Intranasal administration of vitamin D attenuates blood-brain barrier disruption through endogenous upregulation of osteopontin and activation of CD44/P-gp glycosylation signaling after subarachnoid hemorrhage in rats

Abstract

In this study, we investigated the role of vitamin D3 (VitD3) on endogenous osteopontin (OPN), a neuroprotective glycoprotein, after subarachnoid hemorrhage (SAH). The endovascular perforation SAH model in Sprague-Dawley rats was used to study the effect of intranasal VitD3 (30 ng/kg) before (Pre-SAH + VitD3) and after (Post-SAH + VitD3) subarachnoid hemorrhage. Vitamin D3 (30, 60, 120 ng/kg/day) increased more than one fold endogenous OPN expression in astrocytes and endothelial cells of rat brain. Vitamin D3 significantly decreased brain edema and Evans blue extravasation. In addition, neurobehavioral scores were significantly higher in Pre-SAH + VitD3, but partly higher in Post-SAH + VitD3, group compared with SAH group. These protective effects of vitamin D3 were completely attenuated by intracerebroventricular injection of transcription inhibitor Actinomycin D and significantly inhibited by small interfering ribonucleic acid (siRNA) for vitamin D receptor and OPN in Pre-SAH + VitD3 rats. OPN expression was significantly higher in Pre-SAH + VitD3 rats, specifically A and C, but not B, isomers were upregulated in the astrocytes, leading to CD44 splicing, and P-gp glycosylation in brain endothelial cells. The results show that intranasal vitamin D3 attenuates blood-brain barrier (BBB) disruption through endogenous upregulation of OPN and subsequent CD44 and P-gp glycosylation signals in brain endothelial cells. Furthermore, this study identifies a novel strategy for the cost-effective management of subarachnoid hemorrhage.

Keywords: CD44 splicing; P-glucoprotein glycosylation; Subarachnoid hemorrhage; osteopontin; vitamin D.

Figures

Figure 1.

Intranasal administration of vitamin D3 (VitD3) upregulates endogenous osteopontin (OPN). (a) Representative Western blots of VitD3-induced dose-dependent (30, 60, 120 ng/kg/day) expression of OPN (upper panel) and quantitative analysis (lower panel) 24 h after intranasal administration in brain hemispheres of naïve rats (n = 5 per group). (b) Representative Western blots of time-dependent expression of OPN (upper panel) and quantitative analysis (lower panel) after intranasal administration of 30 ng/kg/h VitD3 in left hemisphere of naïve rats (n = 5 per group). *P < 0.05 vs. naïve rats. (c) Immunohistochemical staining of OPN (red) and astrocytes (GFAP, green) (n = 3 per group). Nuclei are stained with DAPI (blue). Arrows indicate colocalization of OPN and GFAP. Top panel indicates the location of immunohistochemical staining (small black box). Bar = 50 µm. (d) Immunohistochemical staining of OPN (red) and endothelial cells (CD34, green) (n = 3 per group). Nuclei are stained with DAPI (blue). Arrows indicate colocalization of OPN and CD34. Top panel indicates the location of immunohistochemical staining (small black box). Bar = 50 µm.

Figure 2.

Intranasal administration of vitamin D3 (VitD3) improves neurological deficits after subarachnoid hemorrhage (SAH). Pre- and Post-treatment of VitD3 (30 ng/kg) significantly increased the modified Garcia (left), beam balance (middle), and wire hanging test (right) scores 24 h in Pre-SAH + VD3 and Post-SAH + VD3 groups (a, n = 10 per group) and 72 h in Pre-SAH + VD3 group (b, n = 6 per group). Pre-SAH + VitD3, intranasal treatment 30 ng/kg/day VitD3 24 h before SAH; Post-SAH + VitD3, intranasal treatment 30 ng/kg/day VitD3 1 h after SAH. *P < 0.05 vs. sham. #P < 0.05 vs. SAH.

Figure 3.

