A total of 90 clean C57BL/6 mice (weighing 20-25 g, 6-8 weeks old, random selection of 43 males and 47 females) were purchased from the Experimental Animal Center of Chongqing Medical University (Chongqing, China). The mice were housed in an environment with a 12-hlight/dark cycle, 40% humidity at room temperature with free access to the food and water. All experiments were approved by the Ethics Committee of the Key Laboratory of Cell Biology (Kunming, China).

The mice were divided into three groups, namely the control group, γ-ray irradiation group and Rg1 group. The mice in the control group (n=30) were intraperitoneally injected with normal saline and not exposed to γ-ray irradiation. The mice in the γ-ray irradiation group (n=30) were intraperitoneally injected with normal saline for 7 days, followed by exposure to 6.5-Gy γ-ray total-body irradiation. The γ-rays were delivered by a linear accelerator at a dose rate of 57.28 Gy/min (11). The mice in the Rg1 group (n=30) were subjected to the same processes as the γ-ray irradiation group, but with the replacement of normal saline by the same volume of Rg1 at dose of 20 mg/kg/day. The time interval between the final injection and irradiation was 24 h. The specific processes for the treatment of each group were conducted according to a previously published study (11). The Rg1 (cat. no. 060427; purity >95%) was purchased from Jilin Hongjiu Biological Technology Co., Ltd.

Prior to irradiation treatment, the mice were anesthetized by the intraperitoneal injection of 55 mg/kg pentobarbital. Following the 7 day treatment protocol, mice were sacrificed via the intraperitoneal injection of 150 mg/kg pentobarbital (Altana AG) on day 8. The Sca-1⁺ HSC/HPCs were harvested using an immunomagnetic separation method as described in a previous study (21). The harvested Sca-1⁺ HSC/HPCs were used in several experiments, including senescence-associated β-galactosidase (SA-β-Gal) cytochemical staining, flow cytometry (FCM), mixed hematopoietic progenitor cell colony-forming unit (CFU-Mix), reverse transcription-quantitative PCR (RT-qPCR) and western blot assays.

Mice were sacrificed as described above, and blood was collected from the eyeball. Blood routine tests, including white blood cell (WBC), red blood cell (RBC) and blood platelet (PLT) counts were conducted using an XE-2100 hematology analyzer (Sysmex Corporation).

Sca-1⁺ HSC/HPCs were collected on day 7 from every group. The HSC/HPCs were stained using an Senescence β-Galactosidase Staining kit (cat. no. 9860; Cell Signaling Technology, Inc.) according to the manufacturer's instructions. In brief, the purified cells (1x10⁵) were washed three times using PBS for 5 min each time, and fixed using 4% paraformaldehyde (Beyotime Institute of Biotechnology) for 10 min at room temperature. Then, the Sca-1⁺ HSC/HPCs were stained using staining solution at 37˚C for 12 h. The cells were centrifuged at 1,000 x g at room temperature for 10 min and cytospin slides were prepared. Subsequently, the slides were sheet-sealed using 70% glycerol (Sigma-Aldrich; Merck KGaA). In order to observe and calculate the percentage of blue-stained positive cells under bright field illumination, ~1x10⁴ cells were separated on a slide, and 400 cells on each slide were selected randomly and counted.

The CFU-mix assay was performed according to a previously published study (22) with a few modifications. Briefly, the cells (1x10⁴ cells/group) were incubated with DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with horse serum (Gibco; Thermo Fisher Scientific, Inc.), 2-mercaptoethanol (1x10^-4 mol/l; Sigma-Aldrich; Merck KGaA), 3% L-glutamine (Sigma-Aldrich; Merck KGaA), recombinant human erythropoietin (Kyowa Hakko Kirin China Pharmaceutical Co., Ltd.), recombinant human granulocyte macrophage colony stimulating factor (Kyowa Hakko Kirin China Pharmaceutical Co., Ltd.), interleukin-3 (IL-3; Sigma-Aldrich; Merck KGaA) and 2.7% methylcellulose. The components were mixed intensively, seeded into 96-well plates (0.2 ml/well) and then cultured at 37˚C with 5% CO₂ for 7 days. The CFU-Mix and multiple-differentiation capacities were evaluated according to the CFU-Mix numbers and percentage of Sca-1⁺ HSC/HPCs.

