Ginkgolide B promotes the proliferation and differentiation of neural stem cells following cerebral ischemia/reperfusion injury, both in vivo and in vitro

Abstract

Neural stem cells have great potential for the development of novel therapies for nervous system diseases. However, the proliferation of endogenous neural stem cells following brain ischemia is insufficient for central nervous system self-repair. Ginkgolide B has a robust neuroprotective effect. In this study, we investigated the cell and molecular mechanisms underlying the neuroprotective effect of ginkgolide B on focal cerebral ischemia/reperfusion injury in vitro and in vivo. Neural stem cells were treated with 20, 40 and 60 mg/L ginkgolide B in vitro. Immunofluorescence staining was used to assess cellular expression of neuron-specific enolase, glial fibrillary acid protein and suppressor of cytokine signaling 2. After treatment with 40 and 60 mg/L ginkgolide B, cells were large, with long processes. Moreover, the proportions of neuron-specific enolase-, glial fibrillary acid protein- and suppressor of cytokine signaling 2-positive cells increased. A rat model of cerebral ischemia/reperfusion injury was established by middle cerebral artery occlusion. Six hours after ischemia, ginkgolide B (20 mg/kg) was intraperitoneally injected, once a day. Zea Longa's method was used to assess neurological function. Immunohistochemistry was performed to evaluate the proportion of nestin-, neuron-specific enolase- and glial fibrillary acid protein-positive cells. Real-time quantitative polymerase chain reaction was used to measure mRNA expression of brain-derived neurotrophic factor and epidermal growth factor. Western blot assay was used to analyze the expression levels of brain-derived neurotrophic factor and suppressor of cytokine signaling 2. Ginkgolide B decreased the neurological deficit score, increased the proportion of nestin-, neuron-specific enolase- and glial fibrillary acid protein-positive cells, increased the mRNA expression of brain-derived neurotrophic factor and epidermal growth factor, and increased the expression levels of brain-derived neurotrophic factor and suppressor of cytokine signaling 2 in the ischemic penumbra. Together, the in vivo and in vitro findings suggest that ginkgolide B improves neurological function by promoting the proliferation and differentiation of neural stem cells in rats with cerebral ischemia/reperfusion injury.

Keywords: astrocytes; brain-derived neurotrophic factor; bromodeoxyuridine; epidermal growth factor; glial fibrillary acid protein; middle cerebral artery occlusion; nerve regeneration; nestin; neural regeneration; neurological function; neuron-specific enolase; suppressor of cytokine signaling 2.

Figure 1

Effect of ginkgolide B (GKB) on the morphology of neural stem cells. (A–F) Cell morphology of neural stem cells (arrows) at different time points (× 200). (A–C) Neural stem cells after induced differentiation for 3 (A), 7 (B) and 14 (C) days in the control group; (D–F) neural stem cells after induced differentiation for 3 (D), 7 (E) and 14 (F) days in the 40 mg/L GKB group. (G, H) The length of neural stem cell processes 3 days after differentiation. (G) Body area of neural stem cells 14 days after differentiation. (H) Data are presented as the mean ± SD and were tested with analysis of variance (n = 3 per group). *P < 0.05, vs. control group; #P < 0.05, vs. 20 mg/L GKB group.

Figure 2

Effect of ginkgolide B (GKB) on differentiation of neural stem cells. (A–F) Expression of neuron-specific enolase (NSE), glial fibrillary acid protein (GFAP) and suppressor of cytokine signaling 2 (SOCS2) in neural stem cells in the control group (A–C) and the 40 mg/L GKB group (D–F) after induced differentiation for 7 days. The blue color represents cell bodies, and the green color represents processes. Arrows indicate NSE-, GFAP- or SOCS2-positive cells. (G) Percentage of NSE-, GFAP- and SOCS2-positive cells after 7 days of induced differentiation. Data are presented as the mean ± SD (n = 3 per group; independent t-test). *P < 0.05, vs. control group.

Figure 3

Effect of ginkgolide B (GKB) on neurological function in rats with cerebral ischemia/reperfusion injury. Data are presented as the mean ± SD (n = 3 per group; independent t-test). †P < 0.05, vs. day 3; §P < 0.05, vs. middle cerebral artery occlusion (MCAO) group.