Role of vitamin D3 (VitD3), its receptor (VDR), and osteopontin (OPN) in neurological function and BBB disruption. (a) Pre- and Post-treatment of VitD3 (30 ng/kg) decreased brain water content at 24 (n = 8 per group) and (b) decreased brain water content in Pre-SAH + VD3 group at 72 (n = 6 per group) h after SAH. Pre-SAH + VitD3, intranasal treatment 30 ng/kg/day VitD3 24 h before SAH; Post-SAH + VitD3, intranasal treatment 30 ng/kg/day VitD3 1 h after SAH. *P < 0.05 vs. sham. #P < 0.05 vs. SAH. (c) Neurobehavioral tests. siRNA, small interfering ribonucleic acid; ‡P < 0.01 vs. Pre-SAH + VitD3 + Vehicle; †P < 0.05 vs. Pre-SAH + VitD3 + Control siRNA; n = 5-6 per group; ♠P = 0.054 vs. Pre-SAH + VitD3 + Control siRNA. (d) Evans blue extravasation. n = 5 per group.

Figure 4.

Role of vitamin D3 (VitD3) in upregulation of endogenous osteopontin (OPN) expression after subarachnoid hemorrhage (SAH). (a) Pre- and Post-treatment of VitD3 increased VDR brain expression. Pre-SAH + VitD3, intranasal treatment 30 ng/kg/day VitD3 24 h before SAH; Post-SAH + VitD3, intranasal treatment 30 ng/kg/day VitD3 1 h after SAH. *P < 0.05 and †P = 0.057 vs. sham; n = 5 per group. (b) Pre- and Post-treatment of VitD3 increased OPN cerebral expression. *P < 0.05 and **P < 0.01 vs. sham; #P < 0.05 vs. SAH. n = 5–6 per group. (c) Immunohistochemical staining of OPN (green) and astrocytes (GFAP, red). Nuclei are stained with DAPI (blue). Top panel indicates the location of immunohistochemical staining (small black box). Arrows indicate colocalization of OPN and GFAP. n = 3 per group. Bar = 50 µm.

Figure 5.

Vitamin D3 (VitD3) upregulates endogenous osteopontin (OPN) -A and -C isomers via a VDR-dependent pathway after subarachnoid hemorrhage (SAH). (a) Effect of VDR siRNA on the VDR mRNA expression. Pre-SAH + VitD3, intranasal treatment 30 ng/kg/day VitD3 24 h before SAH; siRNA, small interfering ribonucleic acid. (b) Structure of OPN isomers and qRT-PCR universal primer design. (c) VitD3 upregulates endogenous OPN-A and -C isomers. *P < 0.05 vs. sham; #P < 0.05 vs. Pre-SAH + VitD3 + Control siRNA; n = 4–5 per group.

Figure 6.

Vitamin D3 (VitD3) regulates CD44 splicing after subarachnoid hemorrhage (SAH). Representative Western blots of CD44 (a) and quantitative analysis of full length CD44 (b), splice variant (c), and ectodomain (d) of CD44 after 24 h. Pre-SAH + VitD3, intranasal treatment 30 ng/kg/day VitD3 24 h before SAH; siRNA, small interfering ribonucleic acid; *P < 0.05, **P < 0.01, and ***P < 0.001 vs. sham; #P < 0.05 vs. SAH; †P < 0.05, and ††P < 0.01 vs. Pre-SAH + VitD3 + Control siRNA; n = 6 per group.

Figure 7.

Vitamin D3 (VitD3) induces P-gp glycosylation after subarachnoid hemorrhage (SAH). Representative Western blots of P-glycoprotein (P-gp) (a) and quantitative analysis of mature/fully glycosylated P-gp (b) and immature P-gp (c) 24 h after SAH. Pre-SAH + VitD3, intranasal treatment 30 ng/kg/day VitD3 24 hours before SAH; siRNA, small interfering ribonucleic acid; *P < 0.05 vs. sham; #P < 0.05 and ##P < 0.001 vs. SAH; †P < 0.05 vs. Pre-SAH + VitD3 + Control siRNA. Colocalization of OPN (red) with astrocytes (GFAP, green) and endothelial cells (CD34, green) (d), and colocalization of P-gp (red) with endothelial cells (CD34, green), but not astrocytes (GFAP, green) (e) and neurons NeuN, red) (f). Nuclei are stained with DAPI (blue). Top panel indicates the location of immunohistochemical staining (small black box). Arrows indicate colocalization. n = 3 per group. Bar = 50 µm. (g) Proposed mechanism of VitD3 neuroprotection via endogenous OPN/CD44/P-gp glycosylation after SAH. EBI, early brain injury.