Sca-1⁺ HSC/HPCs were collected from all three groups and washed using PBS. Sca-1⁺ HSC/HPCs were incubated with 70% iced ethanol overnight, and washed with PBS three times (5 min/time). The Sca-1⁺ HSC/HPCs were then incubated with bovine pancreatic ribonuclease (cat. no. R2638; Sigma-Aldrich; Merck KGaA; 1 mg/ml; 100 µl) at 37˚C for 30 min. Then, the cells were stained using propidium iodide (PI; 50 µg/ml) for 30 min at room temperature in the dark. Finally, the stained cells were analyzed using FCM with a FACSVantage SE instrument (BD Biosciences). The proportion of cells in each cell cycle phase was analyzed using Cell Quest software version 3.3 (BD Biosciences).

Total cellular RNAs of Sca-1⁺ HSC/HPCs were extracted using TRIzol reagent (cat. no. 15596018; Thermo Fisher Scientific, Inc.). ABeyoRTIII First Strand cDND Synthesis kit (cat. no. D7178S; Beyotime Institute of Biotechnology) was used to synthesize cDNAs from the RNA. All processes of the reverse transcription were conducted according to the manufacturer's protocol. The synthesized cDNAs were amplified using SYBR-Green I as the fluorescent dye. RT-qPCR was performed using a Fast real-time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The following conditions were used for amplification: 94˚C for 4 min, 94˚C for 20 sec, 60˚C for 30 sec and 72˚C for 30 sec, for 35 cycles. The primers for SIRT1, SIRT3, FOXO3, SOD2 and β-actin are listed in Table I. The relative mRNA expression of the target gene was normalized to that of β-actin using the comparative threshold cycle (2^-ΔΔCq) method (23).

Proteins were isolated from Sca-1⁺ HSC/HPCs by lysing cells with lysis buffer (Beyotime Institute of Biotechnology). The concentration of isolated proteins was calculated with a BCA protein quantification kit (Beyotime Institute of Biotechnology). Equal amounts of protein (0.2 µg) were separated using 15% SDS-PAGE (Sigma-Aldrich; Thermo Fisher Scientific, Inc.) and electrotransferred onto PVDF membranes (Pierce; Thermo Fisher Scientific, Inc.). The PVDF membranes were blocked using 5% defatted milk at 4˚C overnight. Then, the PVDF membranes were incubated with rabbit anti-mouse SIRT1 polyclonal antibody (cat. no. sc-15404, 1:3,000; Santa Cruz Biotechnology, Inc.), rabbit anti-mouse SIRT3 polyclonal antibody (cat. no. sc-99143, 1:3,000; Santa Cruz Biotechnology, Inc.), rabbit anti-mouse FOXO3 polyclonal antibody (cat. no. ab47285, 1:3,000; Abcam), rabbit anti-mouse SOD2 polyclonal antibody (cat. no. sc-30080; 1:2,000; Santa Cruz Biotechnology, Inc.) and rabbit anti-mouse GAPDH polyclonal antibody (cat. no. sc-25778; 1:2,000; Santa Cruz Biotechnology, Inc.) for 2 h at room temperature. The PVDF membranes were then washed and incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (cat. no. sc-2030; 1:1,000; Santa Cruz Biotechnology, Inc.) at 37˚C for 1 h. Finally, the western blot bands were visualized using an enhanced chemiluminescence kit (Pierce; Thermo Fisher Scientific, Inc.). The western blotting density was qualified using the NIH ImageJ software version 1.46 (National Institutes of Health).

Data are presented as the mean ± standard deviation. Data were analyzed using SPSS software 11.0 (SPSS, Inc.). All data were obtained from at least three independent tests or experiments. Tukey's post hoc test was used following ANOVA to compare continuous data among multiple groups. P<0.05 was considered to indicate statistical significance.

