BackgroundTo improve the clinical outcome of patients who suffered ischemic stroke, cerebral ischemia-reperfusion (I/R) injury is one of the major concerns that should be conquered. Inflammatory reactions are considered a major contributor to brain injury following cerebral ischemia, and I/R exacerbates these reactions. The aim of this study was to investigate the possible ameliorative effects of progranulin (PGRN) against I/R injury in mice.MethodsIn vivo I/R was induced in four-week-old male ddY mice by 2 h of MCAO (middle cerebral artery occlusion) followed by 22 h of reperfusion. We evaluate expression of PGRN in I/R brain, efficacy of recombinant-PGRN (r-PGRN) treatment and its therapeutic time-window on I/R injury. Two hours after MCAO, 1.0 ng of r-PRGN or PBS was administered via intracerebroventricular. We assess neutrophil infiltration, expression of tumor necrosis factor (TNF)-α, matrix metalloproteinase-9 (MMP-9) and phosphorylation of nuclear factor-κB (NF-κB) by immunofluorescense staining and Western blotting. We also investigate neutrophil chemotaxis and intercellular adhesion molecule-1 (ICAM-1) expression in vitro inflammation models using isolated neutrophils and endothelial cells.ResultsWe found that expression of PGRN was decreased in the I/R mouse brain. r-PGRN treatment at 2 h after MCAO resulted in a reduction in the infarct volume and decreased brain swelling; this led to an improvement in neurological scores and to a reduction of mortality rate at 24 h and 7 d after MCAO, respectively. Immunohistochemistry, Western blotting, and gelatin zymography also confirmed that r-PGRN treatment suppressed neutrophil recruitment into the I/R brain, and this led to a reduction of NF-κB and MMP-9 activation. In the in vitro inflammation models, PGRN suppressed both the neutrophil chemotaxis and ICAM-1 expression caused by TNF-α in endothelial cells.ConclusionsPGRN exerted ameliorative effects against I/R-induced inflammation, and these effects may be due to the inhibition of neutrophil recruitment into the I/R brain.

Accumulating evidence shows that post-ischemic inflammation originated by Toll-like receptors (TLR) plays critical roles in ischemic stroke. However, the functions of other innate immune receptors are poorly understood in cerebral ischemia. Macrophage-inducible C-type lectin, Mincle, is one of the innate immune receptor C-type lectin-like receptor (CLR) to response against dying cells. In the present study, we showed that Mincle, its ligand SAP130, and its downstream phospho-Syk/Syk were upregulated after ischemia, and that Mincle is expressed in immune and non-immune cells in the ischemic brains of mice and human. We treated mice with piceatannol, a Syk inhibitor, and consequently the infarct volume and swelling were suppressed by piceatannol. The levels of phospho-Syk, MMP9 and ICAM-1 were downregulated, and the level of Claudin5 was uplegurated in piceatannol-treated groups. These data indicate that innate immune system, such as Mincle and Syk plays a pivotal role in the pathogenesis after the ischemia and reperfusion.

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Recent data have shown that TLR4 performs a key role in cerebral ischemia-reperfusion injury which serves as the origin of the immunological inflammatory reactions. However, the therapeutic effects of pharmacological inhibitions of TLR4 and its immediate down-stream pathway remain to be uncovered. In the present study, on mice, intracerebroventricular injection of resatorvid (TLR4 signal inhibitor; 0.01 μg) significantly reduced infarct volume and improved neurological score after middle cerebral artery occlusion and reperfusion. The levels of phospho-p38, nuclear factor-kappa B, and matrix metalloproteinase 9 expressions were significantly suppressed in the resatorvid-treated group. In addition, NOX4 associates with TLR4 after cerebral ischemia-reperfusion seen in mice and human. Genetic and pharmacological inhibitions of TLR4 each reduced NOX4 expression, leading to suppression of oxidative/nitrative stress and of neuronal apoptosis. These data suggest that resatorvid has potential as a therapeutic agent for stroke since it inhibits TLR4-NOX4 signaling which may be the predominant causal pathway.

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