| BACKGROUND:Spontaneous intracerebral hemorrhage (ICH) is a devastating stroke subtype, with high morbidity and mortality. Unfortunately, no satisfactory pharmacologic treatments have been found for clinical practice, mainly due to a lack of knowledge underlying the mechanisms of post-ICH brain damage. There is an urgent need to clarify the pathophysiology of this disease to identify effective therapies.Accumulating evidence suggests that innate immunity and inflammatory responses are involved in ICH-induced secondary brain injury. The intracellular Nod-like receptors have recently been shown to play a critical role in the process of innate immunity and inflammatory responses. The NLR family, pyrin domain-containing 3 (NLRP3) inflammasome, the best characterized member of Nod-like receptor family, is a multiprotein complex that contains the adaptor protein apoptosis-associated specklike protein containing a CARD (ASC) and the effector caspase-1. Once activated, caspase-1 can cleave the proforms of interleukin (IL)-1β and IL-18 into their mature and active forms, which leads to the recruitment and activation of other immune cells, such as neutrophils. In this regard, evidence indicates that the NLRP3 inflammasome plays a pivotal role in ICH and other central nervous system (CNS) conditions, but the precise mechanisms associated with inflammasome activation continue to be debated.The role of ATP-gated transmembrane cation channel P2X7R in the signaling cascade has received particular attention due to its widespread involvement as a key regulatory element of NLRP3 inflammasome activation. A growing number of studies have demonstrated the important pathophysiological functions of P2X7R in CNS disorders, including ischemic stroke, subarachnoid hemorrhage, neurotrauma, epilepsy, neuropathic pain, and neurodegenerative illnesses. However, the specific role of P2X7R in ICH has not yet been established, and the interaction between P2X7R and the NLRP3 inflammasome in the development of ICHinduced brain injury remains unclear.In the case of ICH, both NADPH oxidase 2 (NOX2) and inducible nitric oxide synthase (iNOS) have been reported to contribute to brain injury; knockout mice exhibit less brain edema and cell death than wild-type controls following ICH. Superoxide anion (O2-) and nitric oxide (NO), released through NOX and iNOS in activated microglia, act as devastating pro-inflammatory mediators in CNS diseases. More importantly, peroxynitrite (ONOO-), the product of a diffusion-controlled reaction of NO with 02-, is a more potent oxidant species and is involved in the pathologies of ischemic stroke, neurotrauma, and neurodegenerative diseases. We previously demonstrated that abundant ONOO- formed in a hemoglobin (Hb)-induced ICH rat model, but the. exact mechanisms of ONOO- in brain injury after ICH have not been fully characterized. Besides its ability to oxidize or nitrate proteins, lipids, and DNA, ONOO- can also lead to destructive pathological consequences by triggering the activation of several biochemical pathways engaged in the development of neuroinflammation and IL-1β production [22-24]. However, the precise link between ONOO- formation and IL-1β secretion in ICH is unclear.The P2X7R acts as an upstream molecule of NOX2 activation signaling in many in vivo and in vitro disease models. NOX2-mediated oxidative stress was recently proposed to be responsible for activation of the NLRP3 inflammasome and subsequent neurovascular damage in ischemic stroke. Notably, P2X7R-dependent NADPH oxidase activation and ONOO- formation play key roles in caspase-1 and IL-1β processing in endotoxinprimed human monocytes. In an animal model of lipopolysaccharide (LPS)-induced striatum injury, activated P2X7R in microglia was associated with increased iNOS and 3-nitrotyrosine (3-NT, a reliable marker of ONOO-), and this was reversed by