABR-215757 and normal saline were injected (IP) daily for the whole CUMS period. a The body weight of CUMS-treated mice was markedly reduced compared with control group or ABR-215757 group, whereas no significant differences were observed between CUMS + ABR-215757 group and any other group (n = 6–10 mice/group). b, c CUMS + ABR-215757 group displayed higher sucrose preference (n = 10 mice/group) and less immobility duration in tail suspension test (n = 6–9 mice/group). d–i Western blot analyses showed the changes of hippocampal TLR4/NF-κB signaling and RAGE protein (n = 4 mice/group). *p < 0.05, **p < 0.01, ***p < 0.001 between two indicated groups

Fig 3: CUMS induced depressive-like phenotypes and upregulated the expression of Mrp8 and Mrp14 in the hippocampus and serum. a After 4-week chronic unpredictable mild stress (CUMS), mice had a lower body weight than control mice (n = 14–15 mice/group). b, c CUMS resulted in depressive-like behavior, including decreased sucrose preference (n = 10 mice/group) and increased immobility time in tail suspension test (TST) (n = 10 mice/group). d, e Protein levels of Mrp8 (d) and Mrp14 (e) were slightly but statistically significantly increased in the serum of CUMS-exposed mice (n = 10–11 mice/group). f Representative images of western blot. Proteins were extracted from the hippocampus. g, h The quantitative analyses revealed that both Mrp8 (n = 3 mice/group) and Mrp14 (n = 3 mice/group) were increased in the hippocampus of stress mice. *p < 0.05, **p < 0.01 between two groups

Fig 4: Changes in behavioral performance, cytokine expression, and TLR4/NF-κB signaling in response to recombinant proteins. After treatment with recombinant Mrp8, Mrp14, or Mrp8/14, depressive-like behaviors were determined by the sucrose preference test and tail suspension test (TST). a Both Mrp14- and Mrp8/14-treated mice exhibited reduced sucrose preference compared with the vehicle group (n = 10 mice/group). b All of the three recombinant proteins could extend the immobility time in TST compared with vehicle treatment (n = 5–6 mice/group). c The results of RT-PCR of the mRNA changes of proinflammatory cytokines and IBA-1 in the hippocampus. n = 4–5 mice/group for TNF-α, IL-6, and IBA-1, n = 3–5 mice/group for IL-1β. d–i Western blot analyses showed that hippocampal TLR4/NF-κB signaling was activated after these protein treatments, while the RAGE protein was not significantly changed among these groups (n = 4 mice/group). *p < 0.05, **p < 0.01, ***p < 0.001 between two indicated groups. The data are presented as the mean ± SEM

Fig 5: EDB directly binds to S100A9 and inhibits its phosphorylation and self-assembly. A-B WB images (A) and CETSA melting curve (B; means ± SD, two-way ANOVA, F (1,64) = 45.41) of S100A9 with/without EDB. N = 5. C Binding signal between EDA (left) or d-Bor (right) and different concentrations of S100A9 in SPR experiments. D Representative patterns of mouse S100A9 binding with d-Bor or EDA, as predicted by molecular docking analysis. Blue connecting lines represent the hydrogen bond, and dotted lines represent the hydrophobic interaction. E-F WB images (E) and CETSA melting curve (F; means ± SD, two-way ANOVA, F (3,85) = 21.94) of S100A9 mutants with EDB. T19A: Thr19 → Ala; KKEK51-54AAEA: Lys51, 52, 54 → Ala. N = 3. G Co-IP WB images of phosphorylated threonine residues interacting with HA-S100A9. H Normalized ThT fluorescence value during S100A9 incubation with/without EDB. I Representative confocal images showing microglia, S100A9 and phosphorylated threonine. Scale bar, 50 μm. J Representative AFM images of S100A9 with/without EDB after 6 h of incubation. Scale bar, 1 μm. *p < 0.05, **p < 0.01, ***p < 0.001