Ginkgo biloba Extract Inhibits Astrocytic Lipocalin-2 Expression and Alleviates Neuroinflammatory Injury via the JAK2/STAT3 Pathway After Ischemic Brain Stroke

Abstract

Background: Astrogliosis has the potential to lead to harmful effects, namely, neuroinflammation, and to interfere with synapse sprouting. Previous studies have suggested that Lipocalin-2 (LCN2) acts as a key target in regulating the reaction of astrocytes. However, the underlying molecular mechanism is not fully elucidated. In the present study, we examined the neuroprotective and anti-inflammatory effects of Ginkgo biloba extract (EGB), a well-known extract with potential immunoregulatory properties in the nervous system. Methods: Triphenyltetrazolium chloride staining, hematoxylin-eosin staining, electron microscopy, and neurological assessments were performed in a microsphere-embolized rat model. Human astrocytes exposed to oxygen glucose deprivation (OGD) were used for in vitro experiments. Inflammatory cytokines, multi-labeling immunofluorescence, and Western blotting were used to investigate the molecular mechanisms underlying the EGB-mediated anti-inflammatory effects in vivo and in vitro. Results: EGB markedly attenuated cerebral infarction and neuronal apoptosis, reduced the inflammatory cytokine level, and alleviated neurological deficiencies in cerebral ischemic rats. After surgery, EGB significantly inhibited astrocyte activation, reduced the phosphorylation of STAT3 and JAK2 and decreased LCN2 expression. In vitro, EGB blocked OGD-induced STAT3 activation and the generation of pro-inflammatory cytokines in human astrocytes, and these effects were significantly enhanced by LCN2 overexpression. EGB downregulated these effects enhanced by LCN2 overexpression. Conclusion: EGB is demonstrated to mediate neuroinflammation, which protects against ischemic brain injury by inhibiting astrogliosis and suppresses neuroinflammation via the LCN2-JAK2/STAT3 pathway, providing insight into a promising therapeutic strategy for ischemic stroke.

Keywords: Ginkgo biloba extract; Lipocalin-2 (LCN2); astrocyte; cerebral ischemia; neuroinflammation.

FIGURE 1

EGB treatment reduced ischemic infarct volume in the cerebral ischemia model. (A,B) Cerebral infarct volume was assessed via TTC staining 48 h after cerebral ischemia. Neurological score (C) of rats after cerebral ischemia were assessed using a five-point scale system. Data are expressed as mean ± SEM (n = 10). ^∗∗∗p < 0.01 vs. sham group; ^##p < 0.01 vs. the model groups. The ischemic penumbra area in the box (D) was assessed for neuronal apoptosis using HE staining.

FIGURE 2

Ultrastructural characteristics of astrocytes 48 h after cerebral ischemia. Representative transmission electron microscopy images of astrocytes in the peri-infarct area of stroke, at 48 h after cerebral ischemia, in three groups of animals. (A) Normal astrocyte was observed in sham group, (B) Swollen activated perineuronal and perivascular astrocytes were seen in model groups, which demonstrated activation as evidenced by a significant amount of chromatin condensation atop the nuclear membrane and large nucleolus. The astrocytes were characterized by dispersed residual organelles and swollen mitochondria. Astrocyte fusion was often observed (C). (D,E) Less damage was observed in the EGB treatment group. A astrocyte, f—fibrils. Bars: A,C,D,E 2 μm; B 1 μm.

FIGURE 3

Effects of EGB treatment on the level of cytokines/chemokines in the brain after cerebral ischemia. (A–F) Analysis showing the relative levels of the pro-inflammatory mediators IL-1α, IL-6, and CXCL 10 (IP-10), (A–C) was in brain, (D–F) was in serum. Data are expressed as mean ± SEM (n = 5). ^∗p < 0.05, ^∗∗p < 0.01 and ^∗∗∗p < 0.001 vs. sham group; ^#p < 0.05 and ^##p < 0.01 vs. the model groups.

FIGURE 4

Effects of EGB on the activation of astrocytes and the expression of LCN2, p-JAK2, p-STAT3, in cerebral ischemia rats. (A–C) Double-immunofluorescence staining for astrocytic LCN2, p-STAT3, p-JAK2, and GFAP expression in the ischemic penumbra area after cerebral ischemia. The cells indicated with an arrow were magnified. Scale bar = 20 μm. (D–H) Western blots and quantitative analysis of GFAP, LCN2, p-JAK2, and p-STAT3 expression are expressed as mean ± SEM (n = 4). ^∗p < 0.05, ^∗∗p < 0.01 and ^∗∗∗p < 0.001 vs. sham group; ^#p < 0.05, ^##p < 0.01 and ^###p < 0.001 vs. model groups.

FIGURE 5

EGB suppressed OGD-induced inflammation in astrocytes in vitro. (A–C) Analysis showing the relative levels of the pro-inflammatory mediators IL-1β, IL-6, and CXCL10 (IP-10). Data are expressed as mean ± SEM (n = 5). ^∗p < 0.05, ^∗∗p < 0.01 and ^∗∗∗p < 0.001 vs. control group; ^#p < 0.05, ^##p < 0.01 and ^###p < 0.001 and vs. the indicated groups.

FIGURE 6

The effects of EGB on the activation of astrocytes and the expression of astrocytic LCN2, p-JAK2, and p-STAT3 after OGD induction in vitro. (A–H) Double-immunofluorescence staining for GFAP, LCN2, p-JAK2, and p-STAT3 in astrocytes after OGD induction. Scale bar = 20 μm. Western blots and quantitative analysis of GFAP, LCN2, p-JAK2, and p-STAT3 expression. Data are expressed as mean ± SEM (n = 3). ^∗p < 0.05 and ^∗∗p < 0.01 vs. control group; ^#p < 0.05 and ^##p < 0.01 vs. the indicated groups.