P2X7R/cryopyrin inflammasome axis inhibition reduces neuroinflammation after SAH

Abstract

Neuroinflammation contributes to the pathogenesis of early brain injury (EBI) after subarachnoid hemorrhage (SAH). Cytotoxic events following SAH, such as extracellular accumulation of adenosine triphosphate (ATP), may activate the P2X purinoceptor 7 (P2X7R)/cryopyrin inflammasome axis, thus inducing the proinflammatory cytokine IL-1β/IL-18 secretion. We therefore hypothesized that inhibition of P2X7R/cryopyrin inflammasome axis would ameliorate neuroinflammation after SAH. In the present study, SAH was induced by the endovascular perforation in rats. Small interfering RNAs (siRNAs) of P2X7R or cryopyrin were administered intracerebroventricularly 24h before SAH. Brilliant blue G (BBG), a non-competitive antagonist of P2X7R, was administered intraperitoneally 30min following SAH. Post-assessments including SAH severity score, neurobehavioral test, brain water content, Western blot and immunofluorescence, were performed. Administration of P2X7R and cryopyrin siRNA as well as pharmacologic blockade of P2X7R by BBG ameliorated neurological deficits and brain edema at 24h following SAH. Inhibition of P2X7R/cryopyrin inflammasome axis suppressed caspase-1 activation, which subsequently decreased maturation of IL-1β/IL-18. To investigate the link between P2X7R and cryopyrin inflammasome in vivo, Benzoylbenzoyl-ATP (BzATP), a P2X7R agonist, was given to lipopolysaccharide (LPS) primed naive rats with scramble or cryopyrin siRNAs. In LPS-primed naive rats, BzATP induced caspase-1 activation and mature IL-1β release were neutralized by cryopyrin siRNA. Thus, the P2X7R/cryopyrin inflammasome axis may contribute to neuroinflammation via activation of caspase-1 and thereafter mature IL-1β/IL-18 production following SAH. Therapeutic interventions targeting P2X7R/cryopyrin pathway may be a novel approach to ameliorate EBI following SAH.

Keywords: ANOVA; ASC; ATP; BBG; Benzoylbenzoyl-ATP; Brilliant blue G; BzATP; CNS; Cryopyrin; EBI; Early brain injury; Edema; IACUC; IL-1β; Inflammation; Institutional Animal Care and Use Committee; LPS; MPO; NF-κB; NLR; P2X purinoceptor 7; P2X7R; SAH; SD; Sprague–Dawley; Subarachnoid hemorrhage; TLR4; Toll-like receptor 4; adenosine triphosphate; analysis of variance; apoptosis-associated speck-like protein containing a CAED; central nervous system; early brain injury; interleukin-1β; lipopolysaccharide; myeloperoxidase; nuclear factor-kappa B; nucleotide-binding domain, leucine-rich repeat containing; siRNA; small interfering RNA; subarachnoid hemorrhage.

FIGURE 1

Experimental design and animal group classification. SAH = subarachnoid hemorrhage; WB = Western blotting; BWC = brain water content; LPS = Lipopolysaccharide; P2X7R= P2X purinoceptor 7; BzATP= Benzoylbenzoyl ATP; BBG= Brilliant blue G.

FIGURE 2

Expression profile of P2X purinoceptor 7 (P2X7R)/cryopyrin inflammasome components after subarachnoid hemorrhage (SAH). Western blot assay (A) for the expression profiles of cryopyrin, mature IL-1β and P2X7R in the left hemisphere (perforation side) within 72 hours after SAH. Quantification of cryopyrin (B), mature IL-1β (C) and P2X7R (D) expression is shown, respectively. n = 4 rats per group and per time point. Error bars represent mean ± standard error of the mean. *p<0.05 vs. Sham.

