Nicotinamide riboside intervention alleviates hematopoietic system injury of ionizing radiation-induced premature aging mice

Abstract

Radiotherapy destroys cancer cells and inevitably harms normal human tissues, causing delayed effects of acute radiation exposure (DEARE) and accelerating the aging process in most survivors. However, effective methods for preventing premature aging induced by ionizing radiation are lacking. In this study, the premature aging mice of DEARE model was established after 6 Gy total body irradiation (TBI). Then the therapeutic effects and mechanism of nicotinamide riboside on the premature aging mice were evaluated. The results showed that 6 Gy TBI induced premature aging of the hematopoietic system in mice. Nicotinamide riboside treatment reversed aging spleen phenotypes by inhibiting cellular senescence and ameliorated serum metabolism profiles. Further results demonstrated that nicotinamide riboside supplementation alleviated the myeloid bias of hematopoietic stem cells and temporarily restored the regenerative capacity of hematopoietic stem cells probably by mitigating the reactive oxygen species activated GCN2/eIF2α/ATF4 signaling pathway. The results of this study firstly indicate that nicotinamide riboside shows potential as a DEARE therapeutic agent for radiation-exposed populations and patients who received radiotherapy.

Keywords: cellular senescence; ionizing radiation; nicotinamide riboside; radiotherapy.

FIGURE 1

NR relieved IR‐induced premature aging in mice. (a) Schematic of the experimental procedure for mice experiencing IR and NR intervention; (b) Snapshot comparison of mice from control group, IR group, and IR + NR group, respectively (n = 3); (c) Measurement of running distance on the treadmill of experimental mice (n = 4); (d) Quantification of relative NAD⁺ levels in serum (n = 3); (e) Quantification of MDA levels in serum (n = 4); (f) Enzyme activity of CAT in serum (n = 4); (g) Clustering heat map analysis of differential metabolites identified among three groups (n = 5); (h) KEGG pathway analysis of differential metabolites among three groups. Data in bar graphs were shown as mean ± SD. Statistical analyses were performed using unpaired t‐test, *p < 0.05 versus Control, **p < 0.01 versus Control. ****p < 0.0001 versus Control; ^# p < 0.05 versus IR, ^#### p < 0.0001 versus IR.

FIGURE 2

NR alleviated premature aging of the spleen. (a) The spleen index of mice (n = 5); (b) The percentages of CD4⁺ T cells, CD8⁺ T cells, and B cells in spleen (n = 4) (c) Representative images of SA‐β‐Gal staining of spleen of mice (scale bar: 50 μm); (d) Comparative statistics of SA‐β‐Gal staining of spleen (n = 3); (e, f) Protein expressions of phosphorylated p38, p38 and p16^INK4a in spleen which measured by western blotting and their quantification analysis; (g) Quantification analysis of mRNA expression of p38 and p16^INK4a (n = 3); (h, i) Protein expressions of SIRT1, p53 and p21 ^CIP1 in spleen and their quantification analysis; (j) Quantification analysis of mRNA expression of SIRT1, p53 and p21 ^CIP1 (n = 3). The results are expressed as the means ± SD. Statistical analyses were performed using unpaired t‐test, *p < 0.05 versus Control, **p < 0.01 versus Control, ***p < 0.001 versus Control; ^# p < 0.05 versus IR, ^## p < 0.01 versus IR.

FIGURE 3

NR ameliorated the imbalance of myeloid‐lymphoid differentiation. (a–d) The numbers of (a) white blood cells (WBC) and (b) red blood cells (RBC), (c) the level of hemoglobin (HGB), and (d) the percentages of lymphocyte (LY) were analyzed in the peripheral blood (n = 5); (e–g) The percentage of (e) neutrophilic granulocytes (NE), (f) monocytes (MO) and (g) eosinophils (EO) were analyzed in the peripheral blood (n = 5); (h) A representative gating strategy of EO, MO and NE in the peripheral blood analyzed by flow cytometry; (i–k) The percentages of (i) NE, (j) MO and (k) EO in the peripheral blood were analyzed by flow cytometry (n = 4). Data in bar graphs were shown as mean ± SD. Statistical analyses were performed using unpaired t‐test, *p < 0.05 versus Control, **p < 0.01 versus Control, ***p < 0.001 versus Control, ****p < 0.0001 versus Control; ^# p < 0.05 versus IR, ^## p < 0.01 versus IR, ^### p < 0.001 versus IR.

