Ginkgolide B Protects Against Ischemic Stroke Via Modulating Microglia Polarization in Mice

Abstract

Aim: Ginkgolide B (GB) has shown neuroprotective effect in treating ischemic stroke, related to its property of anti-inflammation. Nevertheless, it is unclear whether GB is able to modulate microglia/macrophage polarization, which has recently been proven to be vital in the pathology of ischemic stroke.

Methods: We performed transient middle cerebral artery occlusion (tMCAO) on C57BL/6J male mice and induced cultured BV2 microglia and primary bone marrow-derived macrophages to be M1/2 phenotype by LPS+ interferon-γ and IL-4, respectively. Immunofluorescence and flow cytometry were used for detecting the specialized protein expression of M1/2, such as CD206 and CD16/32. qPCR was utilized to detect the signature gene change of M1/2.

Results: GB significantly reduced cerebral ischemic damage and ameliorated the neurological deficits of mice after tMCAO. More importantly, our experiments proved that GB promoted microglia/macrophage transferring from inflammatory M1 phenotype to a protective, anti-inflammatory M2 phenotype in vivo or vitro. CV3988 and silencing the platelet activator factor (PAF) receptor by siRNA demonstrated that PAF receptor was involved in the modulation of microglia/macrophage polarization.

Conclusion: Our results reveal a novel pharmacological effect of GB in modulating microglia/macrophage polarization after tMCAO, thus deepening our understanding of neuroprotective mechanisms of GB in treatment of ischemic stroke. Furthermore, this new mechanism may allow GB to be used in many other microglia/macrophage polarization-related inflammatory diseases.

Keywords: Ginkgolide B; Microglia/macrophage polarization; Platelet activator factor receptor; Stroke.

Figure 1

Ginkgolide B (GB) significantly attenuates cerebral ischemia and neurological deficits in vivo. (A, B) Representative pictures and quantification for analysis of the effect of GB on infarct volume. Scale: 1 cm. The white brain area presents infarcted tissue. (C) The effect of GB on the cerebral water content. (D) The effect of GB on neurological function. (E) Recovery of motor ability was assessed in ischemic mice by rotarod tests during a 7‐day follow‐up after transient middle cerebral artery occlusion (tMCAO). The brain sections were collected 72 h after I/R. *P < 0.05, **P < 0.01, ***P < 0.001 versus corresponding tMCAO group. Data are shown as mean ± SEM, n = 8–10.

Figure 2

Ginkgolide B (GB) promotes microglia/macrophage polarize toward M2 polarization in transient middle cerebral artery occlusion (tMCAO) model. (A) Representative double‐staining immunofluorescence of CD206 or CD16/32 and Iba1 and on brain sections obtained from ischemic mice at 3 days and 7 days after tMCAO. The observation area was identified according to Figure S1. (B) The immunofluorescence on poststroke 3‐day mice brain slice showed the colocalization rate of CD16/32 or CD206 with Iba1. (C) Similarly, the colocalization rate of CD16/32 or CD206 with Iba1 in poststroke 7‐day slice was shown. (D, E) Representative M2‐related mRNAs change folds at 3 and 7 days after tMCAO, respectively. (F, G) M2‐related cytokines (transforming growth factor‐β, IL‐10) detected by ELISA at 3 and 7 days after tMCAO, respectively. (H, I) Representative M1‐related mRNAs change folds detected by qPCR at 3 and 7 days after tMCAO, respectively. (J, K) M1‐related cytokines (TNF‐α, IL‐6) detected by ELISA at 3 and 7 days after tMCAO, respectively. For immunofluorescences, data are represented as mean ± SEM, n = 6, *P < 0.05, **P < 0.01, ***P < 0.001 versus tMCAO + saline group. For ELISA and qPCR experiment, data are represented as mean ± SEM, n = 3–4, *P < 0.05, **P < 0.01, ***P < 0.001 versus contralateral tissue; #P < 0.05, ##P < 0.01, ###P < 0.001 versus tMCAO + saline group.

Figure 3

Ginkgolide B (GB) facilities M1‐microglia polarizing toward M2 phenotype on BV2 cells. Before M2 induction, BV2 was pretreated by LPS (100 ng/mL) plus IFN‐γ (20 ng/mL) for 24 h to become M1 type. GB is pretreated for 2 h before the cells were stimulated by IL‐4 (20 ng/mL) for 24 h. (A, E) The expression of M2‐related biomarker MGL1/2 and M1‐related biomarker CCR7 detected, respectively, by flow cytometry. (B, F) And GB at 1 μM was found to be effective concentration. (C) Representative M2‐related mRNAs change folds. (D) M2‐related cytokines (transforming growth factor‐β, IL‐10) detected by ELISA. (G) Representative M1‐related mRNAs change folds detected by qPCR. (H) M1‐related cytokines (TNF‐α, IL‐6) detected by ELISA. Data are represented as mean ± SEM from three independent experiments, *P < 0.05, **P < 0.01, ***P < 0.001 versus control group, P### versus IL‐4 or LPS + IFN‐γ.

Figure 4

Platelet activator factor (PAF) receptor is involved in the polarization‐modulating effect of Ginkgolide B (GB). (A–C) CV‐3988 exhibited similar effect with GB on M1/2 gene modulation. CV‐3988(10 μM) is pretreated for 30 min before IL‐4(20 ng/mL) was treated. M2‐related mRNAs and cytokines were detected when BV2 was treated with IL‐4 combined with CV3988. (D, E) CV‐3988 and GB increased M2 cytokines and reduced the production of M1 cytokines, detected by ELISA. (F–H) Silencing PAF receptor showed similar polarization‐modulating effect with GB. PAF receptor targeting siRNA (100 μM) was used to silence PAF receptor, and the M2‐related mRNAs was detected. (I–K) CV‐3988 and GB decreased the expression of M1‐related gene on BV2. (L, M) ELISA showed the cytokines of M1 phenotype were decreased by treating GB or CV3988. (N–P) The results of qPCR showed silencing PAF receptor contributed to the downregulation of M1‐related gene expression, displaying similar effect with GB. Data are represented as mean ± SEM from three independent experiments, *P < 0.05, **P < 0.01, ***P < 0.001 versus control group, P### versus IL‐4 or LPS + interferon‐γ (IFN‐γ).

Figure 5

Ginkgolide B (GB) exhibits protective effect on oxygen glucose deprivation (OGD) neuron via pivoting microglia/macrophage polarization. Primary microglia cultures were induced toward M1 phenotype as described in the methods. The conditioned media was collected for the following neuron treatment. (A) The CM from GB‐pretreated M1 phenotype microglia group improved the MAP2 expression. After 1‐h OGD, primary cortical neurons were incubated with different condition medium of microglia (GB‐M1 or M1), respectively. The MAP2 assay was conducted to show the condition of neurons. (C) The neuritis length was measured by imagej (National Institutes of Health). (B, D) GB improved microglial phagocytosis which was impaired by OGD neuron‐conditioned medium. Fluorescent microspheres were added into microglial cultures for 1 h. Images are representative of three independent experiments. Data are represented as mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001 versus control group; ##P < 0.01, ###P < 0.001 versus M1 microglia CM or OGD neuron CM group.