title: Early activation of nSMase2/ceramide pathway in astrocytes is involved in ischemia-associated neuronal damage via inflammation in rat hippocampi.
abstract: BACKGROUND
  Ceramide accumulation is considered a contributing factor to neuronal dysfunction and damage. However, the underlying mechanisms that occur following ischemic insult are still unclear.
  
  
  METHODS
  In the present study, we established cerebral ischemia models using four-vessel occlusion and oxygen-glucose deprivation methods. The hippocampus neural cells were subjected to immunohistochemistry and immunofluorescence staining for ceramide and neutral sphingomyelinase 2 (nSMase2) levels; immunoprecipitation and immunoblot analysis for nSMase2, receptor for activated C kinase 1 (RACK1), embryonic ectoderm development (EED), p38 mitogen-activated protein kinase (p38MAPK) and phosphorylated p38MAPK expression; SMase assay for nSMase and acid sphingomyelinase (aSMase) activity; real-time reverse transcription polymerase chain reaction for cytokine expression; and Nissl, microtubule-associated protein 2 and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling staining.
  
  
  RESULTS
  We found considerable production of ceramide in astrocytes, but not in neurons, during early cerebral ischemia. This was accompanied by the induction of nSMase (but not aSMase) activity in the rat hippocampi. The inhibition of nSMase2 activity effectively reduced ceramide accumulation in astrocytes and alleviated neuronal damage to some extent. Meanwhile, the expression levels of proinflammatory cytokines, including tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β) and IL-6, were found to be upregulated, which may have played an import role in neuronal damage mediated by the nSMase2/ceramide pathway. Although enhanced binding of nSMase2 with RACK1 and EED were also observed after cerebral ischemia, nSMase2 activity was not blocked by the TNF-α receptor inhibitor through RACK1/EED signaling. p38MAPK, but not protein kinase Cζ or protein phosphatase 2B, was able to induce nSMase2 activation after ischemia. p38MAPK can be induced by A2B adenosine receptor (A2BAR) activity.
  
  
  CONCLUSIONS
  These results indicate that the inhibition of ceramide production in astrocytes by targeting A2BAR/p38MAPK/nSMase2 signaling may represent a viable approach for attenuating inflammatory responses and neuronal damage after cerebral ischemia.
authors.affiliation: Department of Biochemistry and Molecular Biology, Nanjing Medical University, Nanjing 210029, People's Republic of China. Guoj@njmu.edu.cn.
authors.forename: LiZe
authors.initials: L
authors.lastname: Gu
authors.forename: BaoSheng
authors.initials: B
authors.lastname: Huang
authors.forename: Wei
authors.initials: W
authors.lastname: Shen
authors.forename: Li
authors.initials: L
authors.lastname: Gao
authors.forename: ZhengZheng
authors.initials: Z
authors.lastname: Ding
authors.forename: HuiWen
authors.initials: H
authors.lastname: Wu
authors.forename: Jun
authors.initials: J
authors.lastname: Guo
mesh_terms: D000818:Animals; D001253:Astrocytes / Q000378:metabolism*; D002545:Brain Ischemia / Q000378:metabolism* / Q000473:pathology; D002518:Ceramides / Q000378:metabolism*; D004195:Disease Models, Animal; D005455:Fluorescent Antibody Technique; D006624:Hippocampus / Q000378:metabolism* / Q000473:pathology; D015151:Immunoblotting; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D020287:In Situ Nick-End Labeling; D007249:Inflammation / Q000378:metabolism / Q000473:pathology; D008297:Male; D009474:Neurons / Q000378:metabolism / Q000473:pathology; D051381:Rats; D017207:Rats, Sprague-Dawley; D060888:Real-Time Polymerase Chain Reaction; D015398:Signal Transduction / Q000502:physiology; D013108:Sphingomyelin Phosphodiesterase / Q000378:metabolism*
chemical_list: D002518:Ceramides; C107180:Smpd3 protein, rat; D013108:Sphingomyelin Phosphodiesterase