title: Sphingosine kinase 1/sphingosine-1-phosphate regulates the expression of interleukin-17A in activated microglia in cerebral ischemia/reperfusion.
abstract: BACKGROUND
  Microglial activation is one of the causative factors of neuroinflammation in cerebral ischemia/reperfusion (IR). Sphingosine kinase 1 (Sphk1), a key enzyme responsible for phosphorylating sphingosine into sphingosine-1-phosphate (S1P), plays an important role in the regulation of proinflammatory cytokines in activated microglia. Recent research demonstrated that S1P increased IL-17A-secretion and then worsened CNS (central nervous system) inflammation. Thus, in the present study, we sought to use microglial cells as the object of study to discuss the molecular mechanisms in Sphk1/S1P-regulated IL-17A-secretion in IR.
  
  
  METHODS
  We used immunofluorescence and confocal microscopy to detect whether Sphk1 is expressed in microglia after cerebral IR or oxygen-glucose deprivation (OGDR). Western blot analysis was used to estimate the total Sphk1 protein level at different time points after OGDR. To detect cytokine secretion in microglial supernatants in response to OGDR, we measured the concentration of IL-17A in the culture supernatants using an enzyme-linked immunosorbent assay (ELISA). To evaluate whether microglia subjected to OGDR exhibited neuronal injury, we used a commercially available terminal transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) kit to detect apoptotic neurons.
  
  
  RESULTS
  Sphk1 was expressed in microglia in response to cerebral IR or OGDR at appointed time. Pre-injection with PF-543, an inhibitor of Sphk1, before IR clearly reduced the expression of Sphk1 in microglia relative to brain IR alone. The number of TUNEL-positive neurons was also decreased in the PF-543-pretreated animals before IR compared to the animals with IR alone. When S1P was administered in OGDR microglia, IL-17A expression and neuronal apoptosis were increased compared to OGDR alone and the administration of S1P alone. ELISA further confirmed the above results. Moreover, the inhibition of Sphk1 by siRNA reduced IL-17A production and relieved neuronal apoptosis in OGDR microglia.
  
  
  CONCLUSIONS
  These results indicated that Sphk1/S1P regulates the expression of IL-17A in activated microglia, inducing neuronal apoptosis in cerebral ischemia/reperfusion. The microglial Sphk1/S1P pathway may thus be a potential therapeutic target to control neuroinflammation in brain IR.
authors.affiliation: Department of Neurology, The First Affiliated Hospital of Harbin Medical University, 23 Youzheng Str, Nangang District, Harbin, Heilongjiang Province, 150001, People's Republic of China. lvmanhua7266@163.com.
authors.forename: Manhua
authors.initials: M
authors.lastname: Lv
authors.affiliation: School of Mechatronics Engineering, Harbin Institute of Technology, Harbin, Heilongjiang Province, People's Republic of China.
authors.forename: Dayong
authors.initials: D
authors.lastname: Zhang
authors.affiliation: Department of Neurology, The First Affiliated Hospital of Harbin Medical University, 23 Youzheng Str, Nangang District, Harbin, Heilongjiang Province, 150001, People's Republic of China.
authors.forename: Dawei
authors.initials: D
authors.lastname: Dai
authors.affiliation: Department of Neurology, The First Affiliated Hospital of Harbin Medical University, 23 Youzheng Str, Nangang District, Harbin, Heilongjiang Province, 150001, People's Republic of China.
authors.forename: Wei
authors.initials: W
authors.lastname: Zhang
authors.affiliation: Department of Neurology, The First Affiliated Hospital of Harbin Medical University, 23 Youzheng Str, Nangang District, Harbin, Heilongjiang Province, 150001, People's Republic of China.
authors.forename: Liming
authors.initials: L
authors.lastname: Zhang
mesh_terms: D000818:Animals; D017209:Apoptosis; D001921:Brain / Q000378:metabolism; D002545:Brain Ischemia / Q000378:metabolism; D002478:Cells, Cultured; D005947:Glucose / Q000172:deficiency; D002534:Hypoxia, Brain / Q000378:metabolism; D020244:Infarction, Middle Cerebral Artery / Q000378:metabolism*; D020381:Interleukin-17 / Q000378:metabolism*; D008246:Lysophospholipids / Q000378:metabolism*; D008297:Male; D000432:Methanol; D017628:Microglia / Q000187:drug effects / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011759:Pyrrolidines / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000235:genetics; D017207:Rats, Sprague-Dawley; D015427:Reperfusion Injury / Q000378:metabolism*; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000378:metabolism; D013450:Sulfones / Q000494:pharmacology; D000099157:Sphingosine Kinase
chemical_list: D005947:Glucose; D020381:Interleukin-17; D008246:Lysophospholipids; D000432:Methanol; D017853:Phosphotransferases (Alcohol Group Acceptor); D011759:Pyrrolidines; D034741:RNA, Small Interfering; D013110:Sphingosine; D013450:Sulfones; D000099157:Sphingosine Kinase; C573330:PF-543; C060506:sphingosine 1-phosphate
keywords: Cerebral ischemia/reperfusion injury; IL-17A; Microglia; Neuroinflammation; Sphingosine kinase 1; Sphingosine-1-phosphate