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. 2012 May;32(5):835-43.
doi: 10.1038/jcbfm.2011.189. Epub 2012 Jan 11.

"V体育ios版" The flavonoid fisetin attenuates postischemic immune cell infiltration, activation and infarct size after transient cerebral middle artery occlusion in mice

Mathias Gelderblom  1 , Frank Leypoldt, Jan Lewerenz, Gabriel Birkenmayer, Denise Orozco, Peter Ludewig, John Thundyil, Thiruma V Arumugam, Christian Gerloff, Eva Tolosa, Pamela Maher, Tim Magnus
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The flavonoid fisetin attenuates postischemic immune cell infiltration, activation and infarct size after transient cerebral middle artery occlusion in mice

Mathias Gelderblom et al. J Cereb Blood Flow Metab. 2012 May.

Abstract

The development of the brain tissue damage in ischemic stroke is composed of an immediate component followed by an inflammatory response with secondary tissue damage after reperfusion VSports手机版. Fisetin, a flavonoid, has multiple biological effects, including neuroprotective and antiinflammatory properties. We analyzed the effects of fisetin on infarct size and the inflammatory response in a mouse model of stroke, temporary middle cerebral artery occlusion, and on the activation of immune cells, murine primary and N9 microglial and Raw264. 7 macrophage cells and human macrophages, in an in vitro model of inflammatory immune cell activation by lipopolysaccharide (LPS). Fisetin not only protected brain tissue against ischemic reperfusion injury when given before ischemia but also when applied 3 hours after ischemia. Fisetin also prominently inhibited the infiltration of macrophages and dendritic cells into the ischemic hemisphere and suppressed the intracerebral immune cell activation as measured by intracellular tumor necrosis factor α (TNFα) production. Fisetin also inhibited LPS-induced TNFα production and neurotoxicity of macrophages and microglia in vitro by suppressing nuclear factor κB activation and JNK/Jun phosphorylation. Our findings strongly suggest that the fisetin-mediated inhibition of the inflammatory response after stroke is part of the mechanism through which fisetin is neuroprotective in cerebral ischemia. .

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"V体育平台登录" Figures

Figure 1
Figure 1
Dose-dependent protection from ischemic stroke by fisetin. (A) Representative vital-stained (2,3,5-triphenyl-2-hydroxy-tetrazolium chloride (TTC)) brain sections of placebo and fisetin (50 mg/kg bw posttreatment) treated mice 3 days after ischemia. (B, C) Fisetin treatment 20 minutes before (A) or 3 hours after onset of ischemia (B). Infarct size as edema corrected infarct volume in μL. Scatter plot with mean. (D, E) Time until animals stopped circling (reached score 1) in days in fisetin pretreatment (D) and fisetin posttreatment (E). Error bars indicate s.d. Significance by one-way analysis of variance (ANOVA) and Bonferroni post hoc test (A, C) or one-sided Student's t-test (B, D). *P<0.05. (F) Systemic blood pressure is not influenced by fisetin. Tail cuff measurements after intraperitoneal injection show no significant changes (two-way ANOVA).
Figure 2
Figure 2
Decreased inflammatory cells in postischemic brains of fisetin pretreated mice. (A) Absolute numbers of microglia per hemisphere by flow cytometry 3 days after ischemia are not different between fisetin and vehicle pretreated mice. (B–D) Significant reduction of ischemic brain infiltrating CD45high leukocytes, macrophages, dendritic cell (DC), and lymphocytes relative to numbers of microglia 3 days after stroke (n=3). Error bars indicate s.d. Significance by two-sided Student's t-test. n.s., not significant, *P<0.05, **P<0.05.
Figure 3
Figure 3
Fisetin pretreatment and posttreatment reduces tumor necrosis factor α (TNFα) production in the brain. Flow cytometry of microglia (A) and macrophages (B) isolated from ipsilesional brain hemispheres 3 days and 7 days after stroke following pretreatment or posttreatment with fisetin 50 mg/kg body weight. Staining for intracellular TNFα shows attenuation of TNFα production in fisetin pretreated mice in the brain-derived microglia and macrophages at day 3 and macrophages at day 7 after stroke. The same effect is observed in mice treated with fisetin 3 hours after ischemia. Percentages relative to gated cells. Representative experiments with pooled hemispheres from three mice are shown. TNFα-positive cells are defined by isotype control (not shown).
Figure 4
Figure 4
Fisetin does not exhibit systemic effects on tumor necrosis factor α (TNFα) production. Flow cytometry of macrophages isolated from spleen following stroke (A, B) or sham-operated mice and pretreatment (A) or posttreatment (B) with fisetin or placebo at day 3 or 7 (fisetin 50 mg/kg body weight). Staining for intracellular TNFα does not show differences in TNFα production in spleen macrophages between fisetin and placebo. Of note, no difference in TNFα production between stroke and sham-operated mice is seen (A–C). Percentages relative to gated cells. TNFα-positive cells are defined by isotype control (not shown). Representative experiments with pooled spleens from three mice are shown.
Figure 5
Figure 5
Fisetin attenuates lipopolysaccharide (LPS)-stimulated tumor necrosis factor α (TNFα) production and LPS-induced neurotoxicity of murine macrophage and microglia as well as human macrophages. Reduced TNFα production in murine macrophage cell lines (A; Raw264.7, n=3), murine microglia cell lines (B; N9, n=3) and human monocyte-derived macrophages (C; n=3) on fisetin treatment. Percentage of TNFα production relative to LPS-stimulated cells. (D) Reduced microglial neurotoxicity after fisetin pretreatment of microglia. Murine primary microglia pretreated with fisetin or dimethyl sulfoxide (DMSO) together with or in the absence of LPS and incubated with primary cortical neurons. Percentage of neuronal death by trypan blue exclusion. Error bars indicate s.d. Significance by one-way analysis of variance (ANOVA) and Bonferroni post hoc test. *P<0.05, **P<0.01, ***P<0.001. LPS=10 ng/mL for (A, B) and 1 μg/mL for (C). Fisetin dose in μg/mL final concentration.
Figure 6
Figure 6
Fisetin blocks phosphorylation of JNK, c-Jun, and IκB. (A) Representative western blots of fisetin inhibition of lipopolysaccharide (LPS)-induced phosphorylation of IκB, c-Jun, and JNK isoforms in microglial (N9) and macrophage (Raw264.7) cell lines. Fisetin 2.9 μg/mL (10 μmol). (B) Quantification of the reduction of phosphorylation (in percentage; LPS=100%); Fisetin 2.9 μg/mL (10 μmol). Error bars indicate s.d. Significance by one-way analysis of variance (ANOVA) and Bonferroni post hoc test. **P=0.002, ***P<0.0001; n=3. LPS=10 ng/mL.
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