In order to investigate Sca-1⁺ HSC/HPC-induced hematopoietic functions, the WBC, RBC and PLT levels in the peripheral blood of the mice were examined. The results indicated that the WBC, RBC and PLT counts in the γ-ray irradiation group were significantly decreased compared with those in the control group (P<0.01; Table II). Notably, Rg1 treatment significantly increased the WBC, RBC and PLT counts compared with those in the γ-ray irradiation group (P<0.05; Table II). However, the WBC, RBC and PLT counts in the Rg1 group were significantly lower compared with those in the control group (P<0.01; Table II).

SA-β-Gal is an extensively applied biomarker for aging cells (24). A decreased CFU-Mix formation capacity is also an evaluative marker for the senescence of HSCs (25). Therefore, an SA-β-Gal staining assay and CFU-Mix formation assay were performed to evaluate the effects of Rg1 on the senescence of Sca-1⁺ HSC/HPCs.

The results showed that γ-ray irradiation induced a significant increase in the percentage of SA-β-Gal stained Sca-1⁺ HSC/HPCs (P<0.01; Fig. 1A) and a significant reduction in the CFU-Mix counts of Sca-1⁺ HSC/HPCs, compared with the control group (P<0.01; Fig. 1B). In the Rg1 group, the proportion of SA-β-Gal stained HSC/HPCs was significantly decreased (Fig. 1A; P<0.05) and the CFU-Mix count was significantly increased (Fig. 1B; P<0.05), compared with the respective values in the γ-ray irradiation group. However, the percentage of SA-β-Gal stained HSC/HPCs and CFU-Mix numbers in the γ-ray irradiation group were also significantly higher and significantly lower, respectively, compared with those in the control group (Fig. 1; P<0.05).

Cell proliferation-associated SIRT1 and SIRT3 molecules were examined using RT-qPCR. Compared with the control group, SIRT1 (Fig. 2A) and SIRT3 (Fig. 2B) mRNA levels in the γ-ray irradiation group were significantly decreased (both P<0.01). However, Rg1 treatment (Rg1 group) significantly upregulated SIRT1 (Fig. 2A) and SIRT3 (Fig. 2B) mRNA levels compared with those in the γ-ray irradiation group (P<0.05).

The downstream molecule of SIRT1 (FOXO3) and the downstream molecule of SIRT3 (SOD2) were also examined. The results indicated that FOXO3 (Fig. 2C) and SOD2 (Fig. 2D) mRNA levels were significantly decreased in the γ-ray irradiation group compared with the control group (both P<0.01). However, Rg1 treatment significantly increased the mRNA levels of FOXO3 (Fig. 2C) and SOD2 (Fig. 2D) compared with those in the γ-ray irradiation group (both P<0.05).

The SIRT1/SIRT3 signaling pathway-associated molecules, namely SIRT1, SIRT3, FOXO3 and SOD2, were also examined using western blotting (Fig. 3A). The results showed that SIRT1, SIRT3, FOXO3 and SOD2 expression levels in the γ-ray irradiation group were significantly decreased compared with their respective levels in the control group (Fig. 3B; all P<0.01). However, when compared with the γ-ray irradiation group, the SIRT1, SIRT3, FOXO3 and SOD2 expression levels in the Rg1 group were significantly increased (Fig. 3B; all P<0.05).

Previous studies (26,27) reported that SIRT1 and SIRT3 participate in cell proliferation and cell cycle regulation. Furthermore, HSCs undergoing aging are arrested at the G1 stage (28). Therefore, the cell cycle distribution of Sca-1⁺ HSC/HPCs was analyzed using FCM. The significant G1 phase arrest of Sca-1⁺ HSC/HPCs was observed, and the percentages of cells in the S and M phases were significantly decreased in the γ-ray irradiation and Rg1 groups, compared with those in the control group (Fig. 4; all P<0.05). However, the percentage of Sca-1⁺ HSC/HPCs arrested at the G1 phase in the Rg1 group was significantly decreased compared with that in the γ-ray irradiation group (Fig. 4; P<0.05).

The proliferative index (PI; S + G2/M) in the γ-ray irradiation group was significantly decreased compared with that in the control group (Fig. 4; P<0.01). However, the PI of Sca-1⁺ HSC/HPCs in the Rg1 group was significantly increased compared with that in the γ-ray irradiation group (Fig. 4; P<0.05).