the P2X7R antagonist oxidized ATP (oxATP). Despite this knowledge, the potential roles of P2X7R and NLRP3 inflammasomes and NOX2/iNOS-dependent ONOO- formation in the development of ICH-induced brain damage remain to be clarified.OBJECT:We hypothesized that ONOO-, formed from NOX2-derived 02- and iNOS-derived NO, may be involved in transducing P2X7R-mediated IL-1β/IL-18 production and brain injury via NLRP3 inflammasome activation after ICH. We first investigated the expression profiles of P2X7R and the NLRP3 inflammasome components. Next, a mixed small interfering (si) RNA was applied to knockdown P2X7R in vivo, and alterations in NLRP3 inflammasome components and functional outcomes were measured. We then explored the therapeutic effect of the selective P2X7R antagonist, blue brilliant G (BBG). Additionally, we observed iNOS- and NOX2-dependent formation of ONOO- and their alterations in ICH rats following BBG treatment. Finally, to determine whether ONOO- is involved in P2X7R-mediated NLRP3 inflammasome activation, we used an ONOO-decomposition catalyst in vivo and measured the expression levels of P2X7R and NLRP3 inflammasome components.METHODS:1. Experiment designation and groups(1) Thirty-six rats were divided into six groups (Sham, and 6,12,24,48, and 72 h after ICH). The expression levels of P2X7R, NLRP3, ASC, and caspase-1 were detected by western blot. The tissue for immunofluorescence (IF) was collected 24 h after ICH induction.(2) Eighty-eight rats were randomized into four groups:Sham, Vehicle (ICH+ saline, intracerebroventricular injection), Scramble small interfering RNA (siRNA) (1000 pmol,2 μl, ICH+scramble siRNA), and P2X7R siRNA (1000 pmol,2 μl, ICH +P2X7R siRNA). siRNA silencing efficacy was assessed by western blot. Brain water content and modified Neurological Severity Score (mNSS) were also measured.(3) One hundred and thirty-two rats were randomized into four groups:Sham, Vehicle (ICH+saline, intraperitoneal injection), BBG (50 mg/kg), and BBG (100 mg/kg). Brain edema was evaluated by the wet weight/dry weight method and hematoxylin and eosin (H&E) staining; Neurological deficits were blindly evaluated by mNSS; Neuronal apoptosis was evaluated by terminal deoxynucleotidyl transferase mNSS dUTP nick end labeling (TUNEL). The expression levels of P2X7R, NLRP3, ASC, and caspase-1 were detected by western blot. NOX2,iNOS and ONOO-was detected by IF.(4) Thirty-three rats were randomized into three groups:Sham, Vehicle (ICH+ saline, intraperitoneal injection), and FeTPPS (30 mg/kg). Western blotting was performed 24 h after ICH induction.2. ICH modelSprague-Dawley (SD) male rats weighing 280-320 g were prepared. ICH was induced by stereotaxic infusion of bacterial collagenase VII-S (0.25U in 1.0 μl sterile saline). In the Sham group, rats were subjected to only a needle insertion in the same way.3. RT-PCRRats were anesthetized and decapitated. Lesioned tissues (about 40 mg) were obtained and stored at -80 ℃. Total RNA was extracted from the tissue with GeneJETTM RNA Purification Kit according to the instruction. RNA (1 μg) was reverse-transcribed to cDNA with high capacity (Life Technologies, Carlsbad, CA, USA). RT-PCR was performed in an ABI Prism 7500 sequence detection system after reverse-transcribing.4. Western blot analysisRats were anesthetized and decapitated. Lesioned tissues (about 100 mg) were obtained and stored at -80 ℃. Protein concentrations were measured with the BCA Protein Assay Kit after homogenization and centrifugation. Protein samples were transferred, incubated with primary and secondary antibody, separately. Immunoblots were visualized using a ECL Plus Western Blotting Detection Reagents. Densitometry analysis was performed with the use of the ImageJ software.5. Paraffin section preparationsAfter anesthetization, rats were transcardially perfused with 200 ml saline followed by 