FIGURE 4

NR affected the bone marrow hematopoietic differentiation. (a) The number of BMNCs (n = 4); (b–d) The percentages of (b) T, (c) B, and (d) myeloid cells of BMNCs (n = 3); (e) Representative flow cytometric dot plots for LSK (Lin⁻Sca‐1⁺c‐Kit⁺), HPCs (Lin⁻Sca‐1⁻c‐Kit⁺), CLPs (Lin⁻Sca‐1⁻c‐Kit⁻CD127⁺ CD135⁺), CMPs (Lin⁻Sca‐1⁻c‐Kit⁺CD34⁺CD16/32⁻), GMPs (Lin⁻Sca‐1⁻c‐Kit⁺CD34⁺CD16/32⁺), and MEPs (Lin⁻Sca‐1⁻c‐Kit⁺CD34⁻CD16/32⁻) in the BM; (f) Frequencies of LSK, HPCs, CLPs, CMPs, GMPs, and MEPs which analyzed by flow cytometry (n = 4); (g) Numbers of LSK, HPCs, CLPs, CMPs, GMPs, and MEPs which analyzed by flow cytometry (n = 4); (h) Representative analysis of ROS level in LSKs and HPCs by flow cytometry; (i) The MFI of ROS level in LSKs and HPCs (n = 3). The results are expressed as the means ± SD. Statistical analyses were performed using unpaired t‐test, *p < 0.05 versus Control, ****p < 0.0001 versus Control; ^# p < 0.05 versus IR, ^## p < 0.01 versus IR.

FIGURE 5

NR attenuated the LSKs senescence. (a) Representative images of SA‐β‐Gal staining of LSKs (scale bar: 20 μm); (d) Comparative statistics of SA‐β‐Gal staining of LSKs (n = 3); (c–h) Representative analysis of (c) p‐GCN2, (e) p‐eIF2α, and (g) ATF4 in LSKs, and the MFI of (d) p‐GCN2, (f) p‐eIF2α, and (h) ATF4 in LSKs detected by flow cytometry (n = 4); (i) Representative images of p‐GCN2 and p‐eIF2α in LSKs (red, p‐GCN2; green, p‐eIF2α; blue, DAPI); (j) Representative images of ATF4 in LSKs (red, ATF4; blue, DAPI), Scale bars: 10 or 5 mm (inset). The results are expressed as the means ± SD. Statistical analyses were performed using unpaired t‐test, *p < 0.05 versus Control, **p < 0.01 versus Control; ^# p < 0.05 versus IR, ^## p < 0.01 versus IR.

FIGURE 6

NR enhanced the multilineage engraftment and reconstitution potential of HSCs. (a) Scheme of the strategy for quantifying HSCs multilineage engraftment and reconstitution potential by competitive repopulation assay. (b) Representative flow cytometry plots showing percentage of donor (CD45.1⁺) cell engraftment, donor‐derived T cells (CD45.1⁺ CD3⁺), B cells (CD45.1⁺ B220⁺), and myeloid cells (CD45.1⁺ CD11⁺/Gr‐1⁺); (c) Donor‐derived cell multilineage engraftments in peripheral blood at 4 weeks after transplantation in lethally irradiated recipients; (d) The percentage of donor‐derived T, B, and myeloid cells in peripheral blood at 4 weeks after transplantation in lethally irradiated recipients; (e) Donor‐derived cell multilineage engraftments in peripheral blood at 8 weeks after transplantation in lethally irradiated recipients; (f) The percentage of donor‐derived T, B, and myeloid cells in peripheral blood at 8 weeks after transplantation in lethally irradiated recipients. (g) Donor‐derived cell multilineage engraftments in BM at 8 weeks after transplantation in lethally irradiated recipients; (h) The percentage of donor‐derived T, B, and myeloid cells in BM at 8 weeks after transplantation in lethally irradiated recipients. Results are presented as mean ± SD (n = 3). Statistical analyses were performed using unpaired t‐test, **p < 0.01 versus Control, ***p < 0.001 versus Control, ****p < 0.0001 versus Control; ^# p < 0.05 versus IR, ^## p < 0.01 versus IR.