Background Activated microglia play a key role in initiating the inflammatory cascade following ischemic stroke and exert proinflammatory or anti-inflammatory effects, depending on whether they are polarized toward the M1 or M2 phenotype. The present study investigated the regulatory effect of icaritin (ICT) on microglial polarization in rats after cerebral ischemia/reperfusion injury (CI/RI) and explored the possible anti-inflammatory mechanisms of ICT. Methods A rat model of transient middle cerebral artery occlusion (tMCAO) was established. Following treatment with ICT, a G protein-coupled estrogen receptor (GPER) inhibitor or an extracellular signal-regulated kinase (ERK) inhibitor, the Garcia scale and rotarod test were used to assess neurological and locomotor function. 2,3,5-Triphenyltetrazolium chloride (TTC) and Fluoro-Jade C (FJC) staining were used to evaluate the infarct volume and neuronal death. The levels of inflammatory factors in the ischemic penumbra were evaluated using enzyme-linked immunosorbent assays (ELISAs). In addition, western blotting, immunofluorescence staining and quantitative PCR (qPCR) were performed to measure the expression levels of markers of different microglial phenotypes and proteins related to the GPER–ERK–nuclear factor kappa B (NF-κB) signaling pathway. Results ICT treatment significantly decreased the cerebral infarct volume, brain water content and fluorescence intensity of FJC; improved the Garcia score; increased the latency to fall and rotation speed in the rotarod test; decreased the levels of interleukin-1 beta (IL-1β), tumor necrosis factor-alpha (TNF-α), Iba1, CD40, CD68 and p-P65-NF-κB; and increased the levels of CD206 and p-ERK. U0126 (an inhibitor of ERK) and G15 (a selective antagonist of GPER) antagonized these effects. Conclusions These findings indicate that ICT plays roles in inhibiting the inflammatory response and achieving neuroprotection by regulating GPER–ERK–NF-κB signaling and then inhibiting microglial activation and M1 polarization while promoting M2 polarization, which provides a new therapeutic for against cerebral ischemic stroke. Graphical Abstract

Protecting the blood–brain barrier (BBB) is a potential strategy to treat cerebral ischaemic injury. We previously reported that hypertonic sodium chloride hydroxyethyl starch 40 (HSH) treatment alleviates brain injury induced by transient middle cerebral artery occlusion (tMCAO). However, other fluids, including 20% mannitol (MN), 3% hypertonic sodium chloride (HTS) and hydroxyethyl starch 130/0.4 solution (HES), have the same effect as HSH in cerebral ischaemia/reperfusion injury (CI/RI) remains unclear. The present study evaluated the protective effects of these four fluids on the BBB in tMCAO rats. Sprague–Dawley (SD) rats were randomly assigned to six groups. A CI/RI rat model was established by tMCAO for 120 min followed by 24 h of reperfusion. The sham and tMCAO groups were treated with normal saline (NS), whereas the other four groups were treated with the four fluids. After 24 h of reperfusion, neurological function, brain oedema, brain infarction volume, permeability of the BBB, cortical neuron loss and protein and mRNA expression were assessed. The four fluids (especially HSH) alleviated neurological deficits and decreased the infarction volume, brain oedema, BBB permeability and cortical neuron loss induced by tMCAO. The expression levels of GFAP, IL‐1β, TNF‐α, MMP‐9, MMP‐3, AQP4, MMP‐9, PDGFR‐β and RGS5 were decreased, whereas the expression levels of laminin and claudin‐5 were increased. These data suggested that small‐volume reperfusion using HSH, HES, MN and HTS ameliorated CI/RI, probably by attenuating BBB disruption and postischaemic inflammation, with HSH exerting the strongest neuroprotective effect.

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BackgroundActivated microglia play a key role in initiating the in ammatory cascade following ischemic stroke and exert proin ammatory or anti-in ammatory effects depending on whether they are polarized toward the M1 or M2 phenotype. The present study investigated the regulatory effect of Icaritin (ICT) on microglial polarization in rats after cerebral ischemia/reperfusion injury (CI/RI) and explored the possible antiin ammatory mechanisms of ICT. MethodsA rat model of transient middle cerebral artery occlusion (tMCAO) was established. Following treatment with ICT, a G protein coupled estrogen receptor (GPER) inhibitor or an extracellular signal regulated kinase (ERK) inhibitor, the Garcia scale and rotarod test were used to assess neurological and locomotor function. 2,3,5-Triphenyltetrazolium chloride (TTC) and Fluoro-Jade C (FJC) staining were used to evaluate the infarct volume and neuronal death. The levels of in ammatory factors in the ischemic penumbra were evaluated by enzyme-linked immunosorbent assay (ELISA). In addition, Western blotting, immuno uorescence and quantitative PCR (qPCR) were used to measure the expression levels of markers of different microglial phenotypes and proteins related to the GPER-ERK-NF-κB signaling pathway. ResultsWe found that ICT treatment signi cantly decreased the cerebral infarct volume, brain water content and uorescence intensity of FJC; improved the Garcia score; increased the latency to fall and rotation speed in the rotarod test; inhibited the expression of IL-1β, TNF-α, Iba1, CD40, CD68 and p-P65-NF-κB; and increased the levels of CD206 and p-ERK. U0126 (an inhibitor of ERK) and G15 (a selective antagonist of GPER) antagonized these effects. ConclusionsThese ndings indicate that ICT plays roles in inhibiting the in ammatory response and achieving neuroprotection by regulating GPER-ERK-NF-κB signaling and then inhibits microglial activation and M1 polarization while promoting M2 polarization, which provides new therapeutic strategy against cerebral ischemia stroke.

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