Ginsenoside Rg1 enhances the resistance of hematopoietic stem/progenitor cells to radiation-induced aging in mice

Abstract

Aim: To investigate the effects of ginsenoside Rg1 on the radiation-induced aging of hematopoietic stem/progenitor cells (HSC/HPCs) in mice and the underlying mechanisms.

Methods: Male C57BL/6 mice were treated with ginsenoside Rg1 (20 mg·kg(-1)·d(-1), ip) or normal saline (NS) for 7 d, followed by exposure to 6.5 Gy X-ray total body irradiation. A sham-irradiated group was treated with NS but without irradiation. Sca-1(+) HSC/HPCs were isolated and purified from their bone marrow using MACS. DNA damage was detected on d 1. The changes of anti-oxidative activities, senescence-related markers senescence-associated β-galactosidase (SA-β-gal) and mixed colony-forming unit (CFU-mix), P16(INK4a) and P21(Cip1/Waf1) expression on d 7, and cell cycle were examined on d 1, d 3, and d 7.

Results: The irradiation caused dramatic reduction in the number of Sca-1(+) HSC/HPCs on d 1 and the number barely recovered until d 7 compared to the sham-irradiated group. The irradiation significantly decreased SOD activity, increased MDA contents and caused DNA damage in Sca-1(+) HSC/HPCs. Moreover, the irradiation significantly increased SA-β-gal staining, reduced CFU-mix forming, increased the expression of P16(INK4a) and P21(Cip1/Waf1) in the core positions of the cellular senescence signaling pathways and caused G1 phase arrest of Sca-1(+) HSC/HPCs. Administration of ginsenoside Rg1 caused small, but significant recovery in the number of Sca-1(+) HSC/HPCs on d 3 and d 7. Furthermore, ginsenoside Rg1 significantly attenuated all the irradiation-induced changes in Sca-1(+) HSC/HPCs, including oxidative stress reaction, DNA damage, senescence-related markers and cellular senescence signaling pathways and cell cycle, etc.

Conclusion: Administration of ginsenoside Rg1 enhances the resistance of HSC/HPCs to ionizing radiation-induced senescence in mice by inhibiting the oxidative stress reaction, reducing DNA damage, and regulating the cell cycle.

Figure 1

The effect of ginsenoside Rg1 on the number of Sca-1⁺ HSC/HPCs per femur of irradiated mouse (d 0 n=3, others n=10). Mice were excuted at the desired time. The femurs were collected and the Sca-1⁺ HSCs were isolated. The number of the HSC/HPCs in each group were analyzed. Data in d 0 represents cells colleceted before TBI. ^cP<0.01 vs the sham-irradiated control group. ^fP<0.01 vs the irradiated group.

Figure 2

The effect of ginsenoside Rg1 on the Sca-1⁺ HSC/HPCs senescence of irradiated mouse. The Sca-1⁺ HSC/HPCs were collected on d 7 following TBI. (A) The senescence-associated β-galactosidase (SA-β-gal) staining was carried out. The aged cells are stained in blue in the cytoplasm (arrow). (B) The the capacity of Sca-1⁺ HSC/HPCs to form hematopoietic progenitor colonies were evaluated by CFU-mix culture. (C) The correlation analysis between the percentage of SA-β-gal positive cells and the number of CFU-mix.

Figure 3

The effect of ginsenoside Rg1 on the DNA damage of Sca-1⁺ HSC/HPCs in the irradiated mice. The Sca-1⁺ HSC/HPCs in each group were collected on the d 1 following TBI. The comet assay was used to determine the DNA damage present in the cells. Enlarged images shown in the left top corner are from the framed region. (A) The sham+irradiated control group (×100). (B) The irradiated group (×100). (C) The irradiated+Rg1 group (×100).

Figure 4

The effect of ginsenoside Rg1 on the mRNA and protein expression of p16^INK4a and p21^Cip1/Waf1 in the irradiated mice. The mRNA of Sca-1⁺ HSC/HPCs (1×10⁶) from each group were collected on d 7 following TBI. Gapdh was used as the internal control. The experiments were performed three times with similar results. (A) The sham+irradiated control group. (B) The irradiated group. (C) The irradiated+Rg1 group.

Figure 5

The effect of ginsenoside Rg1 on the cell cycle distribution of Sca-1⁺ HSC/HPCs in the irradiated mice (n=10). The Sca-1⁺ HSC/HPC cells from each group were collected on d 1 (A), d 3 (B), and d 7 (C) following TBI. Cell cycle was measured by flow cytometry, and ≥2×10⁴ cells were analyzed for each sample. ^cP<0.01 vs the sham-irradiated control group. ^fP<0.05 vs the irradiated group.