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P2X7R blockade prevents NLRP3 inflammasome activation and brain injury in a rat model of intracerebral hemorrhage: involvement of peroxynitrite

P2X7R blockade prevents NLRP3 inflammasome activation and brain injury in a rat model of... Background: The NLR family, pyrin domain-containing 3 (NLRP3) inflammasome plays a key role in intracerebral hemorrhage (ICH)-induced inflammatory injury, and the purinergic 2X7 receptor (P2X7R) is upstream of NLRP3 activation. This study aimed to investigate how P2X7R functions in ICH-induced inflammatory injury and how the receptor interacts with the NLRP3 inflammasome. Methods: Rats were treated with P2X7R small interfering RNA (siRNA) 24 h before undergoing collagenase-induced ICH. A selective P2X7R inhibitor (blue brilliant G, BBG) or a peroxynitrite (ONOO ) decomposition catalyst (5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrinato iron(III) [FeTPPS]) was injected 30 min after ICH. Brain water content, hemorrhagic lesion volume, and neurological deficits were evaluated, and western blot, immunofluorescence, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) were carried out. Results: Striatal P2X7R and NLRP3 inflammasomes were activated after ICH. Gene silencing of P2X7R suppressed NLRP3 inflammasome activation and interleukin (IL)-1β/IL-18 release and significantly ameliorated brain edema and phox neurological deficits. Additionally, enhanced NADPH oxidase 2 (NOX2, gp91 ) and inducible nitric oxide synthase (iNOS), as well as their cytotoxic product (ONOO ) were markedly attenuated by BBG treatment following ICH. This was accompanied by downregulations of the inflammasome components, IL-1β/IL-18 and myeloperoxidase (MPO, a neutrophil marker). Most importantly, inflammasome activation and IL-1β/IL-18 release were significantly inhibited by ONOO decomposition with FeTPPS. Conclusions: Our findings implicate that P2X7R exacerbated inflammatory progression and brain damage in ICH rats possibly via NLRP3 inflammasome-dependent IL-1β/IL-18 release and neutrophil infiltration. ONOO , a potential downstream signaling molecule of P2X7R, may play a critical role in triggering NLRP3 inflammasome activation. Keyword: P2X7R, NLRP3, Peroxynitrite, Intracerebral hemorrhage, NOX2, IL-1β Background ICH brain damage. There is an urgent need to clarify the Spontaneous intracerebral hemorrhage (ICH) is a devas- pathophysiology of this disease to identify effective tating stroke subtype, with high morbidity and mortality therapies. [1]. Unfortunately, no satisfactory pharmacologic treat- Accumulating evidence suggests that innate immunity ments have been found for clinical practice, mainly due to and inflammatory responses are involved in ICH-induced a lack of knowledge underlying the mechanisms of post- secondary brain injury [1–3]. The intracellular Nod-like receptors have recently been shown to play a critical role * Correspondence: [email protected] in the process of innate immunity and inflammatory The National Key Clinical Specialty, The Engineering Technology Research responses [4]. The NLR family, pyrin domain-containing 3 Center of Education Ministry of China, Guangdong Provincial Key Laboratory (NLRP3) inflammasome, the best characterized member on Brain Function Repair and Regeneration, Department of Neurosurgery, Zhujiang Hospital, Southern Medical University, Guangzhou 510282, China of Nod-like receptor family, is a multiprotein complex that Full list of author information is available at the end of the article © 2015 Feng et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Feng et al. Journal of Neuroinflammation (2015) 12:190 Page 2 of 17 contains the adaptor protein apoptosis-associated speck- In the case of ICH, both NADPH oxidase 2 (NOX2) and like protein containing a CARD (ASC) and the effector inducible nitric oxide synthase (iNOS) have been reported caspase-1. Once activated, caspase-1 can cleave the pro to contribute to brain injury; knockout mice exhibit less forms of interleukin (IL)-1β and IL-18 into their mature brain edema and cell death than wild-type controls follow- and active forms, which leads to the recruitment and acti- ing ICH [15, 16]. Superoxide anion (O ) and nitric oxide vation of other immune cells, such as neutrophils [5]. In (NO), released through NOX and iNOS in activated this regard, evidence indicates that the NLRP3 inflamma- microglia, act as devastating pro-inflammatory mediators some plays a pivotal role in ICH [2] and other central ner- in CNS diseases [17]. More importantly, peroxynitrite vous system (CNS) conditions [6–11], but the precise (ONOO ), the product of a diffusion-controlled reaction mechanisms associated with inflammasome activation of NO with O , is a more potent oxidant species and is continue to be debated. involved in the pathologies of ischemic stroke, neuro- The role of ATP-gated transmembrane cation channel trauma, and neurodegenerative diseases [18–20]. We pre- P2X7R in the signaling cascade has received particular viously demonstrated that abundant ONOO formed in a attention due to its widespread involvement as a key hemoglobin (Hb)-induced ICH rat model [21], but the regulatory element of NLRP3 inflammasome activation exact mechanisms of ONOO in brain injury after ICH [12]. A growing number of studies have demonstrated have not been fully characterized. Besides its ability to the important pathophysiological functions of P2X7R in oxidize or nitrate proteins, lipids, and DNA, ONOO can CNS disorders, including ischemic stroke, subarachnoid also lead to destructive pathological consequences by trig- hemorrhage, neurotrauma, epilepsy, neuropathic pain, gering the activation of several biochemical pathways en- and neurodegenerative illnesses [13, 14]. However, the gaged in the development of neuroinflammation and IL- specific role of P2X7R in ICH has not yet been estab- 1β production [22–24]. However, the precise link between lished, and the interaction between P2X7R and the ONOO formation and IL-1β secretion in ICH is unclear. NLRP3 inflammasome in the development of ICH- The P2X7R acts as an upstream molecule of NOX2 induced brain injury remains unclear. activation signaling in many in vivo and in vitro disease Fig. 1 Expression profile of P2X7R and its cellular location after collagenase-induced intracerebral hemorrhage (ICH). Western blot analysis (a) for the time course of P2X7R expression in the ipsilateral hemisphere of Sham and ICH rats within 72 h. Quantification of P2X7R (b)expression is shown, n = 4 rats per group and time point. Confocal images (c) of double immunofluorescence for P2X7R expression in Iba-1-positive microglia 24 h * ** following ICH, n = 6 rats per group. Scale bar = 12.5 μm. Data represent means ± SD. P < 0.05 vs. Sham, P <0.01 vs. Sham. GAPDH glyceraldehyde 3-phosphate dehydrogenase, ICH intracerebral hemorrhage Feng et al. Journal of Neuroinflammation (2015) 12:190 Page 3 of 17 models [25–27]. NOX2-mediated oxidative stress was re- ONOO is involved in P2X7R-mediated NLRP3 inflam- cently proposed to be responsible for activation of the masome activation, we used an ONOO decomposition NLRP3 inflammasome and subsequent neurovascular catalyst in vivo and measured the expression levels of damage in ischemic stroke [7]. Notably, P2X7R-dependent P2X7R and NLRP3 inflammasome components. NADPH oxidase activation and ONOO formation play key roles in caspase-1 and IL-1β processing in endotoxin- Materials and methods primed human monocytes [28]. In an animal model of Animals lipopolysaccharide (LPS)-induced striatum injury, acti- Sprague–Dawley (SD) male rats weighing 280–320 g vated P2X7R in microglia was associated with increased were purchased from the Animal Experiment Center of iNOS and 3-nitrotyrosine (3-NT, a reliable marker of Southern Medical University (Guangzhou, China). All ONOO ), and this was reversed by the P2X7R antagonist experimental procedures and animal care were approved oxidized ATP (oxATP) [29]. Despite this knowledge, the by the Southern Medical University Ethics Committee. potential roles of P2X7R and NLRP3 inflammasomes and NOX2/iNOS-dependent ONOO formation in the devel- opment of ICH-induced brain damage remain to be ICH model clarified. After anesthetization (0.3 ml/100 g, 10 % chloral hy- We hypothesized that ONOO , formed from NOX2- drate, Sigma-Aldrich, St. Louis, MO, USA), an incision derived O and iNOS-derived NO, may be involved in was made on the skin along the sagittal midline to ex- transducing P2X7R-mediated IL-1β/IL-18 production and pose the skull. A burr hole (1 mm) was drilled 3 mm lat- brain injury via NLRP3 inflammasome activation after eral and 1 mm anterior to the bregma, then a 30-gauge ICH. We first investigated the expression profiles of needle was inserted through the burr hole into the stri- P2X7R and the NLRP3 inflammasome components. Next, atum (6 mm ventral from the skull surface), and ICH a mixed small interfering (si) RNA was applied to knock was induced by stereotaxic infusion of bacterial collage- down P2X7R in vivo, and alterations in NLRP3 inflamma- nase VII-S (0.25U in 1.0 μl sterile saline, Sigma-Aldrich) some components and functional outcomes were mea- over a 10-min period. In the Sham group, rats were sub- sured. We then explored the therapeutic effect of the jected to only a needle insertion as described above. The selective P2X7R antagonist, blue brilliant G (BBG). Add- needle was kept in situ for another 10 min to prevent itionally, we observed iNOS- and NOX2-dependent for- backflow and then slowly removed. The craniotomies mation of ONOO and their alterations in ICH rats were sealed with bone wax. Rats were allowed to recover following BBG treatment. Finally, to determine whether in separate cages with free access to food and water. Fig. 2 Expression profiles of NLRP3, ASC, and caspase-1 p20 subunit after ICH. Western blot analysis (a)for thetimecourse expressions of NLRP3 (b), ASC (c), and caspase-1 p20 subunit (d) in the ipsilateral hemisphere of Sham and ICH rats within 72 h. Quantification of NLRP3, ASC, and caspase-1 p20 subunit expression is shown, respectively, n = 4 rats per group and time point. Data represent means ± SD. P <0.05 ** vs. Sham, P < 0.01 vs. Sham. ASC adaptor protein apoptosis-associated speck-like protein containing a CARD, GAPDH glyceraldehyde 3-phosphate dehydrogenase, ICH intracerebral hemorrhage, NLRP3 pyrin domain-containing 3 Feng et al. Journal of Neuroinflammation (2015) 12:190 Page 4 of 17 Experimental protocol the 72-h study, BBG was administered daily by intraperi- Four separate experiments were conducted as shown in toneal injection. Western blot, hematoxylin and eosin Additional file 1. (H&E) staining, immunofluorescence (IF), and terminal deoxynucleotidyl transferase dUTP nick end labeling Experiment 1 (TUNEL) were measured 24 h after ICH induction; Thirty-six rats were divided into six groups (Sham, and mNSS and brain water content were detected at both 24 6, 12, 24, 48, and 72 h after ICH). The expression levels and 72 h. of P2X7R, NLRP3, ASC, and caspase-1 were detected by western blot. The tissue for immunofluorescence (IF) Experiment 4 was collected 24 h after ICH induction. Thirty-three rats were randomized into three groups: Sham, Vehicle (ICH + saline, intraperitoneal injection), Experiment 2 and FeTPPS (30 mg/kg). Western blotting was performed Eighty-eight rats were randomized into four groups: 24 h after ICH induction. Sham, Vehicle (ICH + saline, intracerebroventricular in- jection), Scramble small interfering RNA (siRNA) (1000 siRNA and drug delivery pmol, 2 μl, ICH + scramble siRNA), and P2X7R siRNA BBG (Sigma-Aldrich) was diluted at 50 and 