title: Anandamide induces cell death through lipid rafts in hepatic stellate cells.
abstract: OBJECTIVE
  Anandamide (AEA), the most extensively studied endocannabinoid, and its putative cannabinoid receptors, CB1 and CB2, exert a variety of physiological and pharmacological effects in chronic liver diseases, such as hyperdynamic circulation. Anandamide selectively blocks proliferation and induces cell death in hepatic stellate cells (HSC), the key cell type of liver fibrogenesis. However, its precise molecular mechanism in rat HSC has not been fully elucidated.
  
  
  METHODS
  CB1 and CB2 mRNA transcriptions were evaluated by reverse transcription polymerase chain reaction; CB1, CB2, phosphoinositide 3-kinases (PI3K) and protein kinase B (PKB) protein expressions were investigated by western blot and/or immunofluorescence. Cell death was detected by Annexin V-PE/7AAD flow cytometry, lipid raft content by confocal microscopic analysis, cell viability by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, nuclear morphological changes by Hoechst 33258 fluorochrome, and inflammatory cytokines interleukin (IL)-2 and IL-6, and tumor necrosis factor-alpha (TNF-alpha) by enzyme-linked immunosorbent assay.
  
  
  RESULTS
  CB1 and CB2 receptors were detectable in HSC. AEA caused HSC growth inhibition in a concentration-dependent manner. Furthermore, a high concentration of AEA (20 micromol/L) triggered potent cell death-induced necrosis but not apoptosis. None of these effects were blocked by CB1 or CB2 receptor antagonist, but by methyl-beta-cyclodextrin (MCD; 10 mmol/L), a cholesterol depletory agent. AEA significantly inhibited PI3K/PKB activity, and increased IL-2, IL-6 and TNF-alpha release.
  
  
  CONCLUSIONS
  These results demonstrated that AEA induced HSC necrosis through lipid rafts: a possible role of PI3K/PKB signaling pathway downregulation and inflammatory factors production. Cholesterol depletion abolished the effects of AEA on HSC necrosis.
authors.affiliation: Institute of Liver Diseases, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
authors.forename: Qiao
authors.initials: Q
authors.lastname: Yang
authors.forename: Hong Yan
authors.initials: HY
authors.lastname: Liu
authors.forename: Yao Wen
authors.initials: YW
authors.lastname: Zhang
authors.forename: Wen Jie
authors.initials: WJ
authors.lastname: Wu
authors.forename: Wang Xian
authors.initials: WX
authors.lastname: Tang
mesh_terms: D000818:Animals; D001095:Arachidonic Acids / Q000494:pharmacology*; D015153:Blotting, Western; D002460:Cell Line; D053144:Cell Nucleus Shape / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002784:Cholesterol / Q000172:deficiency; D004305:Dose-Response Relationship, Drug; D063388:Endocannabinoids; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D055166:Hepatic Stellate Cells / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D018836:Inflammation Mediators / Q000378:metabolism; D007376:Interleukin-2 / Q000378:metabolism; D015850:Interleukin-6 / Q000378:metabolism; D021962:Membrane Microdomains / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D018613:Microscopy, Confocal; D009336:Necrosis; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D053284:Polyunsaturated Alkamides / Q000494:pharmacology*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D043884:Receptor, Cannabinoid, CB1 / Q000235:genetics / Q000378:metabolism; D043885:Receptor, Cannabinoid, CB2 / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
chemical_list: D001095:Arachidonic Acids; C469526:Cnr2 protein, rat; D063388:Endocannabinoids; D018836:Inflammation Mediators; D007376:Interleukin-2; D015850:Interleukin-6; D053284:Polyunsaturated Alkamides; D012333:RNA, Messenger; D043884:Receptor, Cannabinoid, CB1; D043885:Receptor, Cannabinoid, CB2; D014409:Tumor Necrosis Factor-alpha; D002784:Cholesterol; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; C078814:anandamide