| Ischemic stroke that accounts for approximate 85%of the stroke is the second leading cause of death and leads to severe sequelae like paresis and speech defects with limited effective therapies in the clinical treatment. Cerebral ischemia followed by a cascade of energy failure and excitatory toxicity, especially inflammation, which triggers a severe loss of neurons and the massive activation of the resident microglia and infiltrated macrophages. Recent studies have demonstrated that these immune cells serve as the first defensive line to modulate central nervous system repair and also mediate inflammatory cascades after ischemia. Therefore, the activated microglia/macrophage play an irreplaceable role in the pathological progress of ischemia and act as an important therapeutic target of ischemic stroke.Microglial activation can be classified into two major phenotypes defined as ’classical activation’(also termed M1 phenotype or overactivated phenotype) and ’alternative activation’(M2 phenotype). M1 microglia polarization is associated with the production and release of multiple proinflammatory cytokines such as tumor necrosis factor (TNF-a) and interleukin-6 (IL-6). In general, the released factors act in tissue defense and promote the destruction of pathogens. However, the extensive production of inflammatory cytokines by overactivated or dysregulated microglia is constantly involved in the pathogenesis of stroke and induces more widespread damage to ischemic penumbra neurons. In contrast to the M1 phenotype, M2 microglia execute an anti-inflammatory effect and promote wound healing and tissue repair. The major anti-inflammatory cytokines, such as transforming growth factor-β (TGF-β) and interleukin-10 (IL-10), alleviates pro-inflammatory responses and enhance the expression of genes that are involved in tissue recovery and repair. In addition, it has been known that microglia/macrophage polarization changes dynamically after the stroke. After tMCAO, the microglia/macrophage mobile quickly to the injury site, displaying M2 phenotype at the post-stroke initial 3 days, and then the deleterious M1 type will be dominated from post-stroke 7 days. Thus, switching microglia from M1 type toward M2 type may be an effective therapeutic strategy for treatment of ischemic stroke. And finding drugs which are able to switching the phenotypes will broaden our strategy on treating ischemic stroke.GB, an essential abstract of Gink biloba, is a potent PAF receptor antagonist. GB has extensive biological and pharmacological effects, such as anti-allergy, anti-inflammation, and anti-oxidation. Recent studies have demonstrated that GB exhibits neuroprotection in Parkinson’s disease (PD) via anti-inflammation and in ischemic stroke by reducing the degradation of membrane phospholipids. In clinical therapeutics, GB is commonly used for improving the functional recovery of patients with ischemic stroke during the recovery phase, but the underlying mechanisms of GB against ischemic damage have not yet been fully explored.In the present study, we explored the neuroprotective mechanism of GB in tMCAO model. We demonstrated that GB promoted microglia/macrophage transition towards M2 phenotype. Consistently, in vitro experiments showed GB increase the expression of M2 relative the signature genes and the cytokines on BV2 and BMDM. Moreover, this modulation effect is related to inhibiting PAF receptor. These results revealed a novel pharmacological mechanism of GB that promoting microglia/macrophage to M2 phenotype in ischemic stroke and allows it to be used in other polarization related inflammatory diseases.AIM:Ginkgolide B (GB) has been 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.METHOD:1) The modified method of Zea Longa was used to establish the transient middle artery occlusion (tMCAO) model in mice. GB was injected intraperitoneally two times a day to mice from the first hour after tMCAO with different dosages (1.75 mg/kg,3.5 mg/kg, and 7.0 mg/kg). The infarct volume was evaluated with using triphenyl-2,3,4-tetrazolium-chloride (TTC; Sigma, St Louis, MO, USA) histology. The neurobehavioral evaluation was performed at 24 h after surgery, according to the Benderson scores.2) Immunofluorescence, ELISA and Real-time quantitative PCR are utilized to identify the phenotypes of the microglia/macrophage after tMCAO.3) We cultured the microglia cell line BV2 and the bone marrow derived macrophage to confirm the polarization modulating effect of GB in vitro. Flow cytometry, ELISA and Real-time quantitative PCR are used to identify the phenotypes of the microglia/macrophage on cells.3) The post-OGD primary cortical neuron was incubated with