Fig 1: CHMP4B inhibited the neuronal toxicity of activated microglia. There are four treatment groups take part in the experiments: control, Glu, Glu + vector, Glu + CHMP4B (#P: Glu groups compared with Ctrl groups; *P: Glu + CHMP4B groups compared with the Glu + vector groups). A‐C, MCM levels of TNF‐α (A), IL‐1β (B) and IL‐6 (C). D, Representative images of TUNEL staining after injury in each group (Scale bar = 150 μm). E, Statistical analysis of the positive cells shown in A (n = 3; data are presented as the means ± SEM). F, Representative flow cytometry images of neuron stained with Annexin V‐APC /7‐AAD. G, The apoptosis activation was represented by ratio of APC + (n = 3; data are presented as the means ± SEM). H, CCK‐8 assay tested primary neurons viability (n = 5; data are presented as the means ± SEM). I, Lactate dehydrogenase release assay (n = 3; data are presented as the means ± SEM). J, Representative images of neurons growth one week after different treatments (scale bar = 20 μm). *P < .05; **P < .01; #P < .05, ##P < .01, ###P < .001
Fig 2: There is necroptosis after traumatic brain injury and its level changes with time. A, Protein levels of RIP3 and p‐MLKL obtained from epileptic patients and severe trauma patients. GAPDH was used as the loading control. B, Bar graphs show the results of analysis (by band density analysis) of RIP3 and p‐MLKL (trauma patients = 3, epileptic patients = 3; data are presented as the means ± SEM). C, Immunohistochemistry of RIP3 and p‐MLKL in TBI and Ctrl. (Scale bar = 100 μm.) D, Protein levels of RIP3 and p‐MLKL obtained from sham mice and TBI mice at 6 h, 12 h and 1 d. GAPDH was used as the loading control. E, Bar graphs show the results of analysis (by band density analysis) of RIP3 and p‐MLKL (TBI = 5, sham = 5; data are presented as the means ± SEM). F, Protein levels of CHMP4B obtained from sham mice and TBI mice at 6 h. GAPDH was used as the loading control. G, Bar graphs show the results of analysis (by band density analysis) of CHMP4B (TBI = 5, sham = 5; data are presented as the means ± SEM). H, Immunohistochemistry of CHMP4B in sham and TBI at 6 h. (Scale bar = 50 μm.) *P < .05; **P < .01
Fig 3: FOXO1 regulates CHMP4B transcription and expression. A, The relative luciferase activity in BV2 cells after co‐transfected plasmids containing transcription factors and CHMP4B. B and C, Expression of nuclear FOXO1 was detected by laser scanning confocal microscopy (scale bar = 20 μm). D, Protein level of FOXO1. GAPDH was used as the loading control. E, Bar graphs show the results of analysis (by band density analysis) of FOXO1 (n = 3; data are presented as the means ± SEM). F, qRT‐PCR analysis of FOXO1 expression in Ctrl groups and Glu groups (n = 3; data are presented as the means ± SEM). G‐L. BV2 cells were transfected with FOXO1 plasmid or siRNA for 2 d, and then added the Glu. The protein and mRNA levels of CHMP4B were determined by Western blot and qRT‐PCR (n = 3; data are presented as the means ± SEM; *P: Glu groups compared with Ctrl groups; #P: Glu + FOXO1 groups compared with the Glu + vector groups). *P < .05; **P < .01; #P < .05
Fig 4: CHMP4B improves memory and motor ability and reduces the number of cell death and the size of oedema area after TBI. There are four treatment groups take part in the experiments: sham, TBI, TBI + vector and TBI + CHMP4B (#P: TBI groups compared with sham groups; *P: TBI + CHMP4B groups compared with the TBI + vector groups). A, Mean latency to the platform of each group over 6 d (n = 10 mice; data are presented as the means ± SEM). B and C, Mean swimming speed (B) and distance (C) of each group on the day 6 (n = 10 mice; data are presented as the means ± SEM). D and E, Time (s) spent in the target quadrant (D) and the number of times the mouse crossed the target platform location (E) during the probe trials on day 7 (n = 10 mice; data are presented as the means ± SEM). F, The time mice can stay on the rotarod (n = 10 mice; data are presented as the means ± SEM). G, Brain water content after TBI (n = 3 mice; data are presented as the means ± SEM). H, Representative images of TUNEL staining after TBI in each group (Scale bar = 100 μm). I, Statistical analysis of the positive cell shown in G (n = 5 mice; data are presented as the means ± SEM). J, Mouse brain tissue MRI T2W1 images in each group. *P < .05; #P < .05, ##P < .01
Fig 5: FOXO1 binds the CHMP4B promoter region. A, Schematic diagram revealing the mouse CHMP4B promoter region and FOXO1 potential binding site. B, ChIP assay results determined the relative enrichment of FOXO1 on promoter region of CHMP4B. C, Schematic view represented WT and mutant Rip3 promoter. And luciferase reporter assay was performed in BV2 cells cotransfected FOXO1 or si‐FOXO1 with WT and mutant Rip3 promoter (n = 3; data are presented as the means ± SEM). *P < .05
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