Fig 2: S1PR3 inhibition reduces the expression of AQP4 in the mouse brain tissue after tMCAO. Protein (A) and mRNA (B) levels of AQP4 and MMP9 in the sham group, 24 h tMCAO group, CAY-10444 + 24 h tMCAO group, and V+24 h tMCAO group (n = 4). (C,D) Representative images of double-immunofluorescence staining (n = 4) showing the colocalization of DAPI (blue)/AQP4 (green) (C) and MMP9 (green) (D)/CD31 (red). (E,F) Immunofluorescence statistics of AQP4 and MMP9 (n = 4). Data are presented as the means ± SEM. p-values were determined using ANOVA followed by the Tukey post hoc test. *p < 0.05; **p < 0.01; ***p < 0.001; and N.S. not significant compared to the sham group. Scale bar = 100 µm.

Fig 3: Inhibition of S1PR3 increases the expression of occludin and ZO1 in the brain tissue of mice after tMCAO. The protein (A) and mRNA (B) levels of occludin and ZO1 in the sham group, 24 h tMCAO group, CAY-10444 + 24 h tMCAO group, and V+24 h tMCAO group (n = 4). (C,D) Representative images of double-immunofluorescence staining showing the colocalization of DAPI (blue)/occludin (green) (C) and ZO1 (green) (D)/CD31 (red) (n = 4). (E,F) Immunofluorescence statistics of ZO1 and occludin (n = 4). Data are presented as the means ± SEM. p-values were determined using ANOVA followed by the Tukey post hoc test. *p < 0.05; **p < 0.01; ***p < 0.001; and N.S. not significant compared to the sham group. Scale bar = 100 µm.

Fig 4: TREM2 depletion abolished the effects of COG1410 on vascular phenotypes and microglial states. A Representative images of Claudin-5 (green) and CD31 (red) surrounding the lesion sites at 3 d after CCI. Nuclei were stained with DAPI (blue). Scale bar = 100 µm. B Quantitative analysis of the relative Claudin-5 to CD31 fluorescence intensity in different groups. *p < 0.05 vs. WT CCI + COG1410; #p < 0.05 vs. WT CCI + Vehicle; ns p > 0.05 KO CCI + Vehicle vs. KO CCI + COG1410, n = 6 per group. C Representative Western blot bands of ZO-1, Occludin, Claudin-5, and ß-Actin at the lesion sites after CCI. D Quantitative analysis of relative ZO-1, Occludin, and Claudin-5 density. *p < 0.05 vs. WT CCI + COG1410; #p < 0.05 vs. WT CCI + Vehicle; ns p > 0.05 KO CCI + Vehicle vs. KO CCI + COG1410, n = 6 per group. E Representative images of microglia (Iba1, red) and CD86-positive microglia (CD86, green) surrounding the lesion sites at 3 d after CCI. Nuclei were stained with DAPI (blue). Scale bar = 100 µm. F Representative images of microglia (Iba1, red) and CD206-positive microglia (CD206, green) surrounding the lesion sites at 3 d after CCI. Nuclei were stained with DAPI (blue). Scale bar = 100 µm. G Quantitative analysis of the percentage of colocalized CD86-Iba1+ cells and the percentage of colocalized CD206-Iba1+ cells in different groups at 3 d after CCI. *p < 0.05 vs. WT CCI + COG1410; #p < 0.05 vs. WT CCI + Vehicle; ns p > 0.05 KO CCI + Vehicle vs. KO CCI + COG1410, n = 6 per group

Fig 5: The immune intervention with the Ox-ApoB peptide fragment protects the vascular endothelial barrier connection, reduces the expression of LOX-1 and cell apoptosis in the plaque, and increases the clearance of Ox-LDL by liver macrophages. (a, d) Representative images of aortic root sections obtained from the control and immunization groups stained with ZO-1 and CD31 (a). Nuclei were stained with DAPI (blue). Compared with the control group (set as 1), the relative ratio of CD31 that was occupied by ZO-1 is presented among the indicated groups (d). (b, e, f) Representative images of aortic root sections obtained from the control and immunization groups stained with TUNEL and LOX-1 (b). Nuclei were stained with DAPI (blue). The percentage of the plaque area that was occupied by TUNEL and LOX-1 cells is presented among the indicated groups (e, f). (c, g) Representative images of liver sections obtained from the control and immunization groups stained with ABCA1 and F4/80 (c). Compared with the control group (set as 1), the relative ratio of F4/80 occupied by ABCA1 is presented among the indicated groups (g). Data are expressed as the mean ± SD. *P < 0.05, **P < 0.01.