100 mg/kg (1000 pmol, 2 μl, ICH + P2X7R siRNA). siRNA silencing in Vehicle (saline) solution. Rats were treated intraperi- efficacy was assessed by western blot. Brain water con- toneally with either BBG or Vehicle immediately after tent and modified Neurological Severity Score (mNSS) ICH induction and at 12, 36, and 60 h. were also measured. For in vivo siRNA administration, P2X7R siRNA or non-silencing RNA (Sigma-Aldrich) was applied 24 h Experiment 3 before ICH by intracerebroventricular injection as previ- One hundred and thirty-two rats were randomized into ously described [2]. A cranial burr hole (1 mm) was four groups: Sham, Vehicle (ICH + saline, intraperitoneal drilled, and a 30-gauge needle was inserted stereotaxic- injection), BBG (50 mg/kg), and BBG (100 mg/kg). For ally into the right lateral ventricle. To improve the gene Fig. 3 Effects of P2X7R small interfering RNA (siRNA) treatment in ICH rats. RT-PCR of P2X7R after siRNA treatment 24 h following ICH, n = 6 rats per group (a). Western blot assay and quantification of P2X7R (b) after siRNA treatment 24 h following ICH, n = 4 rats per group. Brain edema (c)at 24 ** @ h after ICH, n = 6 rats per group. mNSS (d) at 24 and 72 h after ICH, n = 6 rats per group. Data represent means ± SD. P < 0.01 vs. Sham, P <0.05 vs. Vehicle, P < 0.05 vs. ICH + Scramble siRNA. GAPDH glyceraldehyde 3-phosphate dehydrogenase, mNSS modified Neurological Severity Score, siRNA small interfering RNA Feng et al. Journal of Neuroinflammation (2015) 12:190 Page 5 of 17 silence efficiency, two different sequences targeting RT-PCR P2X7R siRNA were combined (a)sense:5′-CAGUGAA Rats were anesthetized and decapitated. Lesioned tissues UGAGUACUACUA-3′;antisense:5′-UAGUAGUACU (about 40 mg) were obtained, and total RNA was ex- CAUUCACUG-3′;(b)sense:5′-CUCUUGAGGAGCGC tracted from the tissue with GeneJET™ RNA Purification CGAAA-3′;antisense:5′-UUUCGGCGCUCCUCAAGA Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA). G-3′. siRNA was dissolved in RNA free water. Scrambled RNA (1 μg) was reverse-transcribed to cDNA with high control siRNA (1000 pmol, 2 μl), P2X7R siRNA (1000 capacity (Life Technologies, Carlsbad, CA, USA). RT-PCR pmol, 2 μl), or RNA free water (2 μl) was delivered intra- was performed in an ABI Prism 7500 sequence detection cerebroventricularly for 2 min. The needle was left in system (Applied Biosystems, Foster City, CA, USA) using place for an additional 10 min after injection and then specific primers designed from known sequences. GAPDH slowly withdrawn. was used as an endogenous control gene. Sequence- FeTPPS (Millipore, Billerica, MA, USA) was diluted specific primers for P2X7R, NLRP3, and GADPH were as to 30 mg/kg in Vehicle (saline) solution. Rats were follows: treated intraperitoneally with either FeTPPS or Vehicle P2X7R, 5′-CTACTCTTCGGTGGGGGCTT-3′ immediately and at 12 h after ICH induction. The (forward primer), FeTPPS dose was selected based on our previous re- P2X7R, 5′-CTCTGGATCCGGGTGACTTT-3′ ports that showed that it is effectively protected against (reverse primer); injury [30]. NLRP3, 5′-CTGCATGCCGTATCTGGTTG-3′ Fig. 4 Effects of P2X7R siRNA on NLRP3 inflammasome activation and IL-1β/IL-18 maturation after ICH. RT-PCR of NLRP3 after P2X7R siRNA treatment 24 h following ICH (a), n = 6 rats per group. Western blot assay (b) and quantification of NLRP3 (c), ASC (d), caspase-1 p20 subunit (e), IL-1β (f), and IL-18 (g) after P2X7R siRNA treatment 24 h following ICH, n = 4 rats per group. Data represent means ± SD. ** @ # P <0.01 vs. Sham, P <0.05 vs. Vehicle, P < 0.05 vs. ICH + Scramble siRNA. ASC adaptor protein apoptosis-associated speck-like protein containing a CARD, GAPDH glyceraldehyde 3-phosphate dehydrogenase, ICH intracerebral hemorrhage, IL interleukin, NLRP3 The NLR family, pyrin domain-containing 3 Feng et al. Journal of Neuroinflammation (2015) 12:190 Page 6 of 17 (forward primer), Western blot NLRP3, 5′-GCTGAGCAAGCTAAAGGCTTC-3′ Western blotting was performed as described previously (reverse primer); [30]. The following primary antibodies were used: rabbit GAPDH, 5′-AGACAGCCGCATCTTCTTGT-3′ polyclonal anti-P2X7R (1:1000, Alomone Labs, Jerusalem, (forward primer), Israel), rabbit polyclonal anti-NLRP3 antibody (1:1000, GAPDH, 5′- TGATGGCAACAATGTCCACT-3’ Santa Cruz, Biotechnology, Santa Cruz, CA, USA), rabbit (reverse primer); polyclonal anti-ASC antibody (1:500, Abclonal, Cambridge, Fig. 5 Effects of BBG on brain edema in ICH rats. BBG treatment evidently reduced brain edema at 24 (d) and at 72 h (h) after ICH. Hematoxylin and eosin (H&E) showed the hemorrhagic lesion volume alteration after BBG treatment at 24 (a, b) and at 72 h (e, f) following ICH. Tissue * ** damage after BBG treatment at 24 (c) and at 72 h (g) following ICH. n = 6 rats per group. Data represent means ± SD. P < 0.05, P < 0.01. BBG brilliant blue G, ICH intracerebral hemorrhage Feng et al. Journal of Neuroinflammation (2015) 12:190 Page 7 of 17 MA, USA), mouse monoclonal anti-caspase-1 p20 antibody vitrification, they were embedded in paraffin, and 3-μm (1:1000, Santa Cruz Biotechnology), rabbit polyclonal anti- sections were prepared. Sections were dewaxed, rehy- IL-1β antibody (1:1000, Millipore), rabbit monoclonal anti- drated, and then processed for IF and TUNEL. IL-18 antibody (1:1000, Abcam, Cambridge, UK), mouse monoclonal anti-nitrotyrosine antibody (1:1000, Abcam), Histological examination mouse monoclonal anti-iNOS antibody (1:200, Santa Cruz Coronal sections (1 mm apart) [31] were prepared accord- phox Biotechnology), mouse monoclonal anti-gp91 antibody ingly and then stained with H&E. Hemorrhagic volumes (1:2000, BD Transduction Laboratories, San Jose, CA, were calculated using Image Pro Plus 6.0 software (Media USA), and rabbit polyclonal anti-myeloperoxidase antibody Cybernetics, USA) to span the entire hematoma [32]. (MPO, 1:500, Abcam). GAPDH (1:1000, Cell Signaling Technology, Danvers, MA, USA) was employed as the IF loading control. Blot bands were quantified by densitometry Antigen retrieval was performed by heat treatment in a with ImageJ software (National Institutes of Health, microwave oven for 21 min in Tris–EDTA buffer solu- Baltimore, MD, USA). tion (0.05 mol/L Tris, 0.001 mol/L EDTA; pH 8.5). Sections were incubated for 30 min in 5 % bovine serum Paraffin section preparations albumin (BSA) and then incubated at 4 °C overnight The sections were processed as previously described [30] with primary antibodies (rabbit polyclonal anti-P2X7R, with minor modifications. After anesthetization, rats 1:500, Alomone labs; mouse monoclonal anti-3- were transcardially perfused with 200 ml saline followed Nitrotyrosine, 1:400, Abcam; rabbit polyclonal anti- by 400 ml 4 % paraformaldehyde solution. Brain tissues nitrotyrosine antibody, 1:200, Millipore; rabbit polyclonal were then removed and fixed by immersion in the same anti-MPO antibody, 1:50, Abcam; rabbit polyclonal anti- solution at 4 °C for 24 h. After dehydration and iNOS antibody, 1:40, Santa Cruz Biotechnology; rabbit Fig. 6 Effects of BBG on neuronal apoptosis and neurological outcomes in ICH rats. BBG significantly reduced the number of apoptotic neurons (a, b) 24 h following ICH, n = 6 rats per group. BBG significantly improved neurological deficits (c) at 24 and at 72 h after ICH, n = 6 rats per * ** group. Scale bar = 50 μm. Data represent means ± SD. P < 0.05, P < 0.01. BBG brilliant blue G Feng et al. Journal of Neuroinflammation (2015) 12:190 Page 8 of 17 polyclonal anti-Iba-1 antibody, 1:600, WAKO, Osaka, Brain water content measurement Japan; goat polyclonal anti-Iba-1 antibody, 1:300, Abcam; Brain edema was evaluated by a common wet/dry phox mouse monoclonal gp91 antibody, 1:400, BD Trans- method as previously described [33]. Briefly, at 24 or 72 duction Laboratories). For double-staining experiments, h post-ICH, rats were anesthetized and decapitated. The primary antibodies were separately incubated overnight brains were removed and immediately separated into at 4 °C. After they were washed with phosphate-buffered contralateral and ipsilateral hemispheres and the cere- saline (PBS), sections were then incubated with second- bellum and wet weighed. The cerebellum was used as an ary antibodies. Images were obtained using confocal mi- internal control. Brain specimens were dried in an oven croscopes (FV10i-W, Olympus, Tokyo, Japan; LSM780, at 100 °C for 24 h to obtain the dry weight. The water Zeiss, Oberkochen, Germany). content was expressed as a percentage of the wet weight: ([wet weight] – [dry weight]) / (wet weight) × 100 %. TUNEL Behavioral testing At 24 h after ICH, TUNEL staining was performed with Behavioral tests were assessed with mNSS at 24 and 72 an in situ apoptosis detection kit (Roche, Basel, h after ICH by an investigator who was blinded to the Switzerland) according to the manufacturer’s instruction. experimental groups [34]. For NeuN and TUNEL co-staining, the sections were first labeled with a NeuN antibody (1:400, Abcam), Statistical analysis followed by TUNEL. The slides were analyzed using a Data are shown as mean ± SD. Statistical analysis was fluorescence microscope (Bx51, Olympus). performed using SPSS 13.0 (SPSS Inc., Chicago, IL, USA). Fig. 7 Effects of BBG on NLRP3 inflammasome activation and IL-1β/IL-18 maturation. RT-PCR of NLRP3 after BBG treatment 24 h following ICH, n =6 rats pergroup (a). Representative western blot (b) and therapeutic effects of BBG on P2X7R (c), NLRP3 (d), ASC (e), caspase-1 p20 subunit (f), mature IL-1β (g), and mature IL-18 (h) levels in the ipsilateral hemisphere 24 h after ICH, n = 4 rats per group. Data * ** represent means ± SD. P < 0.05, P < 0.01. ASC adaptor protein apoptosis-associated speck-like protein containing a CARD, BBG brilliant blue G, GAPDH glyceraldehyde 3-phosphate dehydrogenase, ICH intracerebral hemorrhage, IL interleukin, NLRP3 pyrin domain-containing 3 Feng et al. Journal of Neuroinflammation (2015) 12:190 Page 9 of 17 Comparison between groups was determined by Student’s t but still remained higher than those in the Sham group at tests or one-way analysis of variance (ANOVA) followed by 48 h (P < 0.05 vs. Sham) and 72 h (P < 0.05 vs. Sham). least significant difference (LSD) tests with multiple compari- sons. The statistically significant level was P <0.05. P2X7R RNA interference reduced brain water content and improved neurological outcomes Results We next explored whether P2X7R is involved in brain injury P2X7R was increased and mainly expressed in microglia following ICH. Two P2X7R siRNA mixtures were applied 24 cells following ICH h before ICH induction. Silencing efficacy by RT-PCR dem- Protein content was analyzed at different time points onstrated a significant inhibitory effect of P2X7R siRNA on after injection to investigate whether P2X7R would re- itsmRNAlevels(P<0.01) (Fig.3a).Western blot (Fig.3b) spond to collagenase-induced ICH. As shown by western showed 41.3 and 40.7 % reductions of P2X7R in the P2X7R blot (Fig. 1a, b), P2X7R levels were significantly elevated siRNA group compared with the Vehicle and Scramble at 6 h after ICH (P < 0.05 vs. Sham) and peaked at siRNA groups, respectively (both P < 0.05), at 24 h after ICH. around 24 h (P < 0.01 vs. Sham) when P2X7R levels were Brain water content in the ipsilateral hemisphere was signifi- nearly 4.5 times more than those in the Sham group. cantly increased in the Vehicle (82.56 ± 0.72 % vs. Sham, Following the peak, P2X7R levels decreased, returning 79.40 ± 0.44 %, P < 0.01) and Scramble siRNA (82.44 ± 0.75 close to baseline levels at 72 h. %vs. Sham,79.40 ±0.44%, P < 0.01) groups at 24 h post- Double immunolabeling was performed to identify the ICH, while that in P2X7R siRNA