Figure 4

Effect of ginkgolide B (GKB) on nestin immunoreactivity in the penumbra of rats with cerebral ischemia/reperfusion injury. (A) Nestin expression (arrows) in the brain of rats at 3, 7 and 14 days after middle cerebral artery occlusion (MCAO) (immunohistochemistry; scale bars: 50 μm). (B) Quantitative analysis of nestin immunoreactivity. Data are presented as the mean ± SD (n = 6 per group; two-way analysis of variance with Bonferroni correction for pairwise comparison). ‡P < 0.05, vs. sham group; $P < 0.05, vs. day 7.

Figure 5

Effect of ginkgolide B (GKB) on neuron-specific enolase (NSE) immunoreactivity in the penumbra of rats with cerebral ischemia/reperfusion injury. (A) NSE expression (arrows) in the brain of rats at 3, 7 and 14 days after reperfusion (immunohistochemistry; scale bars: 50 μm). (B) Quantitative analysis of NSE immunoreactivity. Data are presented as the mean ± SD (n = 6 per group; two-way analysis of variance with Bonferroni correction for pairwise comparison). ‡P < 0.05, vs. sham group; §P < 0.05, vs. middle cerebral artery occlusion (MCAO) group; †P < 0.05, vs. day 3; $P < 0.05, vs. day 7.

Figure 6

Effect of ginkgolide B (GKB) on glial fibrillary acid protein (GFAP) immunoreactivity in the penumbra of rats with cerebral ischemia/reperfusion injury (A) GFAP expression (arrows) in the brain of rats at 3, 7 and 14 days after reperfusion (immunohistochemistry; scale bars: 50 μm). (B) Quantitative analysis of GFAP immunoreactivity. Data are presented as the mean ± SD (n = 6 per group; two-way analysis of variance with Bonferroni correction for pairwise comparison). ‡P < 0.05, vs. sham group; §P < 0.05, vs. middle cerebral artery occlusion (MCAO) group; $P < 0.05, vs. day 7; *P < 0.05, vs. day 3 within the same group; #P < 0.05, vs. day 7 within the same group.

Figure 7

Effect of ginkgolide B (GKB) on bromodeoxyuridine (BrdU) immunoreactivity in rats with cerebral ischemia/reperfusion injury. (A) BrdU-positive cells (arrows) in the brain of rats at 3, 7 and 14 days after reperfusion (immunohistochemistry; scale bars: 50 μm). (B) Quantitative analysis of BrdU immunoreactivity. Data are presented as the mean ± SD (n = 6 per group; two-way analysis of variance with Bonferroni correction for pairwise comparison). ‡P < 0.05, vs. sham group; §P < 0.05, vs. middle cerebral artery occlusion (MCAO) group.

Figure 8

Effect of ginkgolide B (GKB) on brain-derived neurotrophic factor (BDNF) and epidermal growth factor (EGF) mRNA expression in rats with cerebral ischemia/reperfusion injury. Relative expression of BDNF mRNA (A) and EGF mRNA (B) 3, 7 and 14 days after intervention. Data are presented as the mean ± SD (n = 3 per group; two-way analysis of variance with Bonferroni correction for pairwise comparison). ‡P < 0.05, vs. sham group; §P < 0.05, vs. middle cerebral artery occlusion (MCAO) group.

Figure 9

Effect of ginkgolide B (GKB) on brain-derived neurotrophic factor (BDNF) and suppressor of cytokine signaling 2 (SOCS2) protein expression in rats with cerebral ischemia/reperfusion injury after middle cerebral artery occlusion (MCAO). (A) Expression of BDNF, SOCS2 and β-actin in the brain of rats at 3, 7 and 14 days after reperfusion (western blot assay). (B, C) Quantitative analysis of BDNF protein (B) and SOCS2 protein (C) expression. Relative protein expression was calculated as the ratio of the optical density of the target protein to that of β-actin. Data are presented as the mean ± SD (n = 3 per group; two-way analysis of variance with Bonferroni correction for pairwise comparison). ‡P < 0.05, vs. sham group.