conditioned medium collected from different phenotypes of primary microglia to investigate the link of microglia polarization and neuronal protection.4) We used pharmacological effect to investigate whether PAF receptor is involved in GB’s effect. BV2 cell was cultured and CV3988 (10μM), a PAF receptor antagonist to pre-incubate the cell 30 min before the cell was administrated with polarization stimuli.5) PAF receptor targeting siRNA was synthesized and BV2 cell was transfected with this siRNA (100nM) for 6-8 hours. Western blotting was used to detect the PAF receptor silence efficiency. And ELISA and Real-time quantitative PCR are used to identify the phenotypes of the BV2 cells.RESULTS:1) GB at 3.5 mg/kg significantly ameliorated the infarct volume of tMCAO mice, reduced the water content of brain tissue and the neurological deficits scores.2) CD206, a M2 phenotype signature chemokine, was increased in GB treated group at either 3 or 7 days after tMCAO. At the meantime, CD 16/32 which is the M1 phenotype biomarker, was reduced significantly by treating GB. GB increased M2-related mRNA expression, such as CD206, Arg1 and Ym1 and promoted the levels M2-related cytokines including IL-10 and TGF-β either at 3 or 7 days after the insult. Accordingly, GB decreased M1-related mRNA expression, such as CCL3, iNOS and TNF-α and the release of M2-related cytokines including IL-6 and TNF-α (Fig.2J, K) at 3 or 7 days after tMCAO.3) 1 μM GB-treated group exhibited high mRNA expression of M2 genes and secreted higher M2-related cytokines compared with IL-4 group. Meanwhile, GB not only significantly inhibited expression of M1 signature genes including iNOS, TNF-α and CCL3, but also reduced production of inflammatory cytokines such as IL-6 and TNF-α.4) The conditioned medium from GB-pretreated M1 microglia attenuated OGD-induced neuron damage compared to M1-microglia conditioned medium group.6) Western blotting showed that PAF receptor expression was silenced by 70%.7) It has been demonstrated that PAF receptor is a necessary element of inflammatory cascade, for example PAF is indispensable for NF-κB activation in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD model. Since GB is the selective antagonist of PAF receptor, we investigated whether PAF receptor was involved in the modulating effect of GB on BV2. By compared with CV3988, another PAF receptor antagonist, we found that GB exerted similar effect with CV3988, such as increasing M2 signature genes expression and reducing M1 genes expression. In addition, GB and CV3988 increased TGF-β and IL-10 secretion and decreased IL-6 and TNF-a. This suggested that PAF receptor might be involved in the polarization modulation effect of GB. To verify the modulating role of PAF receptor, we silenced PAF receptor expression on BV2 by siRNA. And as expected, silencing PAF receptor exhibited the M2 promotion effect and M1 inhibition effect, manifested by mRNA expression. Therefore, we drew the conclusion that PAF receptor was involved in the microglia polarization modulation of GB.CONCLUSION:1) Ginkgolide B promoted microglia/macrophage polarization towards M2 polarization in tMCAO model and exhibited neuroprotive effect.2) PAF receptor was involved in the microglia polarization modulating effect of GB.The major contributions of the present study lie in:As the above mentioned, stroke is a severe CNS disease which has limited therapies Up to date, recombinant tissue plasminogen activator is the only clinical treatment that is approved by the United States Food and Drug Administration. Nevertheless, the extremely narrow therapeutic time window and short half-time confines its application. Thus, finding effective drugs for stroke is of emergency. In recent years, increasing evidences suggest that GB plays a neuroprotective role in several CNS disorders, especially in ischemic stroke.With the properties of anti-oxidation, anti-inflammation, GB has been proven to be an effective neuroprotective agent in various ischemic stroke models. In the present study, we have shown that GB exerts neuroprotective effect either during the acute phase (3 days) or recovery phase (7 days) of ischemic stroke. Moreover, this neuroprotection is related to its promotive effects on M2 polarization of resident microglia and infiltrated macrophages. We also have revealed that inhibiting PAF receptor is necessary for GB promoting microglia/macrophage polarization towards M2 phenotype. This finding broadens our understanding on the pharmacological effects of GB, indicating that GB could be utilized on microglia/macrophage polarization related inflammatory diseases. |
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