group was decreased to cell type that expresses P2X7R. The results showed that 81.39 ± 0.58 % (P < 0.05 vs. Vehicle or Scramble siRNA) P2X7R was predominantly expressed in microglia cells (Fig.3c).Consistentwiththe brainedema results, P2X7R (Fig. 1c) and not in other cell types, such as astrocytes or siRNA administration significantly ameliorated neurological neurons (Additional file 2). deficits at 24 h (8.66 ± 1.15 vs. Vehicle, 10.00 ± 1.95, P < 0.05; vs. Scramble siRNA, 10.50 ± 1.26, P<0.05) and NLRP3, ASC, and caspase-1 were upregulated after ICH 72 h (6.33 ± 0.81 vs. Vehicle, 8.10 ± 1.96, P < 0.05; vs. Scram- NLRP3 inflammasome has been proposed to be downstream ble siRNA, 8.16 ± 1.16, P < 0.05) post-ICH (Fig. 3d). of P2X7R [12]. We evaluated the expressions of NLRP3 inflammasome components by western blot (Fig. 2a). NLRP3 P2X7R RNA interference inhibited NLRP3 inflammasome (Fig.2b),ASC (Fig.2c), and cleavedcaspase-1 (Fig.2d) were activation and subsequent IL-1β/IL-18 release significantly upregulated at 6 h (P < 0.05 vs. Sham) and NLRP3 inflammasome is actively involved in brain injury reached their peak at 24 h post-ICH (P < 0.01 vs. Sham). after ICH [2]. We further clarify the role of P2X7R in Following this peak, levels of all three proteins declined NLRP3/ASC/caspase-1 activation and the subsequent Fig. 8 Effects of BBG on neutrophils infiltration after ICH. Representative photographs of immunofluorescence staining (a) for MPO (neutrophil marker)-positive cells in perihematomal area in the Sham, Vehicle, and BBG (50 mg/kg) groups at 24 h following operation, n = 6 rats per group. Representative western blot (b) and effects of BBG on MPO levels (c) at 24 h after ICH, n = 4 rats per group. Scale bar = 50 μm. Data represent * ** means ± SD. P < 0.05, P < 0.01. BBG brilliant blue G, ICH intracerebral hemorrhage Feng et al. Journal of Neuroinflammation (2015) 12:190 Page 10 of 17 processing of IL-1β/IL-18. P2X7R siRNA treatment sig- post-ICH (P < 0.01) (Fig. 5e, f), indicating that BBG could nificantly reduced NLRP3 mRNA expression (P <0.01) promote tissue reconstruction. Meanwhile, the tissue (Fig. 4a). The protein levels of NLRP3 inflammasome damage around the lesion site was evidently mitigated by component and IL-1β/IL-18 production were evidently el- BBG treatment at both 24 and 72 h (Fig. 5c, d, g, h). Con- evated in the Vehicle and Scramble siRNA groups at 24 h sistently, neurological deficits (Fig. 6c) at both 24 h (50 mg/ after ICH (P < 0.01). P2X7R siRNA treatment significantly kg, 8.83 ± 1.64 vs. Vehicle, 10.00 ± 1.95, P < 0.05; 100 mg/ suppressed caspase-1 activation and the subsequent secre- kg, 8.66 ± 1.55 vs. Vehicle, 10.00 ± 1.95, P <0.05) and 72 h tion of mature IL-1β/IL-18 (P < 0.05) (Fig. 4b–f). (50 mg/kg, 6.40 ± 1.64 vs. Vehicle, 8.10 ± 1.96, P <0.05; 100 mg/kg, 7.00 ± 1.78 vs. Vehicle, 8.10 ± 1.96, P <0.05) BBG deceased post-ICH neurological deficits, brain water after ICH were improved by BBG treatment. However, content, and neuronal apoptosis there was no difference between animals that received 50 Next, we investigated the effects of the selective P2X7R and 100 mg/kg doses of BBG treatment with regard to inhibitor, BBG. Both doses (50 and 100 mg/kg) significantly mNSS scores or brain water contents. Thus, the 50 mg/kg attenuated brain water content at 24 h (50 mg/kg, 81.76 ± dose was applied in further studies. 0.32 % vs. Vehicle, 82.54 ± 0.66 %, P < 0.05; 100 mg/kg, The number of apoptotic neurons was significantly in- 81.67 ± 0.43 % vs. Vehicle, 82.54 ± 0.66 %, P <0.05) and 72 creased at 24 h after ICH compared with the Sham h (50 mg/kg, 81.59 ± 1.15 % vs. Vehicle, 82.94 ± 1.00 %, P group (P < 0.01), and BBG treatment significantly re- < 0.05; 100 mg/kg, 81.74 ± 1.12 % vs. Vehicle, 82.94 ± 1.00 duced the number of apoptotic neurons (P < 0.01) rela- %, P < 0.05) after ICH (Fig. 5d, h). The hemorrhagic lesion tive to the Vehicle group (Fig. 6a, b). volume at 24 h post-ICH for the Vehicle and BBG groups were 91.17 ± 23.54 and 82.77 ± 21.31, respectively BBG decreased P2X7R expression, NLRP3/ASC/caspase-1 (P > 0.05) (Fig. 5a, b), indicating that BBG did not affect activation, and subsequent IL-1β/IL-18 production following bleeding. However, the hemorrhagic lesion volume for the ICH BBG group (37.79 + 15.56) was significantly decreased BBG treatment significantly reduced NLRP3 mRNA compared with the Vehicle (73.03 + 19.34) group at 72 h levels (Fig. 7a). Western blot analysis revealed that BBG Fig. 9 Effects of BBG on ICH-induced iNOS expression in the striatum. Most iNOS signals overlapped with Iba-1 positive microglia (a). Western blot (b, c) and immunofluorescence labeling (d) showing that BBG treatment significantly decreased striatal iNOS expression 24 h after ICH compared * ** with the Vehicle group. n =4(b, c)or n =6(a, d) rats per group. Scale bar = 25 μm(a)or 50 μm(d). Data represent means ± SD. P <0.05, P <0.01. BBG brilliant blue G, GAPDH glyceraldehyde 3-phosphate dehydrogenase, ICH intracerebral hemorrhage, iNOS inducible nitric oxide synthase Feng et al. Journal of Neuroinflammation (2015) 12:190 Page 11 of 17 (50 mg/kg) treatment attenuated the expressions of compared with the Sham group (P <0.01 vs. Sham). P2X7R (P < 0.05 vs. Vehicle), NLRP3 (P < 0.05 vs. Ve- BBG (50 mg/kg) significantly suppressed MPO ex- hicle), ASC (P < 0.01 vs. Vehicle), and cleaved caspase-1 pression compared to the Vehicle group (P <0.05 vs. (P < 0.05 vs. Vehicle). Furthermore, the levels of mature Vehicle). IL-1β (P < 0.05 vs. Vehicle) and IL-18 (P < 0.05 vs. Vehicle) were distinctly reduced after BBG treatment (Fig. 7b–h). BBG suppressed ICH-induced iNOS expression iNOS is upregulated in both blood infusion and collagenase- induced ICH rat models [31, 35]. The role of P2X7R in BBG reduced neutrophils infiltration after ICH. iNOS signaling was next investigated using IF and western We detected MPO levels in brain tissue by IF (Fig. 8a) blot analysis. iNOS expression was weak in Sham-operated and western blot (Fig. 8b, c) at 24 h following ICH rats but was dramatically elevated 24 h after ICH induction to determine the effect of P2X7R/NLRP3 inflamma- (P < 0.01 vs. Sham). To further trace the source of iNOS, some axis activation on neutrophil infiltration. Striatal double IF was performed and found that iNOS was mainly MPO levels were evidently increased following ICH expressed in Iba-1-positive microglia (Fig. 9a). Western phox phox phox Fig. 10 Effects of BBG on ICH-induced gp91 expression in the striatum. Gp91 significantly colocalized with Iba-1 (a). Most gp91 signals overlapped with iNOS (b). Western blot (c, d) and immunofluorescence labeling (e) showing that BBG treatment significantly reduced the striatal phox gp91 expression 24 h after ICH compared with the Vehicle group. n =4(d)or n =6 (e) rats per group. Scale bar = 20 μm(a)or25 μm(b)or * ** 50 μm(e). Data represent means ± SD. P < 0.05; P < 0.01. BBG brilliant blue G, GAPDH glyceraldehyde 3-phosphate dehydrogenase, ICH intracerebral hemorrhage, iNOS inducible nitric oxide synthase Feng et al. Journal of Neuroinflammation (2015) 12:190 Page 12 of 17 blots and IF showed that the enhanced iNOS levels were BBG attenuated peroxynitrite formation following ICH markedly attenuated in BBG-treated rats (P <0.05 vs. Ve- The enhancement of iNOS and NOX2 after ICH induction hicle) (Fig. 9b–d). prompted us to examine the involvement of P2X7R in ONOO formation. Double IF demonstrated a high degree BBG reduced ICH-induced NOX2 expression of colocalization with Iba-1 (Fig. 11a). Moreover, 3-NT and − phox NOX2, a primary source of O , is actively involved in gp91 expressions were almost completely overlapped ICH-induced brain injury [15]. We studied the expression (Fig. 11b), implying that peroxynitrite production is NOX2 phox of gp91 , a membrane subunit of NOX2, by IF and dependent. However, BBG treatment significantly down- phox western blotting. Double IF showed that gp91 was also regulated 3-NT overexpression (P < 0.05 vs. Vehicle) mainly expressed in Iba-1-positive areas (Fig. 10a) and (Fig. 11c–e). most overlapped with iNOS expression (Fig. 10b), imply- ing a close connection between them. Consistent with The ONOO decomposition catalyst FeTPPS inhibited phox iNOS, gp91 was significantly increased after ICH in ip- NLRP3/ASC/Caspase-1 activation and subsequent silateral hemisphere brain tissues as compared with the production of mature IL-1β/IL-18 following ICH Sham group (P < 0.01 vs. Sham) at 24 h after ICH. BBG Our findings suggested a pivotal role for microglia- phox − treatment significantly downregulated gp91 overex- expressed P2X7R in mediating ONOO formation in an pression (P < 0.05 vs. Vehicle) (Fig. 10c–e). iNOS and NOX2-dependent way, which further prompted Fig. 11 Effects of BBG on ICH-induced 3-NT expression in the striatum. 3-NT significantly colocalized with Iba-1 (a). Most 3-NT signals overlapped phox with gp91 (b). Western blot (c, d) and immunofluorescence staining (e) showing that BBG treatment significantly suppressed striatal 3-NT expression 24 h after ICH compared with the Vehicle group. n =4 (a, b, e)or n =6 (c, d) rats per group. Bar = 12.5 μm(a, b)or50 μm(e). Data * ** represent means ± SD. P < 0.05, P < 0.01. BBG brilliant blue G, GAPDH glyceraldehyde 3-phosphate dehydrogenase, ICH intracerebral hemorrhage, 3-NT 3-nitrotyrosine Feng et al. Journal of Neuroinflammation (2015) 12:190 Page 13 of 17 us to explore whether ONOO served as the key bridge However, FeTPPS had no influence on P2X7R expressions between P2X7R and NLRP3 inflammasome activation. To (Additional file 3). FeTPPS treatment significantly reduced answer this question, the ONOO decomposition catalyst NLRP3 mRNA expression. Together, these results reveal FeTPPS was applied in vivo. Firstly, FeTPPS significantly that P2X7R-dependent synthesis of ONOO may be a key reduced 3-NT levels (P < 0.01 vs. Vehicle) on western blot activator of the NLRP3 inflammasome. (Fig. 12a, b). Thereafter, the expression levels of P2X7R and NLRP3 inflammasome components were measured by western blot at 24 h following ICH. The results indicated Discussion that FeTPPS significantly downregulated the enhanced Innate immune and inflammatory responses are increas- levels of NLRP3 (P < 0.05 vs. Vehicle), ASC (P <0.05 vs. ingly recognized as important factors in the pathophysi- Vehicle), and cleaved caspase-1 (P < 0.05 vs. Vehicle) after ology of secondary brain injury following ICH. The NLRP3 ICH. Moreover, the upregulation of IL-1β/IL-18 was also inflammasome, the most characterized pattern recognition attenuated by FeTPPS (P < 0.05 vs. Vehicle) (Fig. 12c–h). receptor (PRR) in innate immune response initiation, is Fig. 12 Effects of FeTPPS on NLRP3 inflammasome activation and IL-1β/IL-18 maturation. RT-PCR of NLRP3 after FeTPPS treatment 24 h following ICH, n = 6 rats per group (a). Western blot (d, b) showed that FeTPPS significantly reduced striatal 3-NT expression 24 h after ICH compared with the Vehicle group. Western blot assay (g) and quantification of NLRP3 (c), ASC (e), caspase-1 p20 subunit (f), mature IL-1β (h), and mature * ** IL-18 (j) after FeTPPS treatment 24 h following ICH, n = 5 rats per group. Data represent means ± SD. P < 0.05, P < 0.01. ASC adaptor protein apoptosis-associated speck-like protein containing a CARD, GAPDH glyceraldehyde 3-phosphate dehydrogenase, ICH intracerebral hemorrhage, IL interleukin, NLRP3 pyrin domain-containing 3, 3-NT 3-nitrotyrosine Feng et al. Journal of Neuroinflammation (2015) 12:190 Page 14 of 17 strongly involved in ICH-induced inflammation and pro- neutrophils were also diminished in the BBG group com- duces pro-inflammatory factors such as IL-1β [2]. pared