Figure 1.
Orexin-A promotes cell migration in cultured rat astrocytes.
A: The immunostaining photos indicating the promotion of astrocytes migration induced by orexin-A in different time and concentrations. The confluent monolayer of astrocytes was grown in serum deprived medium for 24 h, then scratched and 3, 10 and 100 nM orexin-A were added, photos were obtained at 0, 24 and 48 h. Astrocytes were immunostained with anti-GFAP. Scale bar = 50 µm. B: Statistical analysis showing the promotion of astrocytes migration induced by orexin-A in different time and concentrations. Data are expressed as means ± SEM. n = 4, *p<0.05 vs. control.
Figure 2.
Both OX1R and OX2R are expressed in primary rat astrocytes.
A: Colocalization of OX1R (Green) and GFAP (Red) in cultured rat astrocytes. n = 4, Scale bar = 50 µm. The white pane indicating enlarged area. B: Colocalization of OX2R (Red) and GFAP (Green) in cultured rat astrocytes. n = 4, Scale bar = 50 µm. The white pane indicating enlarged area. C–D: Representative western blots and statistical analysis showing the effect of siRNA-mediated silencing of OX1R expression in astrocytes. Data are expressed as means ± SEM. n = 3, *p<0.05 vs. control.
Figure 3.
OX1R mediates orexin-A-induced astrocytes migration.
A: Effects of viral vector on astrocytes migration. n = 4, Scale bar = 20 µm. B–C: Representative wound healing images and statistical analysis showing the effects of siRNA-mediated silencing of OX1R on orexin-A induced astrocytes migration. Data are expressed as means ± SEM. n = 4, *p<0.05 vs. null virus+orexin-A. Scale bar = 20 µm. D: Effects of specific OX1R inhibitor SB334867 (10 µM, 24 h) on orexin-A-induced astrocytes migration. n = 4, Scale bar = 20 µm. E: Effects of specific OX2R inhibitor TCS OX2 29 (10 µM, 24 h) on orexin-A-induced astrocytes migration. n = 4, Scale bar = 20 µm. F–G: Statistical analysis showing the effects of OX1R and OX2R inhibitors on orexin-A-induced astrocytes migration. Data are expressed as means ± SEM. n = 4, *p<0.05 vs. control, #p<0.05 vs. TCS OX2 29.
Figure 4.
ERK1/2 signal mediates orexin-A-induced astrocytes migration.
A–B: Representative western blots and statistical analysis showing the effect of orexin-A on p-ERK expression in astrocytes. Data are expressed as means ± SEM. n = 3, *p<0.05 vs. control. C–D: Representative wound healing images and statistical analysis showing the effect of ERK1/2 inhibitor PD98059 (20 µM, 24 h) and U0126 (10 µM, 24 h) on orexin-A-induced astrocytes migration. Data are expressed as means ± SEM. n = 4, *p<0.05 vs. control. Scale bar = 20 µm.
Figure 5.
Orexin-A-induced ERK1/2 phosphorylation is dependent on Ca2+ signaling pathway.
A: Calcium imaging result and statistical analysis showing the effect of orexin-A (10 nM) on intracellular Ca2+ concentration in astrocyte. Data are expressed as means ± SEM (n = 15, *p<0.05 vs. control). B–D: EGTA containing Ca2+-free solution, SR Ca2+-ATPase inhibitor CPA, and IP3Rs, SOC channel inhibitor 2-APB attenuated orexin-A-induced intracellular Ca2+ elevation. Data are expressed as means ± SEM. (B: EGTA, n = 17, **p<0.01 vs. orexin-A; C: CPA 10 µM, n = 15, **p<0.01 vs. orexin-A; D: 2-APB 50 µM, n = 15, **p<0.01 vs. orexin-A). E: Representative western blots and statistical analysis showing the effect of Ca2+ chelator BAPTA-AM (20 µM, 30 min) on orexin-A-mediated upregulation of p-ERK in astrocytes. Data are expressed as means ± SEM. n = 4, *p<0.05, **p<0.01 vs. control. F: Representative western blots and statistical analysis showing the effect of EGTA containing Ca2+-free solution on orexin-A-induced ERK1/2 phosphorylation. n = 3, *p<0.05 vs. control #p<0.05 vs orexin-A. G: Representative western blots and statistical analysis showing the effect of SR Ca2+-ATPase inhibitor CPA (10 µM, 30 min) on orexin-A-induced ERK1/2 phosphorylation. n = 3, *p<0.05 vs control. H: Representative western blots and statistical analysis showing the effect of SOC inhibitor 2-APB (50 µM, 30 min) on orexin-A-induced ERK1/2 phosphorylation. Data are expressed as means ± SEM. n = 3, *p<0.05 vs. control, #p<0.05 vs orexin-A.
Figure 6.
Orexin-A-induced astrocytes migration is dependent on Ca2+ signaling pathway.
A: Representative wound healing images showing the effects of various Ca2+ pathway inhibitors on orexin-A-induced astrocytes migration. B: statistical analysis of wound healing assay. Data are expressed as means ± SEM, n = 5, *p<0.05 vs. control. Scale bar = 20 µm. The images were selected from five individual experiments.
Figure 7.
PLC-Ca2+ pathway mediate orexin-A-induced astrocyte migration.
A–B: Representative western blots and statistical analysis showing the effect of PLC inhibitor U73122 (30 µM, 30 min) on orexin-A-induced ERK1/2 phosphorylation. n = 3, *p<0.05 vs. control. C–D: Representative wound healing images and statistical analysis showing the effect of PLC inhibitor U73122 on orexin-A-induced astrocytes migration. Data are expressed as means ± SEM. n = 3, *p<0.05 vs. control.
Figure 8.
PKCα mediates orexin-A-induced upregulation of p-ERK and cell migration in astrocytes.
A: Effect of PKC inhibitor GF109203X (10 µM, 30 min) on orexin-A-induced ERK1/2 phosphorylation. Data are expressed as means ± SEM. n = 3, *p<0.05 vs. control, #P<0.05 vs. orexin-A (GF: GF109203X). B: Effect of PKCα inhibitor Gö6976 (1 µM, 30 min) on orexin-A-induced ERK1/2 phosphorylation. Data are expressed as means ± SEM. n = 3, *p<0.05 vs. control, #p<0.05 vs. orexin-A (GO: Gö6976). C: Effect of PKCδ inhibitor rottlerin (5 µM, 30 min) on orexin-A-induced ERK1/2 phosphorylation. Data are expressed as means ± SEM. n = 3, **p<0.01 vs. control (RO: Rottlerin). D: Representative wound healing images showing PKCα inhibitor (GO: Gö6976) prevented orexin-A-induced astrocytes migration. E: Statistical analysis of wound healing assay. Data are expressed as means ± SEM, n = 5, *p<0.05 vs. control. Scale bar = 20 µm. The images were selected from five individual experiments.