Sailuotong Capsule Prevents the Cerebral Ischaemia-Induced Neuroinflammation and Impairment of Recognition Memory through Inhibition of LCN2 Expression

Abstract

Background: Astrogliosis can result in astrocytes with hypertrophic morphology after injury, indicated by extended processes and swollen cell bodies. Lipocalin-2 (LCN2), a secreted glycoprotein belonging to the lipocalin superfamily, has been reported to play a detrimental role in ischaemic brains and neurodegenerative diseases. Sailuotong (SLT) capsule is a standardized three-herb preparation composed of ginseng, ginkgo, and saffron for the treatment of vascular dementia. Although recent clinical trials have demonstrated the beneficial effect of SLT on vascular dementia, its potential cellular mechanism has not been fully explored.

Methods: Male adult Sprague-Dawley (SD) rats were subjected to microsphere-embolized cerebral ischaemia. Immunostaining and Western blotting were performed to assess astrocytic reaction. Human astrocytes exposed to oxygen-glucose deprivation (OGD) were used to elucidate the effects of SLT-induced inflammation and astrocytic reaction.

Results: A memory recovery effect was found to be associated with the cerebral ischaemia-induced expression of inflammatory proteins and the suppression of LCN2 expression in the brain. Additionally, SLT reduced the astrocytic reaction, LCN2 expression, and the phosphorylation of STAT3 and JAK2. For in vitro experiments, OGD-induced expression of inflammation and LCN2 was also decreased in human astrocyte by the SLT treatment. Moreover, LCN2 overexpression significantly enhanced the above effects. SLT downregulated these effects that were enhanced by LCN2 overexpression.

Conclusions: SLT mediates neuroinflammation, thereby protecting against ischaemic brain injury by inhibiting astrogliosis and suppressing neuroinflammation via the LCN2-JAK2/STAT3 pathway, providing a new idea for the treatment strategy of ischaemic stroke.

Conflict of interest statement

The authors declare that they have no conflicts of interest.

Copyright © 2019 Yehao Zhang et al.

Figures

Figure 1

SLT treatment reduced ischaemic infarct volume in the cerebral ischaemia model. (a) Timeline depicts the treatment of SLT and assessments of the cognitive functions of rats. The male rats (n = 10) were orally treated with SLT at a daily dose of 16.5 mg/kg and 33 mg/kg for 4 weeks. After surgery for 22 days, memory tests were conducted. The training trial was performed four times a day for 5 days. (b) Cerebral infarct volume was assessed via TTC staining 24 h after cerebral ischaemia. Neurological score (c) of rats after cerebral ischaemia was assessed using a five-point scale system. Data are expressed as the mean ± SEM (n = 10). ∗∗∗P < 0.001 vs. sham group; #P < 0.05, ##P < 0.01, and ###P < 0.001 vs. the model groups.

Figure 2

The ischaemic penumbra area in the box was assessed for neuronal apoptosis using HE staining. Cortex sections and hippocampal regions stained with HE presented with neuronal loss and signs of cerebral edema, and swollen cells were observed in the ipsilateral hippocampus; plentiful apoptotic neurons were observed with karyopyknosis, cell gaps, and debris. SLT (33 mg/kg, daily) significantly alleviated the symptoms of apoptosis in a dose-dependent manner.

Figure 3

Neuroprotective effects of SLT on the Morris water maze (MWM) test: (a) average swimming speed, (b) escape latency, (c) platform crossing, (d) target quadrant time, and (e) trajectory of swimming. Data are expressed as the mean ± SEM (n = 10). ∗∗P < 0.01 and ∗∗∗P < 0.001 vs. sham group; #P < 0.05, ##P < 0.01, and ###P < 0.001 vs. model groups.

Figure 4

Effects of SLT treatment on the level of cytokines/chemokines in the brain after cerebral ischaemia. (a–f) Analysis showing the relative levels of the proinflammatory mediators IL-1α, IL-6, IL-12, and CXCL10 (IP-10) in the brain and in serum; (a–d) was in the brain; (e–h) was in serum. Data are expressed as the mean ± SEM (n = 5). ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 vs. sham group; #P < 0.05, ##P < 0.01, and ###P < 0.001 vs. the model groups.

Figure 5

Effects of SLT on the activation of astrocytes and the expression of LCN2, p-JAK2, and p-STAT3, in cerebral ischaemia rats. (a–c) Double immunofluorescence staining for astrocytic LCN2, p-STAT3, p-JAK2, and GFAP expression in the ischaemic penumbra area after cerebral ischaemia. Scale bar = 20 μm. (d–h) Western blots and quantitative analysis of GFAP, LCN2, p-JAK2, and p-STAT3 expression are expressed as the 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 6

SLT suppressed OGD-induced inflammation in astrocytes in vitro. (a–c) Analysis showing the relative levels of the proinflammatory mediators IL-1β, IL-6, and CXCL10 (IP-10). Data are expressed as the 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 vs. the indicated groups.

Figure 7

The effects of SLT 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 are expressed as the 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.