with the Vehicle group. These results indicate that Several distinct mechanisms have been proposed to ac- P2X7R may be responsible for ICH-induced inflammation, count for NLRP3 activation, including potassium (K )ef- possibly by NLRP3 inflammasome-dependent IL-1β/IL-18 flux, intracellular calcium alteration, ubiquitination, and release and subsequent neutrophil infiltration signals. In reactive oxygen species (ROS) generation [36]. It was accordance with our results, Ma and colleagues [2] found recently reported that extracellular ATP-induced P2X7R that NLRP3 mainly colocalized with microglia but not with activation could directly mediate K efflux through the other cell types, providing further support for a tight rela- hemichannel pannexin 1 to activate the NLRP3 inflamma- tionship between P2X7R and NLRP3. some [5, 28, 36]. However, pannexin 1-deficient mice do In response to microglia activation, large amounts O not show diminished caspase-1 activation [37], implying and NO are released through activated NADPH oxidase that pannexin-1 is dispensable for the assembly of and iNOS [17], both of which play pivotal roles in ICH- caspase-1 activating inflammasome complexes. This raises induced brain damage [15, 16]. In our study, enhanced phox the hypothesis that other signaling pathways may be in- gp91 in microglia largely colocalized with iNOS, volved in P2X7R-mediated NLRP3 inflammasome activa- prompting us to consider ONOO . Double IF revealed that tion or that something besides pannexin 1 is necessary for increased 3-NT was also expressed in microglia and mostly + phox P2X7R-dependent K efflux. NOX2-derived ROS after overlapped with gp91 . Consistently with this, Mander ATP release-mediated P2X7R activation is well established and Brown [47] reported that activation of NOX or iNOS in a growing body of in vitro and in vivo disease models alone was relatively harmless, but their simultaneous acti- [23–25]. Importantly, there are a number of reports sup- vation was lethal because it spurred ONOO production. porting that high ROS levels, particularly NOX2-derived Inhibition of either iNOS [48] or NOX2 [49] significantly ROS, are critical for NLRP3 inflammasome activation reduced ONOO production in CNS disease models. Col- [36]. RNS also play an important role as evidenced by the lectively, we can infer that NOX2-derived O and iNOS- − − fact that both an ONOO scavenger and NOX2 inhibitor derived NO may contribute to ONOO formation upon suppressed nigericin-induced caspase-1 activation and IL- microglia activation after ICH. 1β secretion in human monocytes [28]. Additionally, K We also found that ICH-induced upregulations of efflux can be positively regulated by ROS [14] and RNS NOX2, iNOS, and ONOO were diminished in BBG (e.g., ONOO ) [38]. In this regard, we propose that treated ICH rats, indicating that they may be downstream NOX2-mediated ONOO formation may link P2X7R and of P2X7R as previously reported [26, 29]. We next explored NLRP3 inflammasome activation. theroleofONOO in NLRP3 inflammasome activation. P2X7R activation in the ICH brain is a novel finding As a highly active and relatively specific ONOO decom- of this study. ATP is released in large quantities follow- position catalyst, FeTPPS exerts neuroprotective effects in ing any kind of cell injury [39], and cell death occurs 3 many CNS disease models [20, 50] because it catalyzes per- to 6 h after collagenase- induced ICH [40]. Elevated oxynitrite to become a harmless nitrate [51]. Our results extracellular ATP is necessary for P2X7R activation [13]. show that overexpression of NLRP3 inflammasome compo- In the present study, increased P2X7R was observed as nents and mature IL-1β/IL-18 was reduced by FeTPPS. early as 6 h after ICH in the perihematomal tissue along These findings suggest that ONOO maybeinvolvedin with expression of the microglia marker Iba-1, suggest- inflammasome activation following ICH. Consistent with ing that P2X7R may act as a signal in the process of our results, an early report found that FeTPPS inhibited microglia activation as previously reported [41]. nigericin-induced caspase-1 activation and IL-1β secretion Microglia are believed to be one of the first non- in human monocytes [28]. neuronal cells to respond to brain injury [42], much earlier Although we did not attempt to explore the exact mech- than neutrophil invasion [43]. Activated microglia then re- anisms of ONOO in modulating NLRP3 inflammasome lease pro-inflammatory cytokines, such as IL-1β [44] and activation, ONOO may act as a key mediator for inflam- IL-18 [45], which recruit leukocytes especially neutrophils. masome activation through several mechanisms. Firstly, The infiltrated neutrophils amplify neuroinflammation by direct oxidation of mitochondria and the release of mito- releasing and expressing neurotoxic factors or even by chondrial DNA (mtDNA) activate the NLRP3 inflamma- stimulating microglia to secrete neurotoxic factors, creat- some; a recent study showed that oxidized mtDNA ing a vicious cycle [46]. In the present study, upregulated released into the cytosol from injured mitochondria could P2X7R was closely associated with increased NLRP3/ASC/ bind to and activate the NLRP3 inflammasome [52]. caspase-1 inflammasome expression; siRNA interference ONOO is a potent ROS with strong abilities to oxidize or pharmacological blockage of P2X7R impaired inflam- and nitrate proteins, lipids, and DNA. Mitochondria are a masome activation and IL-1β/IL-18 secretion, which had a primary target for peroxynitrite [18]. Secondly, nitration pronounced neuroprotective effect. Moreover, infiltrated can inactivate thioredoxin (Trx), leading to the Feng et al. Journal of Neuroinflammation (2015) 12:190 Page 15 of 17 dissociation of thioredoxin interaction protein (TXNIP) Additional file 2: No evident immunostaining for P2X7R was found from Trx. Zhou et al. [53] reported that TXNIP could dis- in astrocytes and neurons. Double immunostaining showed that P2X7R was not expressed in GFAP positive astrocytes (A). Double sociate from Trx in an ROS-sensitive way, allowing it to immunostaining showed that P2X7R was not expressed in NeuN positive bind and activate NLRP3. Thirdly, ONOO may mediate neurons (B). (TIFF 4781 kb) + + K efflux and then activate NLRP3. K efflux is a well- Additional file 3: FeTPPS treatment had no influence on P2X7R characterized activator for NLRP3 inflammasome, and expressions. Western blot (A,B) showed that FeTPPS did not affect the * ** − + protein expressions of P2X7R. P < 0.05, P < 0.01 (TIFF 494 kb) ONOO is a positive modulator for K efflux [38]. There- fore, ONOO may be responsible for NLRP3 inflamma- Abbreviations some activation. 3-NT: 3-nitrotyrosine; ASC: apoptosis-associated speck-like protein containing Of translational significance, we investigated the effect a CARD; ATP: adenosine triphosphate; BBG: brilliant blue G; CNS: central of BBG in an ICH rat model. Previous studies have dem- nervous system; ICH: intracerebral hemorrhage; IL: interleukin; iNOS: inducible nitric oxide synthase; LPS: lipopolysaccharide; mNSS: modified Neurological onstrated a neuroprotective effect of BBG in many acute Severity Score; MPO: myeloperoxidase; mtDNA: mitochondrial DNA; CNS diseases such as ischemic stroke [54], subarachnoid NLRP3: NLR family, pyrin domain-containing 3; NO: nitric oxide; − − hemorrhage [6], traumatic brain injury [55], and spinal NOX2: NADPH oxidase 2; O : superoxide anion; ONOO : peroxynitrite; P2X7R: purinergic 2X7 receptor; RNS: reactive nitrogen species; ROS: reactive cord injury [56]. BBG is a derivative of the widely used oxygen species; SD: Sprague–Dawley; Sham: sham-operated animals; and Food and Drug Administration-approved food addi- siRNA: small interfering RNA; TUNEL: terminal deoxynucleotidyl transferase- tive FD&C Blue number 1 [57]. With its low toxicity and mediated dUTP nick 3′-end labeling. high selectivity, BBG is considered to be an attractive drug Competing interests for CNS diseases [56]. We found that BBG treatment The authors declare that they have no competing interests. inhibited the inflammatory response via the P2X7R/ Authors’ contributions NLRP3 axis following ICH, and this was associated with Conceived and designed the experiments: YZC and LF. Performed the significantly improved neurological function and less brain experiments: LF. Analyzed the data: YZC and LF. Wrote the paper: LF. Paper edema. revision: YZC. RD, ZHF, SY, XQD, and JZ provided experimental technical support and assisted in completing the study at different stages. All authors Several potential limitations deserve mention. Firstly, read and approved the final manuscript. although we employed the most commonly used ICH model and injected sterile-filtered collagenase, the extra- Acknowledgements This study was supported by the National Natural Science Foundation of inflammatory responses induced by the collagenase itself China (Nos. 81271314 and 30500526), Natural Science Foundation of seemed unavoidable. Secondly, our observation period Guangdong (No. 5300468), Special Project on the Integration of Industry, was 72 h but it appears that ASC and caspase-1 activa- Education and Research of Guangdong Province and Ministry of Education (No. 2012B091100154), Natural Science Foundation of Guangdong tion may undergo a second peak at 72 h; we speculate (No.2014A030313346), and The Guangdong Provincial Clinical Medical Centre that other signaling pathways may be involved in ASC For Neurosurgery (No. 2013B020400005). and caspase-1 activation in a later phase. Finally, al- Author details though it was significant, the effect of P2X7R siRNA and The National Key Clinical Specialty, The Engineering Technology Research BBG on changes in brain water content and neurobehav- Center of Education Ministry of China, Guangdong Provincial Key Laboratory ioral scores was relatively small. Thus, further experi- on Brain Function Repair and Regeneration, Department of Neurosurgery, Zhujiang Hospital, Southern Medical University, Guangzhou 510282, China. ments are necessary to address these problems. Department of Neurosurgery, Jingmen No. 1 People’s Hospital, Jingmen 448000Hubei, China. Department of Neurosurgery, The Fifth Affiliated Hospital of Southern Medical University, Guangzhou 510900, China. Conclusion Department of Neurosurgery, Gaoqing Campus of Central Hospital of Zibo, In summary, our results indicate that P2X7R contributes Gaoqing People’s Hospital, Gaoqing, Zibo 256300 Shandong, China. Department of Neurosurgery, 999 Brain Hospital, Jinan University, to NLRP3 inflammasome activation and subsequent IL- Guangzhou 510510 Guangdong, China. 1β/IL-18 release to drive brain inflammation and neuronal damage in an ICH rat model. NOX2/iNOS-dependent Received: 18 June 2015 Accepted: 5 October 2015 ONOO formation, a potential downstream signaling component of P2X7R, may be a key trigger of NLRP3 References inflammasome activation. Thus, inhibition of P2X7R or 1. Keep RF, Hua Y, Xi G. Intracerebral haemorrhage: mechanisms of injury and therapeutic targets. Lancet Neurol. 2012;11:720–31. ONOO could be a potential therapeutic target for sec- 2. Ma Q, Chen S, Hu Q, Feng H, Zhang JH, Tang J. NLRP3 inflammasome ondary brain injury accompanying ICH. contributes to inflammation after intracerebral hemorrhage. Ann Neurol. 2014;75:209–19. 3. Wang YC, Zhou Y, Fang H, Lin S, Wang PF, Xiong RP, et al. 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P2X7R blockade prevents NLRP3 inflammasome activation and brain injury in a rat model of intracerebral hemorrhage: involvement of peroxynitrite

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Springer Journals
Copyright
Copyright © 2015 by Feng et al.