400 ml 4%paraformaldehyde solution. Brain tissues were then removed and fixed by immersion in the same solution at 4 ℃ for 24 h. After dehydration and vitrification, they were embedded in paraffin, and 3-μm sections were prepared. Sections were dewaxed, rehydrated, and then processed for IF and TUNEL.6. Histological examination and Hemorrhagic volumes calculationCoronal sections (1 mm apart) were prepared accordingly and then stained with H&E. Hemorrhagic volumes were calculated using Image Pro Plus 6.0 software to span the entire hematoma.7. IFAntigen retrieval was performed by heat treatment in amicrowave oven for 21 min in Tris-EDTA buffer solution (0.05 mol/L Tris,0.001 mol/L EDTA; pH 8.5). Sections were incubated for 30 min in 5% bovine serum albumin (BSA) and then incubated at 4 ℃ overnight with primary antibodies. For doublestaining experiments, primary antibodies were separately incubated overnight at 4 ℃. After they were washed with phosphate-buffered saline (PBS), sections were then incubated with secondary antibodies. Images were obtained using confocal microscopes.8. TUNEL stainingTUNEL staining was performed with an in situ apoptosis detection kit (Roche, Basel, Switzerland) according to the manufacturer’s instruction. For NeuN and TUNEL co-staining, the sections were first labeled with a NeuN antibody, followed by TUNEL. The slides were analyzed using a fluorescence microscope.9. Brain water content measurementBriefly, at 24 or 72h post-ICH, rats were anesthetized and decapitated. The brains were removed and immediately separated into contralateral and ipsilateral hemispheres and the cerebellum, and wet weighed. The cerebellum was used as an internal control. Brain specimens were dried in an oven at 100℃ for 24 h to obtain the dry weight. The water content was expressed as a percentage of the wet weight: ([wet weight] — [dry weight])/(wet weight) × 100%.10. Behavioral testingBehavioral tests were assessed with mNSS at 24 and 72 h after ICH by an investigator who was blinded to the experimental groups.11. Statistical analysisData are shown as mean ± SD. Statistical analysis was performed using SPSS 13.0 (SPSS Inc, Chicago, IL, USA). Comparison between groups was determined by Student’s t tests or one-way analysis of variance (ANOVA) followed by least significant difference (LSD) tests with multiple comparisons. The statistically significant level was P<0.05.RESULTS:1. P2X7R was increased and mainly expressed in microglia cells following ICHProtein content was analyzed at different time points after injection to investigate whether P2X7R would respond to collagenase-induced ICH. As shown by western blot, P2X7R levels were significantly elevated at 6 h after ICH (P< 0.05 vs. Sham) and peaked at around 24 h (P< 0.01 vs. Sham) when P2X7R levels were nearly 4.5 times more than those in the Sham group. Following the peak, P2X7R levels decreased, returning close to baseline levels at 72 h. Double immunolabeling was performed to identify the cell type that expresses P2X7R. The results showed that P2X7R was predominantly expressed in microglia cells and not in other cell types, such as astrocytes or neurons.2. NLRP3, ASC, and caspase-1 were upregulated after ICHNLRP3 inflammasome has been proposed to be downstream of P2X7R. We evaluated the expressions of NLRP3 inflammasome components by western blot. NLRP3, ASC, and cleaved caspase-1 were significantly upregulated at 6 h (P< 0.05 vs. Sham) and reached their peak at 24 h post-ICH (P< 0.01 vs. Sham). Following this peak, levels of all three proteins declined but still remained higher than those in the Sham group at 48 h (P<0.05 vs. Sham) and 72 h (P<0.05 vs. Sham).3. P2X7R RNA interference reduced brain water content and improved neurological outcomesWe next explored whether P2X7R is involved in brain injury following ICH. Two P2X7R siRNA mixtures were applied 24 h