title: P2X7R blockade prevents NLRP3 inflammasome activation and brain injury in a rat model of intracerebral hemorrhage: involvement of peroxynitrite.
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 neurological deficits. Additionally, enhanced NADPH oxidase 2 (NOX2, gp91(phox)) 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.
authors.affiliation: The National Key Clinical Specialty, The Engineering Technology Research Center of Education Ministry of China, Guangdong Provincial Key Laboratory on Brain Function Repair and Regeneration, Department of Neurosurgery, Zhujiang Hospital, Southern Medical University, Guangzhou, 510282, China. doctor_fengliang@163.com.
authors.forename: Liang
authors.initials: L
authors.lastname: Feng
authors.affiliation: The National Key Clinical Specialty, The Engineering Technology Research Center of Education Ministry of China, Guangdong Provincial Key Laboratory on Brain Function Repair and Regeneration, Department of Neurosurgery, Zhujiang Hospital, Southern Medical University, Guangzhou, 510282, China. yizhao_chen@hotmail.com.
authors.forename: Yizhao
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Department of Neurosurgery, Jingmen No. 1 People's Hospital, Jingmen, 448000, Hubei, China. rui_ding@hotmail.com.
authors.forename: Rui
authors.initials: R
authors.lastname: Ding
authors.affiliation: Department of Neurosurgery, The Fifth Affiliated Hospital of Southern Medical University, Guangzhou, 510900, China. 2474030376@qq.com.
authors.forename: Zhenghao
authors.initials: Z
authors.lastname: Fu
authors.affiliation: Department of Neurosurgery, Gaoqing Campus of Central Hospital of Zibo, Gaoqing People's Hospital, Gaoqing, Zibo, 256300, Shandong, China. 343039756@qq.com.
authors.forename: Shuo
authors.initials: S
authors.lastname: Yang
authors.affiliation: Department of Neurosurgery, 999 Brain Hospital, Jinan University, Guangzhou, 510510, Guangdong, China. 1476494570@qq.com.
authors.forename: Xinqing
authors.initials: X
authors.lastname: Deng
authors.affiliation: The National Key Clinical Specialty, The Engineering Technology Research Center of Education Ministry of China, Guangdong Provincial Key Laboratory on Brain Function Repair and Regeneration, Department of Neurosurgery, Zhujiang Hospital, Southern Medical University, Guangzhou, 510282, China. 2405428@qq.com.
authors.forename: Jun
authors.initials: J
authors.lastname: Zeng
mesh_terms: D000818:Animals; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism; D001929:Brain Edema / Q000209:etiology; D001930:Brain Injuries / Q000188:drug therapy / Q000209:etiology* / Q000473:pathology / Q000517:prevention & control*; D053468:CARD Signaling Adaptor Proteins; D002352:Carrier Proteins / Q000378:metabolism*; D020170:Caspase 1 / Q000378:metabolism; D002543:Cerebral Hemorrhage / Q000150:complications*; D016207:Cytokines / Q000378:metabolism; D004195:Disease Models, Animal; D008297:Male; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism; D000071199:NLR Family, Pyrin Domain-Containing 3 Protein; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism; D009195:Peroxidase / Q000378:metabolism; D030421:Peroxynitrous Acid / Q000378:metabolism*; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D058486:Receptors, Purinergic P2X7 / Q000378:metabolism*; D013997:Time Factors; D015854:Up-Regulation / Q000187:drug effects / Q000502:physiology
chemical_list: D051017:Apoptosis Regulatory Proteins; D053468:CARD Signaling Adaptor Proteins; D002352:Carrier Proteins; D016207:Cytokines; D008562:Membrane Glycoproteins; D000071199:NLR Family, Pyrin Domain-Containing 3 Protein; C573346:Nlrp3 protein, rat; C498535:Pycard protein, rat; D000644:Quaternary Ammonium Compounds; D058486:Receptors, Purinergic P2X7; D030421:Peroxynitrous Acid; D009195:Peroxidase; D052247:Nitric Oxide Synthase Type II; C500593:Cybb protein, rat; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; D020170:Caspase 1; C006798:brilliant green