Subject
Biomedicine; Neurosciences; Neurology; Neurobiology; Immunology
eISSN
1742-2094
DOI
10.1186/s12974-015-0409-2
pmid
26475134
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Abstract

Background: The NLR family, pyrin domain-containing 3 (NLRP3) inflammasome plays a key role in intracerebral hemorrhage (ICH)-induced inflammatory injury, and the purinergic 2X7 receptor (P2X7R) is upstream of NLRP3 activation. This study aimed to investigate how P2X7R functions in ICH-induced inflammatory injury and how the receptor interacts with the NLRP3 inflammasome. Methods: Rats were treated with P2X7R small interfering RNA (siRNA) 24 h before undergoing collagenase-induced ICH. A selective P2X7R inhibitor (blue brilliant G, BBG) or a peroxynitrite (ONOO ) decomposition catalyst (5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrinato iron(III) [FeTPPS]) was injected 30 min after ICH. Brain water content, hemorrhagic lesion volume, and neurological deficits were evaluated, and western blot, immunofluorescence, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) were carried out. Results: Striatal P2X7R and NLRP3 inflammasomes were activated after ICH. Gene silencing of P2X7R suppressed NLRP3 inflammasome activation and interleukin (IL)-1β/IL-18 release and significantly ameliorated brain edema and phox neurological deficits. Additionally, enhanced NADPH oxidase 2 (NOX2, gp91 ) and inducible nitric oxide synthase (iNOS), as well as their cytotoxic product (ONOO ) were markedly attenuated by BBG treatment following ICH. This was accompanied by downregulations of the inflammasome components, IL-1β/IL-18 and myeloperoxidase (MPO, a neutrophil marker). Most importantly, inflammasome activation and IL-1β/IL-18 release were significantly inhibited by ONOO decomposition with FeTPPS. Conclusions: Our findings implicate that P2X7R exacerbated inflammatory progression and brain damage in ICH rats possibly via NLRP3 inflammasome-dependent IL-1β/IL-18 release and neutrophil infiltration. ONOO , a potential downstream signaling molecule of P2X7R, may play a critical role in triggering NLRP3 inflammasome activation. Keyword: P2X7R, NLRP3, Peroxynitrite, Intracerebral hemorrhage, NOX2, IL-1β Background ICH brain damage. There is an urgent need to clarify the Spontaneous intracerebral hemorrhage (ICH) is a devas- pathophysiology of this disease to identify effective tating stroke subtype, with high morbidity and mortality therapies. [1]. Unfortunately, no satisfactory pharmacologic treat- Accumulating evidence suggests that innate immunity ments have been found for clinical practice, mainly due to and inflammatory responses are involved in ICH-induced a lack of knowledge underlying the mechanisms of post- secondary brain injury [1–3]. The intracellular Nod-like receptors have recently been shown to play a critical role * Correspondence: [email protected] in the process of innate immunity and inflammatory The National Key Clinical Specialty, The Engineering Technology Research responses [4]. The NLR family, pyrin domain-containing 3 Center of Education Ministry of China, Guangdong Provincial Key Laboratory (NLRP3) inflammasome, the best characterized member on Brain Function Repair and Regeneration, Department of Neurosurgery, Zhujiang Hospital, Southern Medical University, Guangzhou 510282, China of Nod-like receptor family, is a multiprotein complex that Full list of author information is available at the end of the article © 2015 Feng et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Feng et al. Journal of Neuroinflammation (2015) 12:190 Page 2 of 17 contains the adaptor protein apoptosis-associated speck- In the case of ICH, both NADPH oxidase 2 (NOX2) and like protein containing a CARD (ASC) and the effector inducible nitric oxide synthase (iNOS) have been reported caspase-1. Once activated, caspase-1 can cleave the pro to contribute to brain injury; knockout mice exhibit less forms of interleukin (IL)-1β and IL-18 into their mature brain edema and cell death than wild-type controls follow- and active forms, which leads to the recruitment and acti- ing ICH [15, 16]. Superoxide anion (O ) and nitric oxide vation of other immune cells, such as neutrophils [5]. In (NO), released through NOX and iNOS in activated this regard, evidence indicates that the NLRP3 inflamma- microglia, act as devastating pro-inflammatory mediators some plays a pivotal role in ICH [2] and other central ner- in CNS diseases [17]. More importantly, peroxynitrite vous system (CNS) conditions [6–11], but the precise (ONOO ), the product of a diffusion-controlled reaction mechanisms associated with inflammasome activation of NO with O , is a more potent oxidant species and is continue to be debated. involved in the pathologies of ischemic stroke, neuro- The role of ATP-gated transmembrane cation channel trauma, and neurodegenerative diseases [18–20]. We pre- P2X7R in the signaling cascade has received particular viously demonstrated that abundant ONOO formed in a attention due to its widespread involvement as a key hemoglobin (Hb)-induced ICH rat model [21], but the regulatory element of NLRP3 inflammasome activation exact mechanisms of ONOO in brain injury after ICH [12]. A growing number of studies have demonstrated have not been fully characterized. Besides its ability to the important pathophysiological functions of P2X7R in oxidize or nitrate proteins, lipids, and DNA, ONOO can CNS disorders, including ischemic stroke, subarachnoid also lead to destructive pathological consequences by trig- hemorrhage, neurotrauma, epilepsy, neuropathic pain, gering the activation of several biochemical pathways en- and neurodegenerative illnesses [13, 14]. However, the gaged in the development of neuroinflammation and IL- specific role of P2X7R in ICH has not yet been estab- 1β production [22–24]. However, the precise link between lished, and the interaction between P2X7R and the ONOO formation and IL-1β secretion in ICH is unclear. NLRP3 inflammasome in the development of ICH- The P2X7R acts as an upstream molecule of NOX2 induced brain injury remains unclear. activation signaling in many in vivo and in vitro disease Fig. 1 Expression profile of P2X7R and its cellular location after collagenase-induced intracerebral hemorrhage (ICH). Western blot analysis (a) for the time course of P2X7R expression in the ipsilateral hemisphere of Sham and ICH rats within 72 h. Quantification of P2X7R (b)expression is shown, n = 4 rats per group and time point. Confocal images (c) of double immunofluorescence for P2X7R expression in Iba-1-positive microglia 24 h * ** following ICH, n = 6 rats per group. Scale bar = 12.5 μm. Data represent means ± SD. P < 0.05 vs. Sham, P <0.01 vs. Sham. GAPDH glyceraldehyde 3-phosphate dehydrogenase, ICH intracerebral hemorrhage Feng et al. Journal of Neuroinflammation (2015) 12:190 Page 3 of 17 models [25–27]. NOX2-mediated oxidative stress was re- ONOO is involved in P2X7R-mediated NLRP3 inflam- cently proposed to be responsible for activation of the masome activation, we used an ONOO decomposition NLRP3 inflammasome and subsequent neurovascular catalyst in vivo and measured the expression levels of damage in ischemic stroke [7]. Notably, P2X7R-dependent P2X7R and NLRP3 inflammasome components. NADPH oxidase activation and ONOO formation play key roles in caspase-1 and IL-1β processing in endotoxin- Materials and methods primed human monocytes [28]. In an animal model of Animals lipopolysaccharide (LPS)-induced striatum injury, acti- Sprague–Dawley (SD) male rats weighing 280–320 g vated P2X7R in microglia was associated with increased were purchased from the Animal Experiment Center of iNOS and 3-nitrotyrosine (3-NT, a reliable marker of Southern Medical University (Guangzhou, China). All ONOO ), and this was reversed by the P2X7R antagonist experimental procedures and animal care were approved oxidized ATP (oxATP) [29]. Despite this knowledge, the by the Southern Medical University Ethics Committee. potential roles of P2X7R and NLRP3 inflammasomes and NOX2/iNOS-dependent ONOO formation in the devel- opment of ICH-induced brain damage remain to be ICH model clarified. After anesthetization (0.3 ml/100 g, 10 % chloral hy- We hypothesized that ONOO , formed from NOX2- drate, Sigma-Aldrich, St. Louis, MO, USA), an incision derived O and iNOS-derived NO, may be involved in was made on the skin along the sagittal midline to ex- transducing P2X7R-mediated IL-1β/IL-18 production and pose the skull. A burr hole (1 mm) was drilled 3 mm lat- brain injury via NLRP3 inflammasome activation after eral and 1 mm anterior to the bregma, then a 30-gauge ICH. We first investigated the expression profiles of needle was inserted through the burr hole into the stri- P2X7R and the NLRP3 inflammasome components. Next, atum (6 mm ventral from the skull surface), and ICH a mixed small interfering (si) RNA was applied to knock was induced by stereotaxic infusion of bacterial collage- down P2X7R in vivo, and alterations in NLRP3 inflamma- nase VII-S (0.25U in 1.0 μl sterile saline, Sigma-Aldrich) some components and functional outcomes were mea- over a 10-min period. In the Sham group, rats were sub- sured. We then explored the therapeutic effect of the jected to only a needle insertion as described above. The selective P2X7R antagonist, blue brilliant G (BBG). Add- needle was kept in situ for another 10 min to prevent itionally, we observed iNOS- and NOX2-dependent for- backflow and then slowly removed. The craniotomies mation of ONOO and their alterations in ICH rats were sealed with bone wax. Rats were allowed to recover following BBG treatment. Finally, to determine whether in separate cages with free access to food and water. Fig. 2 Expression profiles of NLRP3, ASC, and caspase-1 p20 subunit after ICH. Western blot analysis (a)for thetimecourse expressions of NLRP3 (b), ASC (c), and caspase-1 p20 subunit (d) in the ipsilateral hemisphere of Sham and ICH rats within 72 h. Quantification of NLRP3, ASC, and caspase-1 p20 subunit expression is shown, respectively, n = 4 rats per group and time point. Data represent means ± SD. P <0.05 ** vs. Sham, P < 0.01 vs. Sham. ASC adaptor protein apoptosis-associated speck-like protein containing a CARD, GAPDH glyceraldehyde 3-phosphate dehydrogenase, ICH intracerebral hemorrhage, NLRP3 pyrin domain-containing 3 Feng et al. Journal of Neuroinflammation (2015) 12:190 Page 4 of 17 Experimental protocol the 72-h study, BBG was administered daily by intraperi- Four separate experiments were conducted as shown in toneal injection. Western blot, hematoxylin and eosin Additional file 1. (H&E) staining, immunofluorescence (IF), and terminal deoxynucleotidyl transferase dUTP nick end labeling Experiment 1 (TUNEL) were measured 24 h after ICH induction; Thirty-six rats were divided into six groups (Sham, and mNSS and brain water content were detected at both 24 6, 12, 24, 48, and 72 h after ICH). The expression levels and 72 h. of P2X7R, NLRP3, ASC, and caspase-1 were detected by western blot. The tissue for immunofluorescence (IF) Experiment 4 was collected 24 h after ICH induction. Thirty-three rats were randomized into three groups: Sham, Vehicle (ICH + saline, intraperitoneal injection), Experiment 2 and FeTPPS (30 mg/kg). Western blotting was performed Eighty-eight rats were randomized into four groups: 24 h after ICH induction. Sham, Vehicle (ICH + saline, intracerebroventricular