before ICH induction. Silencing efficacy by RT-PCR demonstrated a significant inhibitory effect of P2X7R siRNA on its mRNA levels (P< 0.01). Western blot showed 41.3 and 40.7% reductions of P2X7R in the P2X7R siRNA group compared with the Vehicle and Scramble siRNA groups, respectively (both P<0.05), at 24 h after ICH. Brain water content in the ipsilateral hemisphere was significantly increased in the Vehicle (82.56 ± 0.72% vs. Sham,79.40 ± 0.44%, P< 0.01) and Scramble siRNA (82.44 ± 0.75% vs. Sham, 79.40 ± 0.44%, P< 0.01) groups at 24 h post-ICH, while that in P2X7R siRNA group was decreased to 81.39 ± 0.58%(P< 0.05 vs. Vehicle or Scramble siRNA). Consistent with the brain edema results, P2X7R siRNA administration significantly ameliorated neurological 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 72 h (6.33 ± 0.81 vs. Vehicle,8.10 ± 1.96, P< 0.05; vs. Scramble siRNA,8.16 ± 1.16, P< 0.05) post-ICH.4. P2X7R RNA interference inhibited NLRP3 inflammasome activation and subsequent IL-1β/IL-18 releaseNLRP3 inflammasome is actively involved in brain injury after ICH. We further clarify the role of P2X7R in NLRP3/ASC/caspase-1 activation and the subsequent processing of IL-1β/IL-18. P2X7R siRNA treatment significantly reduced NLRP3 mRNA expression (P< 0.01. The protein levels of NLRP3 inflammasome component and IL-1β/IL-18 production were evidently elevated in the Vehicle and Scramble siRNA groups at 24 h after ICH (P< 0.01). P2X7R siRNA treatment significantly suppressed caspase-1 activation and the subsequent secretion of mature IL-1β/IL-18(P<0.05).5. BBG deceased post-ICH neurological deficits, brain water content, and neuronal apoptosisNext, we investigated the effects of the selective P2X7R inhibitor, BBG. Both doses (50 and 100 mg/kg) significantly attenuated brain water content at 24 h (50 mg/kg,81.76 ± 0.32% vs. Vehicle,82.54 ± 0.66%, P< 0.05; 100 mg/kg,81.67 ± 0.43% vs. Vehicle,82.54 ± 0.66%, P< 0.05) and 72 h (50 mg/kg,81.59 ± 1.15% vs. Vehicle,82.94 ± 1.00%, P< 0.05; 100 mg/kg,81.74 ± 1.12% vs. Vehicle,82.94 ± 1.00%, P<0.05) after ICH. The hemorrhagic lesion volume at 24 h post-ICH for the Vehicle and BBG groups were 91.17 ± 23.54 and 82.77 ±21.31, respectively (P> 0.05), indicating that BBG did not affect bleeding. However, the hemorrhagic lesion volume for the BBG group (37.79 ± 15.56) was significantly decreased compared with the Vehicle (73.03 ± 19.34) group at 72 h post-ICH (P< 0.01), indicating that BBG could promote tissue reconstruction. Meanwhile, the tissue damage around the lesion site was evidently mitigated by BBG treatment at both 24 and 72 h. Consistently, neurological deficits at both 24 h (50 mg/kg,8.83 ± 1.64 vs. Vehicle, 10.00 ± 1.95, P< 0.05; 100 mg/kg,8.66 ± 1.55 vs. Vehicle,10.00 ± 1.95, P< 0.05) and 72 h (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) after ICH were improved by BBG treatment. However, there was no difference between animals that received 50 and 100 mg/kg doses of BBG treatment with regard to mNSS scores or brain water contents. Thus, the 50 mg/kg dose was applied in further studies. The number of apoptotic neurons was significantly increased at 24 h after ICH compared with the Sham group (P< 0.01), and BBG treatment significantly reduced the number of apoptotic neurons (P< 0.01) relative to the Vehicle group.6. BBG decreased P2X7R expression, NLRP3/ASC/caspase-1 activation, and subsequent IL-1β/IL-18 production following ICHBBG treatment significantly reduced NLRP3 mRNA levels. Western blot analysis revealed that BBG (50 mg/kg) treatment attenuated the expressions of P2X7R (P< 0.05 vs. Vehicle), NLRP3 (P< 0.05 vs. Vehicle), ASC (P< 0.01 vs. Vehicle), and cleaved caspase-1 (P< 0.05 vs. Vehicle). Furthermore, the levels of mature IL-1β (P < 0.05 vs. Vehicle) and IL-18 (P< 0.05 vs. Vehicle) were distinctly reduced after BBG treatment).7. BBG reduced neutrophils