FIGURE 3

Cryopyrin siRNA mixture inhibited inflammation at 24 hours after SAH. Representative (A) Western blot and therapeutic effects of cryopyrin siRNA pre-SAH injection on cryopyrin (B), cleaved caspase-1(P20) (C), mature IL-1β (D) and mature IL-18 (E) levels in left hemisphere at 24 hours following SAH. Error bars represent mean ± standard error of the mean. *p<0.05 vs. Sham; **p<0.01 vs. Sham; #p<0.05 vs. Vehicle; ^@p<0.05 vs. Scramble siRNA.

FIGURE 4

P2X7R siRNA mixture inhibited inflammation at 24 hours after SAH. Representative (A) Western blot and therapeutic effects of P2X7R siRNA pre-SAH injection on P2X7R (B), cleaved caspase-1(P20) (C), mature IL-1β (D) and mature IL-18 (E) levels in left hemisphere 24 hours following SAH. n = 6 rats per group. Error bars represent mean ± standard error of the mean. *p<0.05 vs. Sham; **p<0.01 vs. Sham; ^#p<0.05 vs. Vehicle; ^@p<0.05 vs. Scramble siRNA.

FIGURE 5

Effects of P2X7R/cryopyrin knockdown on neurological functions and brain water content at 24 hours after SAH. Cryopyrin siRNA and P2X7R siRNA improved neurological functions (A), reduced brain edema (B) at 24 hours following SAH. Box=25th-75th interquarile percentiles, horizontal line=median, vertical line=range (A) or Error bars represent mean ± standard error of the mean (B). *p<0.05 vs. Sham; **p<0.01 vs. Sham; ^#p<0.05 vs. Vehicle; ^@p<0.05 vs. Scramble siRNA.

FIGURE 6

Cryopyrin siRNA reduced inflammation induced by Benzoylbenzoyl-ATP (BzATP) in lipopolysaccharide (LPS) primed rats. Representative Western blot (A) and cryopyrin siRNA abolished augment of cleaved caspase-1(P20) and mature IL-1β induced by BzATP in LPS primed model. LPS alone significantly increased cryopyrin level, while failed to induce production of cleaved caspase-1 and mature IL-1β. BzATP enable a large increase in expression of cleaved caspase-1 and mature IL-1β upon LPS primed and cryoprin siRNA abolished the proinflammatory response of BzATP (Fig 6 B–D). n = 6 rats per group. Error bars represent mean ± standard error of the mean. *p<0.05 vs. naive; **p<0.01 vs. naive; &p<0.05 vs. LPS; ^@p<0.05 vs. LPS+BzATP; #p<0.05 vs. LPS+BzATP+Scramble siRNA.

FIGURE 7

Effects of Brilliant blue G (BBG) on neurological functions and brain water content at 24 hours and 72 hours after SAH. BBG significantly improved neurological score (A) and reduced brain edema (B) 24 hours following SAH, but there was no significance difference at 72 hours (C and D). Box=25th–75th interquarile percentiles, horizontal line= median, vertical line=range (A, C). Error bars represent mean ± standard error of the mean (B, D). *p<0.05 vs. Sham; **p<0.01 vs. Sham; ^#p<0.05 vs. Vehicle.

FIGURE 8

BBG inhibited inflammation at 24 hours after SAH. Representative (A) Western blot and therapeutic effects of BBG post-SAH injection on P2X7R (B), cleaved caspase-1(P20) (C), mature IL-1β (D) and mature IL-18 (E) levels in left hemisphere 24 hours following SAH. n = 6 rats per group. Error bars represent mean ± standard error of the mean. *p<0.05 vs. Sham; **p<0.01 vs. Sham; ^#p<0.05 vs. Vehicle.

FIGURE 9

BBG significantly suppressed neutrophil infiltration at 24 hours after SAH. Representative Western blots (A) and effects of BBG on myeloperoxidase (MPO) level (B) in left hemisphere at 24 hours after SAH. n = 6 rats per group. Representative photograph of immunofluorescence staining (C) for MPO showing that the MPO-positive cells were increased in vehicle group and decreased in BBG treatment (30 mg/kg) 24 hours after SAH. Scale bars: 50µm. Error bars represent mean ± standard error of the mean. *p<0.05 vs. Sham; ^#p<0.05 vs. Vehicle.