in- jection), Scramble small interfering RNA (siRNA) (1000 siRNA and drug delivery pmol, 2 μl, ICH + scramble siRNA), and P2X7R siRNA BBG (Sigma-Aldrich) was diluted at 50 and 100 mg/kg (1000 pmol, 2 μl, ICH + P2X7R siRNA). siRNA silencing in Vehicle (saline) solution. Rats were treated intraperi- efficacy was assessed by western blot. Brain water con- toneally with either BBG or Vehicle immediately after tent and modified Neurological Severity Score (mNSS) ICH induction and at 12, 36, and 60 h. were also measured. For in vivo siRNA administration, P2X7R siRNA or non-silencing RNA (Sigma-Aldrich) was applied 24 h Experiment 3 before ICH by intracerebroventricular injection as previ- One hundred and thirty-two rats were randomized into ously described [2]. A cranial burr hole (1 mm) was four groups: Sham, Vehicle (ICH + saline, intraperitoneal drilled, and a 30-gauge needle was inserted stereotaxic- injection), BBG (50 mg/kg), and BBG (100 mg/kg). For ally into the right lateral ventricle. To improve the gene Fig. 3 Effects of P2X7R small interfering RNA (siRNA) treatment in ICH rats. RT-PCR of P2X7R after siRNA treatment 24 h following ICH, n = 6 rats per group (a). Western blot assay and quantification of P2X7R (b) after siRNA treatment 24 h following ICH, n = 4 rats per group. Brain edema (c)at 24 ** @ h after ICH, n = 6 rats per group. mNSS (d) at 24 and 72 h after ICH, n = 6 rats per group. Data represent means ± SD. P < 0.01 vs. Sham, P <0.05 vs. Vehicle, P < 0.05 vs. ICH + Scramble siRNA. GAPDH glyceraldehyde 3-phosphate dehydrogenase, mNSS modified Neurological Severity Score, siRNA small interfering RNA Feng et al. Journal of Neuroinflammation (2015) 12:190 Page 5 of 17 silence efficiency, two different sequences targeting RT-PCR P2X7R siRNA were combined (a)sense:5′-CAGUGAA Rats were anesthetized and decapitated. Lesioned tissues UGAGUACUACUA-3′;antisense:5′-UAGUAGUACU (about 40 mg) were obtained, and total RNA was ex- CAUUCACUG-3′;(b)sense:5′-CUCUUGAGGAGCGC tracted from the tissue with GeneJET™ RNA Purification CGAAA-3′;antisense:5′-UUUCGGCGCUCCUCAAGA Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA). G-3′. siRNA was dissolved in RNA free water. Scrambled RNA (1 μg) was reverse-transcribed to cDNA with high control siRNA (1000 pmol, 2 μl), P2X7R siRNA (1000 capacity (Life Technologies, Carlsbad, CA, USA). RT-PCR pmol, 2 μl), or RNA free water (2 μl) was delivered intra- was performed in an ABI Prism 7500 sequence detection cerebroventricularly for 2 min. The needle was left in system (Applied Biosystems, Foster City, CA, USA) using place for an additional 10 min after injection and then specific primers designed from known sequences. GAPDH slowly withdrawn. was used as an endogenous control gene. Sequence- FeTPPS (Millipore, Billerica, MA, USA) was diluted specific primers for P2X7R, NLRP3, and GADPH were as to 30 mg/kg in Vehicle (saline) solution. Rats were follows: treated intraperitoneally with either FeTPPS or Vehicle P2X7R, 5′-CTACTCTTCGGTGGGGGCTT-3′ immediately and at 12 h after ICH induction. The (forward primer), FeTPPS dose was selected based on our previous re- P2X7R, 5′-CTCTGGATCCGGGTGACTTT-3′ ports that showed that it is effectively protected against (reverse primer); injury [30]. NLRP3, 5′-CTGCATGCCGTATCTGGTTG-3′ Fig. 4 Effects of P2X7R siRNA on NLRP3 inflammasome activation and IL-1β/IL-18 maturation after ICH. RT-PCR of NLRP3 after P2X7R siRNA treatment 24 h following ICH (a), n = 6 rats per group. Western blot assay (b) and quantification of NLRP3 (c), ASC (d), caspase-1 p20 subunit (e), IL-1β (f), and IL-18 (g) after P2X7R siRNA treatment 24 h following ICH, n = 4 rats per group. Data represent means ± SD. ** @ # P <0.01 vs. Sham, P <0.05 vs. Vehicle, P < 0.05 vs. ICH + Scramble siRNA. ASC adaptor protein apoptosis-associated speck-like protein containing a CARD, GAPDH glyceraldehyde 3-phosphate dehydrogenase, ICH intracerebral hemorrhage, IL interleukin, NLRP3 The NLR family, pyrin domain-containing 3 Feng et al. Journal of Neuroinflammation (2015) 12:190 Page 6 of 17 (forward primer), Western blot NLRP3, 5′-GCTGAGCAAGCTAAAGGCTTC-3′ Western blotting was performed as described previously (reverse primer); [30]. The following primary antibodies were used: rabbit GAPDH, 5′-AGACAGCCGCATCTTCTTGT-3′ polyclonal anti-P2X7R (1:1000, Alomone Labs, Jerusalem, (forward primer), Israel), rabbit polyclonal anti-NLRP3 antibody (1:1000, GAPDH, 5′- TGATGGCAACAATGTCCACT-3’ Santa Cruz, Biotechnology, Santa Cruz, CA, USA), rabbit (reverse primer); polyclonal anti-ASC antibody (1:500, Abclonal, Cambridge, Fig. 5 Effects of BBG on brain edema in ICH rats. BBG treatment evidently reduced brain edema at 24 (d) and at 72 h (h) after ICH. Hematoxylin and eosin (H&E) showed the hemorrhagic lesion volume alteration after BBG treatment at 24 (a, b) and at 72 h (e, f) following ICH. Tissue * ** damage after BBG treatment at 24 (c) and at 72 h (g) following ICH. n = 6 rats per group. Data represent means ± SD. P < 0.05, P < 0.01. BBG brilliant blue G, ICH intracerebral hemorrhage Feng et al. Journal of Neuroinflammation (2015) 12:190 Page 7 of 17 MA, USA), mouse monoclonal anti-caspase-1 p20 antibody vitrification, they were embedded in paraffin, and 3-μm (1:1000, Santa Cruz Biotechnology), rabbit polyclonal anti- sections were prepared. Sections were dewaxed, rehy- IL-1β antibody (1:1000, Millipore), rabbit monoclonal anti- drated, and then processed for IF and TUNEL. IL-18 antibody (1:1000, Abcam, Cambridge, UK), mouse monoclonal anti-nitrotyrosine antibody (1:1000, Abcam), Histological examination mouse monoclonal anti-iNOS antibody (1:200, Santa Cruz Coronal sections (1 mm apart) [31] were prepared accord- phox Biotechnology), mouse monoclonal anti-gp91 antibody ingly and then stained with H&E. Hemorrhagic volumes (1:2000, BD Transduction Laboratories, San Jose, CA, were calculated using Image Pro Plus 6.0 software (Media USA), and rabbit polyclonal anti-myeloperoxidase antibody Cybernetics, USA) to span the entire hematoma [32]. (MPO, 1:500, Abcam). GAPDH (1:1000, Cell Signaling Technology, Danvers, MA, USA) was employed as the IF loading control. Blot bands were quantified by densitometry Antigen retrieval was performed by heat treatment in a with ImageJ software (National Institutes of Health, microwave oven for 21 min in Tris–EDTA buffer solu- Baltimore, MD, USA). tion (0.05 mol/L Tris, 0.001 mol/L EDTA; pH 8.5). Sections were incubated for 30 min in 5 % bovine serum Paraffin section preparations albumin (BSA) and then incubated at 4 °C overnight The sections were processed as previously described [30] with primary antibodies (rabbit polyclonal anti-P2X7R, with minor modifications. After anesthetization, rats 1:500, Alomone labs; mouse monoclonal anti-3- were transcardially perfused with 200 ml saline followed Nitrotyrosine, 1:400, Abcam; rabbit polyclonal anti- by 400 ml 4 % paraformaldehyde solution. Brain tissues nitrotyrosine antibody, 1:200, Millipore; rabbit polyclonal were then removed and fixed by immersion in the same anti-MPO antibody, 1:50, Abcam; rabbit polyclonal anti- solution at 4 °C for 24 h. After dehydration and iNOS antibody, 1:40, Santa Cruz Biotechnology; rabbit Fig. 6 Effects of BBG on neuronal apoptosis and neurological outcomes in ICH rats. BBG significantly reduced the number of apoptotic neurons (a, b) 24 h following ICH, n = 6 rats per group. BBG significantly improved neurological deficits (c) at 24 and at 72 h after ICH, n = 6 rats per * ** group. Scale bar = 50 μm. Data represent means ± SD. P < 0.05, P < 0.01. BBG brilliant blue G Feng et al. Journal of Neuroinflammation (2015) 12:190 Page 8 of 17 polyclonal anti-Iba-1 antibody, 1:600, WAKO, Osaka, Brain water content measurement Japan; goat polyclonal anti-Iba-1 antibody, 1:300, Abcam; Brain edema was evaluated by a common wet/dry phox mouse monoclonal gp91 antibody, 1:400, BD Trans- method as previously described [33]. Briefly, at 24 or 72 duction Laboratories). For double-staining experiments, h post-ICH, rats were anesthetized and decapitated. The primary antibodies were separately incubated overnight brains were removed and immediately separated into at 4 °C. After they were washed with phosphate-buffered contralateral and ipsilateral hemispheres and the cere- saline (PBS), sections were then incubated with second- bellum and wet weighed. The cerebellum was used as an ary antibodies. Images were obtained using confocal mi- internal control. Brain specimens were dried in an oven croscopes (FV10i-W, Olympus, Tokyo, Japan; LSM780, at 100 °C for 24 h to obtain the dry weight. The water Zeiss, Oberkochen, Germany). content was expressed as a percentage of the wet weight: ([wet weight] – [dry weight]) / (wet weight) × 100 %. TUNEL Behavioral testing At 24 h after ICH, TUNEL staining was performed with Behavioral tests were assessed with mNSS at 24 and 72 an in situ apoptosis detection kit (Roche, Basel, h after ICH by an investigator who was blinded to the Switzerland) according to the manufacturer’s instruction. experimental groups [34]. For NeuN and TUNEL co-staining, the sections were first labeled with a NeuN antibody (1:400, Abcam), Statistical analysis followed by TUNEL. The slides were analyzed using a Data are shown as mean ± SD. Statistical analysis was fluorescence microscope (Bx51, Olympus). performed using SPSS 13.0 (SPSS Inc., Chicago, IL, USA). Fig. 7 Effects of BBG on NLRP3 inflammasome activation and IL-1β/IL-18 maturation. RT-PCR of NLRP3 after BBG treatment 24 h following ICH, n =6 rats pergroup (a). Representative western blot (b) and therapeutic effects of BBG on P2X7R (c), NLRP3 (d), ASC (e), caspase-1 p20 subunit (f), mature IL-1β (g), and mature IL-18 (h) levels in the ipsilateral hemisphere 24 h after ICH, n = 4 rats per group. Data * ** represent means ± SD. P < 0.05, P < 0.01. ASC adaptor protein apoptosis-associated speck-like protein containing a CARD, BBG brilliant blue G, GAPDH glyceraldehyde 3-phosphate dehydrogenase, ICH intracerebral hemorrhage, IL interleukin, NLRP3 pyrin domain-containing 3 Feng et al. Journal of Neuroinflammation (2015) 12:190 Page 9 of 17 Comparison between groups was determined by Student’s t but still remained higher than those in the Sham group at tests or one-way analysis of variance (ANOVA) followed by 48 h (P < 0.05 vs. Sham) and 72 h (P < 0.05 vs. Sham). least significant difference (LSD) tests with multiple compari- sons. The statistically significant level was P <0.05. P2X7R RNA interference reduced brain water content and improved neurological outcomes Results We next explored whether P2X7R is involved in brain injury P2X7R was increased and mainly expressed in microglia following ICH. Two P2X7R siRNA mixtures were applied 24 cells following ICH h before ICH induction. Silencing efficacy by RT-PCR dem- Protein content was analyzed at different time points onstrated a significant inhibitory effect of P2X7R siRNA on after injection to investigate whether P2X7R would re- itsmRNAlevels(P<0.01) (Fig.3a).Western blot (Fig.3b) spond to collagenase-induced ICH. As shown by western showed 41.3 and 40.7 % reductions of P2X7R in the P2X7R blot (Fig. 1a, b), P2X7R levels were significantly elevated siRNA group compared with the Vehicle and Scramble at 6 h after ICH (P < 0.05 vs. Sham) and peaked at siRNA groups, respectively (both P < 0.05), at 24 h after ICH. around 24 h (P < 0.01 vs. Sham) when P2X7R