Angelica Sinensis Polysaccharide Prevents Hematopoietic Stem Cells Senescence in D-Galactose-Induced Aging Mouse Model

Abstract

Age-related regression in hematopoietic stem/progenitor cells (HSC/HPCs) limits replenishment of the blood and immune system and hence contributes to hematopoietic diseases and declined immunity. In this study, we employed D-gal-induced aging mouse model and observed the antiaging effects of Angelica Sinensis Polysaccharide (ASP), a major active ingredient in dong quai (Chinese Angelica Sinensis), on the Sca-1⁺ HSC/HPCs in vivo. ASP treatment prevents HSC/HPCs senescence with decreased AGEs levels in the serum, reduced SA-β-Gal positive cells, and promoted CFU-Mix formation in the D-gal administrated mouse. We further found that multiple mechanisms were involved: (1) ASP treatment prevented oxidative damage as total antioxidant capacity was increased and levels of reactive oxygen species (ROS), 8-OHdG, and 4-HNE were declined, (2) ASP reduced the expression of γ-H2A.X which is a DNA double strand breaks (DSBs) marker and decreased the subsequent ectopic expressions of effectors in p16^Ink4a-RB and p19^Arf-p21^Cip1/Waf senescent pathways, and (3) ASP inhibited the excessive activation of Wnt/β-catenin signaling in aged HSC/HPCs, as the expressions of β-catenin, phospho-GSK-3β, and TCF-4 were decreased, and the cyto-nuclear translocation of β-catenin was inhibited. Moreover, compared with the positive control of Vitamin E, ASP exhibited a better antiaging effect and a weaker antioxidation ability, suggesting a novel protective role of ASP in the hematopoietic system.

Figure 1

Effect of ASP on Sca-1⁺ HSC/HPCs senescence in D-gal-induced aging mice. The Sca-1+ HSC/HPCs were collected after the treatment. (a) The senescence-associated β-galactosidase (SA-β-gal) staining was carried out. The aged cells are stained in blue in the cytoplasm. Counting analysis of the stained cells was carried out. (b) The capacity of Sca-1+ HSC/HPCs to form hematopoietic progenitor colonies was evaluated by CFU-Mix culture. Counting analysis of the colonies was carried out. Different letters: P < 0.05.

Figure 2

Effect of ASP on oxidative stress and DNA damage of Sca-1⁺ HSC/HPCs in D-gal-induced aging mice. The Sca-1+ HSC/HPCs were collected after the treatment. (a) The level of ROS was determined by DCFH fluorescence through flow cytometry. (b) The protein expression analyses of 4-HNE and γH2A.X by Western blot, β-actin was used as the internal control. (c) The relative protein expression of 4-HNE. (d) The relative protein expression of γH2A.X. Different letters: P < 0.05.

Figure 3

Effect of ASP on mRNA and protein expression of p16^INK4a-Rb and p53-p21^Cip1/Waf1 of Sca-1⁺ HSC/HPCs in D-gal-induced aging mice. The Sca-1+ HSC/HPCs were collected after the treatment. (a) p16 mRNA expression analyses by qRT-PCR. (b) p21 mRNA expression by qRT-PCR. (c) The protein expression analyses of p16^INK4a, Rb, p53, and p21^Cip1/Waf1 by Western blot, β-actin was used as the internal control. (d) The relative protein expression of p16^INK4a-Rb signaling. (e) The relative protein expression of p53-p21^Cip1/Waf1 signaling. Different letters: P < 0.05.

Figure 4

Effect of ASP on β-catenin expression of Sca-1⁺ HSC/HPCs in D-gal- induced aging mice. The Sca-1+ HSC/HPCs were collected after the treatment. (a) The protein expression analyses of β-catenin by Western blot. Proteins were extracted from cellular cytoplasm and nucleus, respectively; β-actin was used as the internal control. (b) The cellular localization of β-catenin by immunofluorescence, PI (red) to visualize nucleus. (c) β-catenin mRNA expression by qRT-PCR, β-actin was used as the internal control. Different letters: P < 0.05.

Figure 5

Effect of ASP on Wnt/β-catenin signaling mediators of Sca-1⁺ HSC/HPCs in D-gal- induced aging mice. The Sca-1+ HSC/HPCs were collected after the treatment. (a) GSK-3β, Ser9-phosphorylated GSK-3β and TCF-4 protein expressions by Western blot. β-actin was used as the internal control. (b) The relative protein expression of GSK-3β. (c) The relative protein expression of Ser9-phosphorylated GSK-3β. (d) The relative protein expression of TCF-4. Different letters: P < 0.05.

Figure 6

The summary of the effects of ASP on HSCs senescence.