Fig 2: The lncRNA H19/miR-766-3p/S1PR3 axis affects cell proliferation and inflammation in psoriasis via the AKT/mTOR pathway. (a-b) Cotransfection of PGMLV-H19 plasmid with miR-766-3p mimics in M5-induced HaCaT cells; expression level of S1PR3 by qRT-PCR (a); and western blot (b). (c) Proliferative effects of lncRNA H19 were assessed by CCK-8. (d) Proliferative effects of cotransfection of PGMLV-H19 plasmid with miR-766-3p mimics were assessed by CCK-8. (e-h) HaCaT cells were transfected with PGMLV-H19/NC (e-f) and cotransfection of PGMLV-H19 plasmid with miR-766-3p mimics (g-h); ELISA showed the expression level of IL-17A (e, g) and IL-22 (f, h). (i) Western blot showed the expression level of p-mTOR, mTOR, AKT, p-AKT in M5-induced HaCaT cells transfected with PGMLV-H19 plasmid and miR-766-3p mimics. (j-l) Cotransfection of miR-766-3p mimics with MK-2206; proliferative effects were assessed by CCK-8 (j); ELISA showed the expression level of IL-17A (k) and IL-22(l). Data were shown as the mean ± SD, *P <0.05, **P <0.01, ***P<0.001. All the experiments were repeated at least three times.

Fig 3: Comparative analysis of S1P signaling between the SHAM group, the OVX group, and the OVX + E group. a and b: expression of SphK at mRNA (n = 4) and protein (n = 4 for SphK1, n = 3 for SphK2) levels respectively; c: the content of aortic S1P between groups (n = 6); d content of aortic C1P between groups (n = 6); e and f: expression of S1P receptors at the mRNA (n = 4) and protein (n = 4 for S1P1, n = 5 for S1P2, n = 3 for S1P3) levels respectively. SphK: sphingosine kinase; S1P: sphingosine-1-phosphate; S1P1/2/3: sphingosine-1-phosphate receptor 1/2/3; SHAM group: sham-operated group; OVX group: ovariectomized group; OVX + E group: OVX group treated with estradiol valerate. Data presented as mean ± standard deviation. * P < 0.05, ** P < 0.01, *** P < 0.001

Fig 4: 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 5: S1PR3 expression detected at different time points in tMCAO mice. Inhibition of S1PR3 ameliorates brain edema and neurological deficits in tMCAO mice. (A) Western blotting was used to detect the SIRT3 protein level (n = 4). (B) Neurological deficit scores of mice recorded 24 h after tMCAO (n = 8). (C) The water content of the right cerebral hemisphere of mice in each group was measured using the wet weight method and dry weight method (n = 4). (D) EB dye (2%; 4 ml/kg of body weight) was injected intraperitoneally immediately after tMCAO, and EB leakage in the brains was analyzed after 24 h of injection (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.