levels were Brain water content in the ipsilateral hemisphere was signifi- nearly 4.5 times more than those in the Sham group. cantly increased in the Vehicle (82.56 ± 0.72 % vs. Sham, Following the peak, P2X7R levels decreased, returning 79.40 ± 0.44 %, P < 0.01) and Scramble siRNA (82.44 ± 0.75 close to baseline levels at 72 h. %vs. Sham,79.40 ±0.44%, P < 0.01) groups at 24 h post- Double immunolabeling was performed to identify the ICH, while that in P2X7R siRNA group was decreased to cell type that expresses P2X7R. The results showed that 81.39 ± 0.58 % (P < 0.05 vs. Vehicle or Scramble siRNA) P2X7R was predominantly expressed in microglia cells (Fig.3c).Consistentwiththe brainedema results, P2X7R (Fig. 1c) and not in other cell types, such as astrocytes or siRNA administration significantly ameliorated neurological neurons (Additional file 2). deficits at 24 h (8.66 ± 1.15 vs. Vehicle, 10.00 ± 1.95, P < 0.05; vs. Scramble siRNA, 10.50 ± 1.26, P<0.05) and NLRP3, ASC, and caspase-1 were upregulated after ICH 72 h (6.33 ± 0.81 vs. Vehicle, 8.10 ± 1.96, P < 0.05; vs. Scram- NLRP3 inflammasome has been proposed to be downstream ble siRNA, 8.16 ± 1.16, P < 0.05) post-ICH (Fig. 3d). of P2X7R [12]. We evaluated the expressions of NLRP3 inflammasome components by western blot (Fig. 2a). NLRP3 P2X7R RNA interference inhibited NLRP3 inflammasome (Fig.2b),ASC (Fig.2c), and cleavedcaspase-1 (Fig.2d) were activation and subsequent IL-1β/IL-18 release significantly upregulated at 6 h (P < 0.05 vs. Sham) and NLRP3 inflammasome is actively involved in brain injury reached their peak at 24 h post-ICH (P < 0.01 vs. Sham). after ICH [2]. We further clarify the role of P2X7R in Following this peak, levels of all three proteins declined NLRP3/ASC/caspase-1 activation and the subsequent Fig. 8 Effects of BBG on neutrophils infiltration after ICH. Representative photographs of immunofluorescence staining (a) for MPO (neutrophil marker)-positive cells in perihematomal area in the Sham, Vehicle, and BBG (50 mg/kg) groups at 24 h following operation, n = 6 rats per group. Representative western blot (b) and effects of BBG on MPO levels (c) at 24 h after ICH, n = 4 rats per group. Scale bar = 50 μm. Data represent * ** means ± SD. P < 0.05, P < 0.01. BBG brilliant blue G, ICH intracerebral hemorrhage Feng et al. Journal of Neuroinflammation (2015) 12:190 Page 10 of 17 processing of IL-1β/IL-18. P2X7R siRNA treatment sig- post-ICH (P < 0.01) (Fig. 5e, f), indicating that BBG could nificantly reduced NLRP3 mRNA expression (P <0.01) promote tissue reconstruction. Meanwhile, the tissue (Fig. 4a). The protein levels of NLRP3 inflammasome damage around the lesion site was evidently mitigated by component and IL-1β/IL-18 production were evidently el- BBG treatment at both 24 and 72 h (Fig. 5c, d, g, h). Con- evated in the Vehicle and Scramble siRNA groups at 24 h sistently, neurological deficits (Fig. 6c) at both 24 h (50 mg/ after ICH (P < 0.01). P2X7R siRNA treatment significantly kg, 8.83 ± 1.64 vs. Vehicle, 10.00 ± 1.95, P < 0.05; 100 mg/ suppressed caspase-1 activation and the subsequent secre- kg, 8.66 ± 1.55 vs. Vehicle, 10.00 ± 1.95, P <0.05) and 72 h tion of mature IL-1β/IL-18 (P < 0.05) (Fig. 4b–f). (50 mg/kg, 6.40 ± 1.64 vs. Vehicle, 8.10 ± 1.96, P <0.05; 100 mg/kg, 7.00 ± 1.78 vs. Vehicle, 8.10 ± 1.96, P <0.05) BBG deceased post-ICH neurological deficits, brain water after ICH were improved by BBG treatment. However, content, and neuronal apoptosis there was no difference between animals that received 50 Next, we investigated the effects of the selective P2X7R and 100 mg/kg doses of BBG treatment with regard to inhibitor, BBG. Both doses (50 and 100 mg/kg) significantly mNSS scores or brain water contents. Thus, the 50 mg/kg attenuated brain water content at 24 h (50 mg/kg, 81.76 ± dose was applied in further studies. 0.32 % vs. Vehicle, 82.54 ± 0.66 %, P < 0.05; 100 mg/kg, The number of apoptotic neurons was significantly in- 81.67 ± 0.43 % vs. Vehicle, 82.54 ± 0.66 %, P <0.05) and 72 creased at 24 h after ICH compared with the Sham h (50 mg/kg, 81.59 ± 1.15 % vs. Vehicle, 82.94 ± 1.00 %, P group (P < 0.01), and BBG treatment significantly re- < 0.05; 100 mg/kg, 81.74 ± 1.12 % vs. Vehicle, 82.94 ± 1.00 duced the number of apoptotic neurons (P < 0.01) rela- %, P < 0.05) after ICH (Fig. 5d, h). The hemorrhagic lesion tive to the Vehicle group (Fig. 6a, b). volume at 24 h post-ICH for the Vehicle and BBG groups were 91.17 ± 23.54 and 82.77 ± 21.31, respectively BBG decreased P2X7R expression, NLRP3/ASC/caspase-1 (P > 0.05) (Fig. 5a, b), indicating that BBG did not affect activation, and subsequent IL-1β/IL-18 production following bleeding. However, the hemorrhagic lesion volume for the ICH BBG group (37.79 + 15.56) was significantly decreased BBG treatment significantly reduced NLRP3 mRNA compared with the Vehicle (73.03 + 19.34) group at 72 h levels (Fig. 7a). Western blot analysis revealed that BBG Fig. 9 Effects of BBG on ICH-induced iNOS expression in the striatum. Most iNOS signals overlapped with Iba-1 positive microglia (a). Western blot (b, c) and immunofluorescence labeling (d) showing that BBG treatment significantly decreased striatal iNOS expression 24 h after ICH compared * ** with the Vehicle group. n =4(b, c)or n =6(a, d) rats per group. Scale bar = 25 μm(a)or 50 μm(d). Data represent means ± SD. P <0.05, P <0.01. BBG brilliant blue G, GAPDH glyceraldehyde 3-phosphate dehydrogenase, ICH intracerebral hemorrhage, iNOS inducible nitric oxide synthase Feng et al. Journal of Neuroinflammation (2015) 12:190 Page 11 of 17 (50 mg/kg) treatment attenuated the expressions of compared with the Sham group (P <0.01 vs. Sham). P2X7R (P < 0.05 vs. Vehicle), NLRP3 (P < 0.05 vs. Ve- BBG (50 mg/kg) significantly suppressed MPO ex- hicle), ASC (P < 0.01 vs. Vehicle), and cleaved caspase-1 pression compared to the Vehicle group (P <0.05 vs. (P < 0.05 vs. Vehicle). Furthermore, the levels of mature Vehicle). IL-1β (P < 0.05 vs. Vehicle) and IL-18 (P < 0.05 vs. Vehicle) were distinctly reduced after BBG treatment (Fig. 7b–h). BBG suppressed ICH-induced iNOS expression iNOS is upregulated in both blood infusion and collagenase- induced ICH rat models [31, 35]. The role of P2X7R in BBG reduced neutrophils infiltration after ICH. iNOS signaling was next investigated using IF and western We detected MPO levels in brain tissue by IF (Fig. 8a) blot analysis. iNOS expression was weak in Sham-operated and western blot (Fig. 8b, c) at 24 h following ICH rats but was dramatically elevated 24 h after ICH induction to determine the effect of P2X7R/NLRP3 inflamma- (P < 0.01 vs. Sham). To further trace the source of iNOS, some axis activation on neutrophil infiltration. Striatal double IF was performed and found that iNOS was mainly MPO levels were evidently increased following ICH expressed in Iba-1-positive microglia (Fig. 9a). Western phox phox phox Fig. 10 Effects of BBG on ICH-induced gp91 expression in the striatum. Gp91 significantly colocalized with Iba-1 (a). Most gp91 signals overlapped with iNOS (b). Western blot (c, d) and immunofluorescence labeling (e) showing that BBG treatment significantly reduced the striatal phox gp91 expression 24 h after ICH compared with the Vehicle group. n =4(d)or n =6 (e) rats per group. Scale bar = 20 μm(a)or25 μm(b)or * ** 50 μm(e). Data represent means ± SD. P < 0.05; P < 0.01. BBG brilliant blue G, GAPDH glyceraldehyde 3-phosphate dehydrogenase, ICH intracerebral hemorrhage, iNOS inducible nitric oxide synthase Feng et al. Journal of Neuroinflammation (2015) 12:190 Page 12 of 17 blots and IF showed that the enhanced iNOS levels were BBG attenuated peroxynitrite formation following ICH markedly attenuated in BBG-treated rats (P <0.05 vs. Ve- The enhancement of iNOS and NOX2 after ICH induction hicle) (Fig. 9b–d). prompted us to examine the involvement of P2X7R in ONOO formation. Double IF demonstrated a high degree BBG reduced ICH-induced NOX2 expression of colocalization with Iba-1 (Fig. 11a). Moreover, 3-NT and − phox NOX2, a primary source of O , is actively involved in gp91 expressions were almost completely overlapped ICH-induced brain injury [15]. We studied the expression (Fig. 11b), implying that peroxynitrite production is NOX2 phox of gp91 , a membrane subunit of NOX2, by IF and dependent. However, BBG treatment significantly down- phox western blotting. Double IF showed that gp91 was also regulated 3-NT overexpression (P < 0.05 vs. Vehicle) mainly expressed in Iba-1-positive areas (Fig. 10a) and (Fig. 11c–e). most overlapped with iNOS expression (Fig. 10b), imply- ing a close connection between them. Consistent with The ONOO decomposition catalyst FeTPPS inhibited phox iNOS, gp91 was significantly increased after ICH in ip- NLRP3/ASC/Caspase-1 activation and subsequent silateral hemisphere brain tissues as compared with the production of mature IL-1β/IL-18 following ICH Sham group (P < 0.01 vs. Sham) at 24 h after ICH. BBG Our findings suggested a pivotal role for microglia- phox − treatment significantly downregulated gp91 overex- expressed P2X7R in mediating ONOO formation in an pression (P < 0.05 vs. Vehicle) (Fig. 10c–e). iNOS and NOX2-dependent way, which further prompted Fig. 11 Effects of BBG on ICH-induced 3-NT expression in the striatum. 3-NT significantly colocalized with Iba-1 (a). Most 3-NT signals overlapped phox with gp91 (b). Western blot (c, d) and immunofluorescence staining (e) showing that BBG treatment significantly suppressed striatal 3-NT expression 24 h after ICH compared with the Vehicle group. n =4 (a, b, e)or n =6 (c, d) rats per group. Bar = 12.5 μm(a, b)or50 μm(e). Data * ** represent means ± SD. P < 0.05, P < 0.01. BBG brilliant blue G, GAPDH glyceraldehyde 3-phosphate dehydrogenase, ICH intracerebral hemorrhage, 3-NT 3-nitrotyrosine Feng et al. Journal of Neuroinflammation (2015) 12:190 Page 13 of 17 us to explore whether ONOO served as the key bridge However, FeTPPS had no influence on P2X7R expressions between P2X7R and NLRP3 inflammasome activation. To (Additional file 3). FeTPPS treatment significantly reduced answer this question, the ONOO decomposition catalyst NLRP3 mRNA expression. Together, these results reveal FeTPPS was applied in vivo. Firstly, FeTPPS significantly that P2X7R-dependent synthesis of ONOO may be a key reduced 3-NT levels (P < 0.01 vs. Vehicle) on western blot activator of the NLRP3 inflammasome. (Fig. 12a, b). Thereafter, the expression levels of P2X7R and NLRP3 inflammasome components were measured by western blot at 24 h following ICH. The results indicated Discussion that FeTPPS significantly downregulated the enhanced Innate immune and inflammatory responses are increas- levels of NLRP3 (P < 0.05 vs. Vehicle), ASC (P <0.05 vs. ingly recognized as important factors in the pathophysi- Vehicle), and cleaved caspase-1 (P < 0.05 vs. Vehicle) after ology of secondary brain injury following ICH. The NLRP3 ICH. Moreover, the upregulation of IL-1β/IL-18 was also inflammasome, the most characterized pattern recognition attenuated by FeTPPS (P < 0.05 vs. Vehicle) (Fig. 12c–h). receptor (PRR) in innate immune response initiation, is Fig. 12 Effects of FeTPPS on NLRP3 inflammasome activation and IL-1β/IL-18 maturation. RT-PCR of NLRP3 after FeTPPS treatment 24 h following ICH, n = 6 rats per group (a). Western blot (d, b) showed that FeTPPS significantly reduced striatal 3-NT expression 24 h