title: Two isomers of HDTIC isolated from Astragali Radix decrease the expression of p16 in 2BS cells.
abstract: BACKGROUND
  Astragali Radix, the root of Astragalus membranceus (Fish) Bunge Var. mongholicus (Bge), is a crude drug considered as one of the effective traditional Chinese anti-ageing material. The two isomers of 4-hydroxy-5-hydroxymethyl-[1, 3] dioxolan-2, 6'-spirane-5', 6', 7', 8'-tetrahydro-indolizine-3'-carbaldehyde (HDTIC), HDTIC-1 and HDTIC-2, were first extracted from the herb in 2002. We demonstrated previously that 0.1 micromol/L HDTIC-1 or 1.0 micromol/L HDTIC-2 strongly delay replicative senescence of human fetal lung diploid fibroblasts (2BS). In this study, we chose them to investigate their effects on the expression of senescence-associated genes to explore the mechanism of how HDTIC delays replicative senescence.
  
  
  METHODS
  The effects of HDTIC-1 and HDTIC-2 on the expression of p16 and p21 were observed in vitro by RT-PCR and Western blot. The anti-oxidative activities of the compounds were also observed by phenotype alteration after treatment with antioxidants.
  
  
  RESULTS
  There was an obvious expression of p16 in the control senescent cells. However, in the 2BS cells, after 56 population doublings (PDs) grown from PD28 in 0.1 micromol/L HDTIC-1 or 1.0 micromol/L HDTIC-2, there was a weak mRNA expression of p16 and no protein expression of p16 was observed. The expression level of p21 increased with cell ageing. Moreover, there was no difference between the expression level of p21 in the control cells and that in the same PD cells cultured with HDTIC compounds. The results also showed that 2BS cells exposed to 100 micromol/L H2O2 for 5 minutes return to their non-senescent phenotype and continue to be confluent after incubating the damaged cells with HDTIC-1 (1.0 micromol/L ) or HDTIC-2 (10 micromol/L ) for 1 hour.
  
  
  CONCLUSIONS
  Expression of p16 by 2BS cells was strongly inhibited by HDTIC compounds, which could contribute to their delayed replicative senescence by the way of p16(INK4a)/Rb/MAPK. The anti-oxidative activities of HDTIC-1 and HDTIC-2, described in this study for the first time, might be indirectly related to their inhibition of p16 expression.
authors.affiliation: Department of Medical Laboratory, Xuanwu Hospital of Capital Medical University, Beijing 100053, China. peichangwang@yahoo.com
authors.forename: Pei-chang
authors.initials: PC
authors.lastname: Wang
authors.forename: Zong-yu
authors.initials: ZY
authors.lastname: Zhang
authors.forename: Jian
authors.initials: J
authors.lastname: Zhang
authors.forename: Tan-jun
authors.initials: TJ
authors.lastname: Tong
mesh_terms: D000975:Antioxidants / Q000494:pharmacology; D027883:Astragalus Plant / Q000737:chemistry*; D002478:Cells, Cultured; D016922:Cellular Senescence / Q000187:drug effects; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000032:analysis / Q000235:genetics*; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000032:analysis / Q000235:genetics; D004148:Dioxolanes / Q000494:pharmacology*; D005260:Female; D005347:Fibroblasts / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D007212:Indolizines / Q000494:pharmacology*; D018517:Plant Roots / Q000737:chemistry*; D012333:RNA, Messenger / Q000032:analysis
chemical_list: C489703:4-hydroxy-5-hydroxymethyl-(1,3)dioxolan-2,6'-spirane-5',6',7',8'-tetrahydro-indolizine-3'-carbaldehyde; D000975:Antioxidants; C493956:CDKN1A protein, human; D019941:Cyclin-Dependent Kinase Inhibitor p16; D050759:Cyclin-Dependent Kinase Inhibitor p21; D004148:Dioxolanes; D007212:Indolizines; D012333:RNA, Messenger