The growth factor progranulin attenuates neuronal injury induced by cerebral ischemia-reperfusion through the suppression of neutrophil recruitment

Abstract

Background: To 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.

Methods: In 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.

Results: We 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.

Conclusions: PGRN 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.

Figure 1

Expression of progranulin in the ischemia-reperfusion injured brain. Progranulin (PGRN) expression was significantly decreased following ischemia-reperfusion (I/R) insults. (A) Representative PGRN bands from the Western blotting analysis of brain tissue taken from sham-operated and I/R animals; ipsilateral and contralateral hemispheres to the middle cerebral artery occlusion (MCAO). (B) Optical densitometry quantification of PGRN protein levels, normalized to β-actin. In the I/R brain, the expression of PGRN was significantly decreased 24 h after the induction of transient cerebral ischemia. **P <0.01 vs. sham contralateral brain; one-way ANOVA followed by Dunnett's test; n = 4 for each group.

Figure 2

r-PGRN treatment reduces cerebral infarct volume and brain edema in transient focal cerebral ischemia. (A) Protocol for surgery and r-PGRN administration. Intracerebroventricular (i.c.v.) injections of either vehicle or r-PGRN (0.1 to 1.0 ng) were administered 2 h after middle cerebral artery occlusion (MCAO). All assessments, with the exception of survival rate evaluation, were performed at 24 h after the induction of 2 h of transient MCAO. (B) Representative photograph showing TTC staining of coronal brain sections 24 h after MCAO in each treatment group. (C) Administration of 1 ng of r-PGRN significantly reduced the infarct volume, (D) and reduced brain edema, compared to the vehicle treatment. Although the 0.1 ng r-PGRN- and 0.3 ng r-PGRN-treated groups tended to experience reduced infarct volume and brain edema, the difference was not statistically significant. * P <0.05 vs. vehicle-treated group; one-way ANOVA followed by Dunnett's test; n = 6 to n = 8 for each group. (E) Only the 1.0 ng r-PGRN-treated group had significantly better neurological function at 24 h after MCAO than at 2 h after MCAO. # P <0.05; Wilcoxon signed-rank test. (F) A higher survival rate was observed throughout the follow-up period in the 1.0 ng r-PGRN-treated group. In contrast, a continuous reduction of the survival rate was observed in the vehicle-treated group. The difference between the groups was statistically significant. † P <0.05; Log-rank test; n = 9 or n =10 for each group. r-PGRN, recombinant-progranulin.

Figure 3

The effects of delayed administration of r-PGRN 6 h after transient MCAO. (A) Protocol for surgery and PGRN administration. Injections (i.c.v.) of either vehicle or r-PGRN (1.0 ng) were administered 6 h after the MCAO procedure. All assessments were performed at 24 h after the induction of 2 h of transient MCAO. (B) Administration of 1 ng of r-PGRN 6 h after MCAO did not reduce the infarct volume assessed at 24 h after the induction of 2 h of MCAO; (C) however, it significantly reduced brain edema. N.S. not significant; * P <0.05 vs. vehicle-treated group; Student's t-test; n = 8 or n = 9 for each group. i.c.v., intracerebroventricular; MCAO, middle cerebral artery occlusion; PGRN, progranulin; r-PGRN, recombinant-progranulin.

Figure 4

r-PGRN treatment significantly suppresses neutrophil recruitment into the I/R brain following MCAO. (A) Representative immunohistochemical staining for myeloperoxidase (MPO) in each of the areas of interest in the sham-operation, vehicle-treated and r-PGRN-treated groups. (B) Quantification of MPO-immunoreactive cells. The number of MPO-positive cells was significantly higher in the vehicle-treated mice than in the r-PGRN-treated mice. Scale bar = 20 μm. ## P <0.01 vs. sham-operation mice; ** P <0.01 vs. vehicle-treated mice; Student's t-test. n = 4 or n = 5 for each group. I/R, ischemia-reperfusion; MCAO, middle cerebral artery occlusion; r-PGRN, recombinant-progranulin.

Figure 5

PGRN inhibits ¹²⁵I-TNF-α binding to neutrophil surfaces and suppresses neutrophil chemotaxis induced by TNF-α. (A) Saturation curve for specific ¹²⁵I-TNF-α binding to neutrophil surfaces was determined, and in accordance with these results, 50 pg/mL of ¹²⁵I-TNF-α was used in the subsequent experiments. (B) The ¹²⁵I-TNF-α binding significantly decreased with increasing concentrations of PGRN. ***P <0.001 vs. 0 ng/mL of PGRN group; one-way ANOVA followed by Dunnett's test. Data were obtained from three independent experiments and presented as mean ± SEM. (C, D, E) Neutrophil chemotaxis was induced by TNF-α, and PGRN was found to significantly suppress this effect in a concentration-dependent manner; at 100 and 250 ng/mL of PGRN attenuates the migration speed and straightness of the route of migration, but did not affect the directionality of migration. ### P <0.001 vs. control group; Student t-test; ** P <0.01, *** P <0.001 vs. TNF-α only group; one-way ANOVA followed by Dunnett's test; n = 5 for each group. PGRN, progranulin; TNF-α, tumor necrosis factor-alpha.

Figure 6

PGRN ameliorates TNF-α-induced inflammation in hBMVECs. (A) Representative bands from the Western blotting analysis of ICAM-1 and β-actin. (B) Optical densitometry quantification of ICAM-1, normalized to β-actin. TNF-α (10 ng/mL) induced an approximately eight-fold increase in ICAM-1 in hBMVECs after a 20-h exposure. ### P <0.001 vs. control group; Student's t-test. PGRN significantly suppressed TNF-α-induced ICAM-1 expression in a concentration-dependent manner. * P <0.05, ** P <0.01 vs. vehicle-treated group; one-way ANOVA followed by Dunnett's test; n = 4 for each group. hBMVECs, human brain microvascular endothelial cells; ICAM-1, intercellular adhesion molecule-1; PGRN, progranulin; TNF-α, tumor necrosis factor-alpha.

Figure 7

PGRN significantly suppresses the expression of MMP-9, and the phosphorylation of NF-κB in I/R brain. (A) Representative bands from Western blotting analysis of phosphorylated and total NF-κB (upper). Optical densitometry quantification for the phosphorylation of NF-κB (p NF-κB), normalized to total NF-κB (tNF-κB) and β-actin (lower). In the I/R brain, phosphorylation of NF-κB was significantly increased. ## P <0.01 vs. sham control group; Student's t-test. PGRN significantly suppressed this increased phosphorylation of NF-κB induced by I/R. * P <0.05 vs. vehicle-treated group; Student t-test. (B) Representative bands from Western blotting analysis of MMP-9 expression (upper). Optical densitometry quantification of MMP-9 expression, normalized to β-actin (lower). MMP-9 expression was significantly increased in the I/R brain. ## P <0.01 vs. sham control group; Student's t-test. PGRN significantly suppressed the expression of MMP-9 induced by I/R. * P <0.05 vs. vehicle-treated group; Student's t-test; n = 5 for each group. (C) Representative bands from gelatin zymography for activated MMP-9 (upper). Optical densitometry quantification of activated MMP-9 (lower). Activated MMP-9 was significantly increased in the I/R brain. ## P <0.01 vs. sham control group; Student's t-test. PGRN significantly suppressed the activation of MMP-9 induced by I/R. * P <0.05 vs. vehicle-treated group; Student's t-test; n = 3 for sham or n = 4 for each treated group. I/R, ischemia-reperfusion; MMP-9, matrix metalloproteinase-9; NF-κB, nuclear factor-κappaB; PGRN, progranulin.