after ICH compared with the Vehicle group. Western blot assay (g) and quantification of NLRP3 (c), ASC (e), caspase-1 p20 subunit (f), mature IL-1β (h), and mature * ** IL-18 (j) after FeTPPS treatment 24 h following ICH, n = 5 rats per group. Data represent means ± SD. P < 0.05, P < 0.01. ASC adaptor protein apoptosis-associated speck-like protein containing a CARD, GAPDH glyceraldehyde 3-phosphate dehydrogenase, ICH intracerebral hemorrhage, IL interleukin, NLRP3 pyrin domain-containing 3, 3-NT 3-nitrotyrosine Feng et al. Journal of Neuroinflammation (2015) 12:190 Page 14 of 17 strongly involved in ICH-induced inflammation and pro- neutrophils were also diminished in the BBG group com- duces pro-inflammatory factors such as IL-1β [2]. pared with the Vehicle group. These results indicate that Several distinct mechanisms have been proposed to ac- P2X7R may be responsible for ICH-induced inflammation, count for NLRP3 activation, including potassium (K )ef- possibly by NLRP3 inflammasome-dependent IL-1β/IL-18 flux, intracellular calcium alteration, ubiquitination, and release and subsequent neutrophil infiltration signals. In reactive oxygen species (ROS) generation [36]. It was accordance with our results, Ma and colleagues [2] found recently reported that extracellular ATP-induced P2X7R that NLRP3 mainly colocalized with microglia but not with activation could directly mediate K efflux through the other cell types, providing further support for a tight rela- hemichannel pannexin 1 to activate the NLRP3 inflamma- tionship between P2X7R and NLRP3. some [5, 28, 36]. However, pannexin 1-deficient mice do In response to microglia activation, large amounts O not show diminished caspase-1 activation [37], implying and NO are released through activated NADPH oxidase that pannexin-1 is dispensable for the assembly of and iNOS [17], both of which play pivotal roles in ICH- caspase-1 activating inflammasome complexes. This raises induced brain damage [15, 16]. In our study, enhanced phox the hypothesis that other signaling pathways may be in- gp91 in microglia largely colocalized with iNOS, volved in P2X7R-mediated NLRP3 inflammasome activa- prompting us to consider ONOO . Double IF revealed that tion or that something besides pannexin 1 is necessary for increased 3-NT was also expressed in microglia and mostly + phox P2X7R-dependent K efflux. NOX2-derived ROS after overlapped with gp91 . Consistently with this, Mander ATP release-mediated P2X7R activation is well established and Brown [47] reported that activation of NOX or iNOS in a growing body of in vitro and in vivo disease models alone was relatively harmless, but their simultaneous acti- [23–25]. Importantly, there are a number of reports sup- vation was lethal because it spurred ONOO production. porting that high ROS levels, particularly NOX2-derived Inhibition of either iNOS [48] or NOX2 [49] significantly ROS, are critical for NLRP3 inflammasome activation reduced ONOO production in CNS disease models. Col- [36]. RNS also play an important role as evidenced by the lectively, we can infer that NOX2-derived O and iNOS- − − fact that both an ONOO scavenger and NOX2 inhibitor derived NO may contribute to ONOO formation upon suppressed nigericin-induced caspase-1 activation and IL- microglia activation after ICH. 1β secretion in human monocytes [28]. Additionally, K We also found that ICH-induced upregulations of efflux can be positively regulated by ROS [14] and RNS NOX2, iNOS, and ONOO were diminished in BBG (e.g., ONOO ) [38]. In this regard, we propose that treated ICH rats, indicating that they may be downstream NOX2-mediated ONOO formation may link P2X7R and of P2X7R as previously reported [26, 29]. We next explored NLRP3 inflammasome activation. theroleofONOO in NLRP3 inflammasome activation. P2X7R activation in the ICH brain is a novel finding As a highly active and relatively specific ONOO decom- of this study. ATP is released in large quantities follow- position catalyst, FeTPPS exerts neuroprotective effects in ing any kind of cell injury [39], and cell death occurs 3 many CNS disease models [20, 50] because it catalyzes per- to 6 h after collagenase- induced ICH [40]. Elevated oxynitrite to become a harmless nitrate [51]. Our results extracellular ATP is necessary for P2X7R activation [13]. show that overexpression of NLRP3 inflammasome compo- In the present study, increased P2X7R was observed as nents and mature IL-1β/IL-18 was reduced by FeTPPS. early as 6 h after ICH in the perihematomal tissue along These findings suggest that ONOO maybeinvolvedin with expression of the microglia marker Iba-1, suggest- inflammasome activation following ICH. Consistent with ing that P2X7R may act as a signal in the process of our results, an early report found that FeTPPS inhibited microglia activation as previously reported [41]. nigericin-induced caspase-1 activation and IL-1β secretion Microglia are believed to be one of the first non- in human monocytes [28]. neuronal cells to respond to brain injury [42], much earlier Although we did not attempt to explore the exact mech- than neutrophil invasion [43]. Activated microglia then re- anisms of ONOO in modulating NLRP3 inflammasome lease pro-inflammatory cytokines, such as IL-1β [44] and activation, ONOO may act as a key mediator for inflam- IL-18 [45], which recruit leukocytes especially neutrophils. masome activation through several mechanisms. Firstly, The infiltrated neutrophils amplify neuroinflammation by direct oxidation of mitochondria and the release of mito- releasing and expressing neurotoxic factors or even by chondrial DNA (mtDNA) activate the NLRP3 inflamma- stimulating microglia to secrete neurotoxic factors, creat- some; a recent study showed that oxidized mtDNA ing a vicious cycle [46]. In the present study, upregulated released into the cytosol from injured mitochondria could P2X7R was closely associated with increased NLRP3/ASC/ bind to and activate the NLRP3 inflammasome [52]. caspase-1 inflammasome expression; siRNA interference ONOO is a potent ROS with strong abilities to oxidize or pharmacological blockage of P2X7R impaired inflam- and nitrate proteins, lipids, and DNA. Mitochondria are a masome activation and IL-1β/IL-18 secretion, which had a primary target for peroxynitrite [18]. Secondly, nitration pronounced neuroprotective effect. Moreover, infiltrated can inactivate thioredoxin (Trx), leading to the Feng et al. Journal of Neuroinflammation (2015) 12:190 Page 15 of 17 dissociation of thioredoxin interaction protein (TXNIP) Additional file 2: No evident immunostaining for P2X7R was found from Trx. Zhou et al. [53] reported that TXNIP could dis- in astrocytes and neurons. Double immunostaining showed that P2X7R was not expressed in GFAP positive astrocytes (A). Double sociate from Trx in an ROS-sensitive way, allowing it to immunostaining showed that P2X7R was not expressed in NeuN positive bind and activate NLRP3. Thirdly, ONOO may mediate neurons (B). (TIFF 4781 kb) + + K efflux and then activate NLRP3. K efflux is a well- Additional file 3: FeTPPS treatment had no influence on P2X7R characterized activator for NLRP3 inflammasome, and expressions. Western blot (A,B) showed that FeTPPS did not affect the * ** − + protein expressions of P2X7R. P < 0.05, P < 0.01 (TIFF 494 kb) ONOO is a positive modulator for K efflux [38]. There- fore, ONOO may be responsible for NLRP3 inflamma- Abbreviations some activation. 3-NT: 3-nitrotyrosine; ASC: apoptosis-associated speck-like protein containing Of translational significance, we investigated the effect a CARD; ATP: adenosine triphosphate; BBG: brilliant blue G; CNS: central of BBG in an ICH rat model. Previous studies have dem- nervous system; ICH: intracerebral hemorrhage; IL: interleukin; iNOS: inducible nitric oxide synthase; LPS: lipopolysaccharide; mNSS: modified Neurological onstrated a neuroprotective effect of BBG in many acute Severity Score; MPO: myeloperoxidase; mtDNA: mitochondrial DNA; CNS diseases such as ischemic stroke [54], subarachnoid NLRP3: NLR family, pyrin domain-containing 3; NO: nitric oxide; − − hemorrhage [6], traumatic brain injury [55], and spinal NOX2: NADPH oxidase 2; O : superoxide anion; ONOO : peroxynitrite; P2X7R: purinergic 2X7 receptor; RNS: reactive nitrogen species; ROS: reactive cord injury [56]. BBG is a derivative of the widely used oxygen species; SD: Sprague–Dawley; Sham: sham-operated animals; and Food and Drug Administration-approved food addi- siRNA: small interfering RNA; TUNEL: terminal deoxynucleotidyl transferase- tive FD&C Blue number 1 [57]. With its low toxicity and mediated dUTP nick 3′-end labeling. high selectivity, BBG is considered to be an attractive drug Competing interests for CNS diseases [56]. We found that BBG treatment The authors declare that they have no competing interests. inhibited the inflammatory response via the P2X7R/ Authors’ contributions NLRP3 axis following ICH, and this was associated with Conceived and designed the experiments: YZC and LF. Performed the significantly improved neurological function and less brain experiments: LF. Analyzed the data: YZC and LF. Wrote the paper: LF. Paper edema. revision: YZC. RD, ZHF, SY, XQD, and JZ provided experimental technical support and assisted in completing the study at different stages. All authors Several potential limitations deserve mention. Firstly, read and approved the final manuscript. although we employed the most commonly used ICH model and injected sterile-filtered collagenase, the extra- Acknowledgements This study was supported by the National Natural Science Foundation of inflammatory responses induced by the collagenase itself China (Nos. 81271314 and 30500526), Natural Science Foundation of seemed unavoidable. Secondly, our observation period Guangdong (No. 5300468), Special Project on the Integration of Industry, was 72 h but it appears that ASC and caspase-1 activa- Education and Research of Guangdong Province and Ministry of Education (No. 2012B091100154), Natural Science Foundation of Guangdong tion may undergo a second peak at 72 h; we speculate (No.2014A030313346), and The Guangdong Provincial Clinical Medical Centre that other signaling pathways may be involved in ASC For Neurosurgery (No. 2013B020400005). and caspase-1 activation in a later phase. Finally, al- Author details though it was significant, the effect of P2X7R siRNA and The National Key Clinical Specialty, The Engineering Technology Research BBG on changes in brain water content and neurobehav- Center of Education Ministry of China, Guangdong Provincial Key Laboratory ioral scores was relatively small. Thus, further experi- on Brain Function Repair and Regeneration, Department of Neurosurgery, Zhujiang Hospital, Southern Medical University, Guangzhou 510282, China. ments are necessary to address these problems. Department of Neurosurgery, Jingmen No. 1 People’s Hospital, Jingmen 448000Hubei, China. Department of Neurosurgery, The Fifth Affiliated Hospital of Southern Medical University, Guangzhou 510900, China. Conclusion Department of Neurosurgery, Gaoqing Campus of Central Hospital of Zibo, In summary, our results indicate that P2X7R contributes Gaoqing People’s Hospital, Gaoqing, Zibo 256300 Shandong, China. 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Journal of Neuroinflammation – Springer Journals

Published: Oct 17, 2015

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