title: Icariin promotes self-renewal of neural stem cells: an involvement of extracellular regulated kinase signaling pathway.
abstract: OBJECTIVE
  To investigate the effects and underlying molecular mechanisms of icariin (ICA) on self-renewal and differentiation of neural stem cells (NSCs).
  
  
  METHODS
  NSCs were derived from forebrains of mice embryos by mechanical dissociation into single cell suspension. The self-renewal of NSCs was measured by neurosphere formation assay. The proliferation of NSCs was detected by water-soluble tetrazolium (WST) and 5-ethynyl-2'-deoxyuridine (EdU) incorporation assay. Protein expression of neuron-specific marker tubulin-βIII(TuJ1) and astrocyte-specific marker glial fibrillary acidic protein (GFAP) were measured by immunofluorescence and Western blotting. Using microarray, the differentially expressed genes (DEGs) were screened between NSCs with or without ICA treatment. The signaling pathways enriched by these DEGs and their role in mediating effects of ICA were analyzed.
  
  
  RESULTS
  ICA significantly promoted neurosphere formation of NSCs cultured in growth protocol in a dose-dependent manner and achieved the maximum effects at 100 nmol/L. ICA also increased optical absorbance value and EdU incorporation into nuclei of NSCs. ICA had no significant effects on the percentage of TuJ1 or GFAP-positive cells, and TuJ1 or GFAP protein expression in NSCs cultured in differentiation protocol. A total of 478 genes were found to be differentially regulated. Among signaling pathways significantly enriched by DEGs, mitogen activated protein kinase (MAPK) pathway was of interest. Blockade of extracellular signal-regulated kinase (ERK)/MAPK, other than p38/MAPK subfamily pathway partially abolished effects of ICA on neurosphere formation and EdU incorporation of NSCs.
  
  
  CONCLUSIONS
  ICA can promote the selfrenewal of NSCs at least partially through ERK/MAPK signaling pathway.
authors.affiliation: Institute of Integrated Traditional Chinese Medicine and Western Medicine, Huashan Hospital, Fudan University, Shanghai, 200040, China.
authors.forename: Jian-hua
authors.initials: JH
authors.lastname: Huang
authors.forename: Wai-jiao
authors.initials: WJ
authors.lastname: Cai
authors.forename: Xin-min
authors.initials: XM
authors.lastname: Zhang
authors.forename: Zi-yin
authors.initials: ZY
authors.lastname: Shen
mesh_terms: D000818:Animals; D002449:Cell Aggregation / Q000187:drug effects / Q000235:genetics; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics; D003857:Deoxyuridine / Q000031:analogs & derivatives / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D005260:Female; D005419:Flavonoids / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects; D020935:MAP Kinase Signaling System / Q000187:drug effects* / Q000235:genetics; D051379:Mice; D058953:Neural Stem Cells / Q000166:cytology* / Q000187:drug effects* / Q000201:enzymology
chemical_list: D005419:Flavonoids; D048049:Extracellular Signal-Regulated MAP Kinases; C031086:5-ethynyl-2'-deoxyuridine; C056599:icariin; D003857:Deoxyuridine