infiltration after ICH.We detected MPO levels in brain tissue by IF and western blot at 24 h following ICH to determine the effect of P2X7R/NLRP3 inflammasome axis activation on neutrophil infiltration. Striatal MPO levels were evidently increased following ICH compared with the Sham group (P<0.01 vs. Sham). BBG (50 mg/kg) significantly suppressed MPO expression compared to the Vehicle group (P< 0.05 vs. Vehicle).8. BBG suppressed ICH-induced iNOS expressioniNOS is upregulated in both blood infusion and collagenase induced ICH rat models. The role of P2X7R in iNOS signaling was next investigated using IF and western blot analysis. iNOS expression was weak in Sham-operated rats but was dramatically elevated 24 h after ICH induction (P< 0.01 vs. Sham). To further trace the source of iNOS, double IF was performed and found that iNOS was mainly expressed in Iba-1-positive microglia. Western blots and IF showed that the enhanced iNOS levels were markedly attenuated in BBG-treated rats (P< 0.05 vs. Vehicle).9. BBG reduced ICH-induced NOX2 expressionNOX2, a primary source of O2-, is actively involved in ICH-induced brain injury. We studied the expression of gp91phox, a membrane subunit of NOX2, by IF and western blotting. Double IF showed that gp91phox was also mainly expressed in Iba-1-positive areas and most overlapped with iNOS expression, implying a close connection between them. Consistent with iNOS, gp91phox was significantly increased after ICH in ipsilateral hemisphere brain tissues as compared with the Sham group (P < 0.01 vs. Sham) at 24 h after ICH. BBG treatment significantly downregulated gp91phox overexpression (p<0.05 vs. Vehicle).10. BBG attenuated peroxynitrite formation following ICHThe enhancement of iNOS and NOX2 after ICH induction prompted us to examine the involvement of P2X7R in ONOO-formation. Double IF demonstrated a high degree of colocalization with Iba-1. Moreover,3-NT and gp91phox expressions were almost completely overlapped, implying that peroxynitrite production is NOX2 dependent. However, BBG treatment significantly downregulated 3-NT overexpression (P<0.05 vs. Vehicle).11. The ONOO- decomposition catalyst FeTPPS inhibited NLRP3/ASC/Caspase-1 activation and subsequent production of mature IL-1β/IL-18 following ICHOur findings suggested a pivotal role for microgliaexpressed P2X7R in mediating ONOO-formation in an iNOS and NOX2-dependent way, which further prompted us to explore whether ONOO- served as the key bridge between P2X7R and NLRP3 inflammasome activation. To answer this question, the ONOO-decomposition catalyst FeTPPS was applied in vivo. Firstly, FeTPPS significantly reduced 3-NT levels (P< 0.01 vs. Vehicle) on western blot. Thereafter, the expression levels of P2X7R and NLRP3 inflammasome components were measured by western blot at 24 h following ICH. The results indicated that FeTPPS significantly downregulated the enhanced levels of NLRP3 (P< 0.05 vs. Vehicle), ASC (P< 0.05 vs. Vehicle), and cleaved caspase-1 (P< 0.05 vs. Vehicle) after ICH. Moreover, the upregulation of IL-1β/IL-18 was also attenuated by FeTPPS (P< 0.05 vs. Vehicle). However, FeTPPS had no influence on P2X7R expressions. FeTPPS treatment significantly reduced NLRP3 mRNA expression. Together, these results reveal that P2X7R-dependent synthesis of ONOO-may be a key activator of the NLRP3 inflammasome.CONCLUSION:In summary, our results indicate that P2X7R contributes to NLRP3 inflammasome activation and subsequent IL-1β/IL-18 release to drive brain inflammation and neuronal damage in an ICH rat model. NOX2/iNOS-dependent ONOO-formation, a potential downstream signaling component of P2X7R, may be a key trigger of NLRP3 inflammasome activation. Thus, inhibition of P2X7R or ONOO- could be a potential therapeutic target for secondary brain injury accompanying ICH. |
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