Two isomers of HDTIC isolated from Astragali Radix decrease the expression of p16 in 2BS cells

Background Astragafi 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 μmol/L HDTIC-1 or 1.0 μmol/L HDTIC-2 strongly delay replicaUve 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 μmol/L HDTIC-1 or 1.0 μmol/L HDTIC-2, there was a weak mRNA expression of p16 and no protein expression of pl 6 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 μmol/L H202 for 5 minutes retum to their non-senescent phenotype and continue to be confluent after incubating the damaged cells with HDTIC-1 （1.0 μmol/L ） or HDTIC-2 （10 μmol/L ） for I 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.