Chronic Low-Dose Alcohol Consumption Attenuates Post-Ischemic Inflammation via PPARγ in Mice

Abstract

Ischemic stroke is one of the leading causes of death and permanent disability in adults. Recently, we found that light alcohol consumption (LAC) suppresses post-ischemic inflammatory response, which plays an important role in ischemic brain damage. Our goal was to determine the role of peroxisome proliferator-activated receptor-gamma (PPARγ) in the anti-inflammatory effect of LAC against transient focal cerebral ischemia. In in vivo study, male C57BL/6J wild type (WT) and endothelial-specific conditional PPARγ knockout mice were gavage fed with 0.7 g/kg/day ethanol or volume-matched water daily for 8 weeks. From the 7th week, 3 mg/kg/day GW9662 (a selective PPARγ antagonist) was intraperitoneally given for two weeks. Cerebral ischemia/reperfusion (I/R) injury and expression of manganese superoxide dismutase (MnSOD) and adhesion molecules, neutrophil infiltration, and microglial activation in the cerebral cortex before and following a 90 min unilateral middle cerebral artery occlusion (MCAO)/24 h reperfusion were evaluated. In in vitro study, the impact of chronic alcohol exposure on expression of PPARγ and MnSOD in C57BL/6J mouse brain microvascular endothelial cells (MBMVECs) was measured. PPARγ and MnSOD were significantly upregulated in the cerebral cortex of ethanol-fed WT mice and low-concentration ethanol-exposed C57BL/6J MBMVECs. GW9662 significantly inhibited alcohol-induced upregulation of MnSOD. Eight-week ethanol feeding significantly reduced cerebral I/R injury and alleviated the post-ischemic inflammatory response (upregulation of intercellular adhesion molecule-1 (ICAM-1) and E-selectin, microglial activation, and neutrophil infiltration). Treatment with GW9662 and endothelial-specific conditional knockout of PPARγ did not alter cerebral I/R injury and the inflammatory response in the control mice but abolish the neuroprotective effect in ethanol-fed mice. In addition, GW9662 and endothelial-specific conditional knockout of PPARγ diminished the inhibitory effect of LAC on the post-ischemic expression of adhesion molecules and neutrophil infiltration. Our findings suggest that LAC may protect against cerebral I/R injury by suppressing the post-ischemic inflammation via activation of PPARγ.

Keywords: PPARγ; alcohol; inflammation; ischemic stroke.

Figure 1

Effect of alcohol on nuclear protein expression and DNA-binding activity of PPARγ and protein expression of MnSOD. (A) Nuclear protein expression of PPARγ in the cerebral cortex of water and 0.7 g/kg/day ethanol-fed mice. Values are means ± SE. * p < 0.05. Analyzed using one-way ANOVA with Dunnett’s post hoc. (B) Nuclear DNA binding activity of PPARγ in the cerebral cortex of water and 0.7 g/kg/day ethanol-fed mice. Values are means ± SE. * p < 0.05. Analyzed using one-way ANOVA with Dunnett’s post hoc. (C) Protein expression of PPARγ in MBMVECs exposed to ethanol for 7 days. Values are means ± SE. * p < 0.05. Analyzed using one-way ANOVA with Dunnett’s post hoc. (D) Protein expression of MnSOD in the cerebral cortex of water and 0.7 g/kg/day ethanol-fed mice with or without GW9662 treatment. Values are means ± SE. * p < 0.05 vs. Water. ^# p < 0.05 vs. Ethanol. Analyzed using two-way ANOVA with Tukey’s post hoc. (E) Protein expression of MnSOD in 10 mM ethanol-exposed mice in the presence and absence of GW9662. Values are means ± SE. * p < 0.05 vs. Control. ^# p < 0.05 vs. Ethanol. Analyzed using two-way ANOVA with Tukey’s post hoc.

Figure 2

Effect of chronic ethanol consumption on cerebral I/R injury in the presence and absence of GW9662 and endothelial-specific conditional PPARγ knockout. (A,D) Representative brain sections stained with cresyl violet. (B,E) Infarct volume in the cerebral cortex and subcortical area. (C,F) Neurological deficit score. Values are means ± SE. ** p < 0.05 vs. Water or Tie2^CreERT2/+/PPARγ^+/+ + Water. ^## p < 0.05 vs. Ethanol or Tie2^CreERT2/+/PPARγ^+/+ + Ethanol. Analyzed using two-way ANOVA with Tukey’s post hoc.

Figure 3

Effect of chronic ethanol consumption on ICAM-1 expression of the cerebral cortex in the presence and absence of GW9662 and endothelial-specific conditional PPARγ knockout. (A,C) Representative Western blots. (B,D) Values are means ± SE. * p < 0.05 vs. Water or Tie2^CreERT2/+/PPARγ^+/+ + Water. ** p < 0.05 vs. Without I/R. ^## p < 0.05 vs. Ethanol or Tie2^CreERT2/+/PPARγ^+/+ + Ethanol. Analyzed using two-way ANOVA with Tukey’s post hoc.

Figure 4

Effect of chronic ethanol consumption on E-selectin expression of the cerebral cortex in the presence and absence of GW9662 and endothelial-specific conditional PPARγ knockout. (A,C) Representative Western blots. (B,D) Values are means ± SE. * p < 0.05 vs. Water or Tie2^CreERT2/+/PPARγ^+/+ + Water. ** p < 0.05 vs. Without I/R. ^## p < 0.05 vs. Ethanol or Tie2^CreERT2/+/PPARγ^+/+ + Ethanol. Analyzed using two-way ANOVA with Tukey’s post hoc.

Figure 5

Effect of chronic ethanol consumption on neutrophil infiltration in the presence and absence of GW9662 and endothelial-specific conditional PPARγ knockout. (A,C) Representative immunohistochemistry staining of MPO (scale bar = 100 μm). (B,D) Values are means ± SE. ** p < 0.05 vs. Water or Tie2^CreERT2/+/PPARγ^+/+ + Water. ^## p < 0.05 vs. Ethanol or Tie2^CreERT2/+/PPARγ^+/+ + Ethanol. Analyzed using two-way ANOVA with Tukey’s post hoc.

Figure 6

Effect of chronic ethanol consumption on microglia activation in the presence and absence of GW9662 and endothelial-specific conditional PPARγ knockout. (A,C) Representative immunohistochemistry staining of Iba1 (scale bar = 100 μm). (B,D) Values are means ± SE. ** p < 0.05 vs. Water or Tie2^CreERT2/+/PPARγ^+/+ + Water. Analyzed using two-way ANOVA with Tukey’s post hoc.