Figure 4.
SCFAs trigger C/EBPβ/AEP activation and cognitive deficits in GF 3xTg mice and inflammation, exacerbated by PGE2-G. (A) Immunoblot showing p-C/EBPβ, C/EBPβ, AEP, APP and tau expression and processing, as well as arachidonic acid metabolism in the brains of GF mice with or without SCFAs administration. (B) AEP activity assay in the brain lysates from GF 3xTg mice with or without SCFAs administration. SCFAs treatment elevates AEP activity in the brains of GF 3xTg mice. Data represent the mean±SEM; representative data of four samples; *p<0.05 compared with control, unpaired t-tests. (C) Proinflammatory cytokine IL1-β, IL-6 and TNFα concentrations in the brain lysates from GF 3xTg mice and SPF 3xTg mice, respectively. Data represent the mean±SEM; representative data of four samples; *p<0.05 compared with control, multiple unpaired T tests. (D) Aβ40 and Aβ42 concentrations in the cortex of GF 3xTg mice with or without SCFAs administration were measured using human aβ40 and Aβ42 ELISA kit. The concentrations of Aβ42 not aβ40 were significantly increased in the cortex regions of the brains from SCFAs-treated GF 3xTg mice compared with vehicle-treated GF 3xTg mice. (N=4 in each group, data are shown as mean±SEM *p<0.05 compared with control, multiple unpaired T tests). (E) Immunofluorescent staining of Aβ (red) and ThS (green), AT8 (green) and T22 (red) in the hippocampus CA1 region of brains from GF 3xTg mice with or without SCFAs administration. Scale bar: 20 μm (F) quantitative analysis of Aβ positive cells, ThS positive cells, AT8 positive cells and T22 positive cells, respectively. The density of Aβ, ThS, AT8 and T22 positive cells was significantly increased in SCFAs-treated GF 3xTg mice brains. (n=8 in each group, data are shown as mean±SEM. *P<0.05, **p<0.01, ***p<0.001, ****p<0.0001 compared with control, unpaired T tests.). (G, H) Y-maze behavioural tests. spontaneous alternation (%) (G), number of arms entered (H); (N=9 in each group, data are shown as mean±SEM. ***P<0.001 compared with control, unpaired T tests). (I) Arachidonic acids and its metabolites concentrations in the faeces of GF mice and SPF mice. (N=3 in each group, data are shown as mean±SEM, *p<0.05, **p<0.01 compared with control, multiple unpaired T tests). (J) Representative images of immunofluorescent staining of Iba-1 (red) in the cortex (upper panel) and 3D reconstruction of Iba-1-stained microglia (lower panel) residing in the cortex of GF 3xTg mice, GF 3xTg mice treated with either PGE2-G or SCFAs, GF 3xTg mice treated with both SCFAs and PGE2-G. (K, L) Quantitative analysis of diameter, number of branch points of microglia residing in the cortex. data represent the mean±SEM; representative data of twelve samples; **p<0.01, ***p<0.001, ****p<0.0001 compared with control, multiple unpaired T tests; #P<0.05, ##P < 0.01, ###P < 0.001, #### P < 0.0001 compared with control, multiple unpaired t tests. Aβ, amyloid-β; AEP, asparagine endopeptidase; GF, germ-free; PGE2-G, prostaglandin E2–1-glyceryl ester; SCFA, short chain fatty acids.