C57BL/6 mice (6–8 weeks; 20–25 g) were bred in-house in a pathogen-free environment at a temperature of 22–25°C. The Institutional Animal Care and Use Committee of Chongqing Medical University (Chongquing, China) approved all animal experiments, which were performed according to Guide for the Care and Use of Laboratory Animals (20). The bone marrow suspension, obtained from the femur and tibia of the C57BL/6 mice with a needle was subjected to Ficoll-Paque density gradient separation to isolate bone mononuclear cells, followed by Sca-1⁺ cell separation using magnetic-activated cell sorting (MACS). The Sca-1⁺ cells were cultured in IMDM supplemented with 10% FBS at 37°C in a humidified atmosphere containing 5% CO₂ prior to drug exposure. Each group contained 5 mice unless otherwise specified.

Briefly, the Sca-1⁺ cells were isolated and purified from male C57BL/6 mice bone marrow mono-nucleated cells using MACS and cultured in IMDM supplemented with 10% fetal bovine serum. A total of 10,000 harvested Sca-1⁺ cells from male C57BL/6 mice were injected into the lateral tail veins of lethally irradiated (8.5 Gy Co⁶⁰γ) female C57BL/6 mice. Mice were irradiated to 8.5 Gy in an Cammacell-40 Exposure Instrument (Atomic Energy of Canada Limited, Gloucester, Canada) at a dose-rate of 1.0 Gy per minute for 8.5 min. At 4 weeks post-transplantation, the recipients were used as donors for a subsequent transplantation cycle and for in vitro assays. Serial transplants were performed in both male and female C57BL/6 mice strains. Engraftment efficiency in the C57BL/6 mice recipients was monitored by detecting the Sry gene of the Y-chromosome using PCR for donor contribution (35 cycles of 94°C for 4 min, 94°C for 20 sec, 60°C for 30 sec and 72°C for 30 sec; primer sequences (Bio-Rad, Hercules, CA, USA), forw ard 5′-GAAAAGCCTTACAGAAGCCGA-3′ and reverse 5′-G TATGTGATGGCATGTGGGTTC-3′; 211 base pairs). Serial transplantation assays were performed three times in independent experiments, with five recipient mice/group/experiment. The animals were monitored daily for the presence of disease, and were sacrificed by decapitation under diethyl ether (Tianhao Chemical Industry, Guangdong, China) anesthetics at designated times-points following transplantation, or when moribund. The experiments were performed following dividing of the 20 adult female C57BL/6 mice into the four following groups, each containing five mice: Group 1, mice were irradiated with total 8.5 Gy Co⁶⁰γ and injected with 0.2 ml PBS via the lateral tail vein, designated the control); group 2, mice were irradiated with total 8.5 Gy Co⁶⁰γ followed by intraperitoneal administration with 20 mg/kg/d Rg1 for 4 weeks as the positive control (designated Rg1); group 3, mice were irradiated with total 8.5 Gy Co⁶⁰γ followed by injection with 10,000 Sca-1⁺ cells via tail vein transplantation, designated the negative control (T) group; group 4, mice were administered intraperitoneally with 20 mg/kg/d Rg1 for 1 month following Sca-1⁺ cell transplantation, designated T+Rg1. The Sca-1⁺ cells isolated from each group of mice were then assessed for aging biomarkers.

The cells (10,000) were fixed with 0.5% glutaraldehyde for 15 min, followed by washing twice with PBS and incubation with 1 ml SA-β-Gal staining solution at 37°C without CO₂ for 24 h. The number of positively stained cells were stochastically counted within 100 cells using a charge-coupled device camera attached to a phase-contrast microscope (CKX41-A32RC; Olympus, Tokyo, Japan). All counting was performed in triplicate.

The cells from all the groups were respectively harvested and plated (3×10³/well) in a 96-well plate, with each well containing 1.1 ml 0.8% methylcellulose (Sigma-Aldrich) in an environment of 37°C, 5% CO₂ for 2 weeks prior to fixation with 4% paraformaldehyde and staining with 0.1% crystal violet. The colony numbers were counted under an optical microscope (CX22; Olympus, Tokyo, Japan). A cell sphere containing >50 cells was considered one colony.

For western blot analysis, cellular proteins were obtained from the total cell lysates resuspended in double distilled H₂O. The cellular proteins (30 µg) were electrophoresed using SDS-poly-acrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride membrane (GE Healthcare Life Sciences, Chalfont, UK). The membrane was blocked with 5% skim milk in PBS for 2 h at room temperature and then incubated with specific antibodies, anti-P16, anti-Rb, anti-CDK2, anti-CDK4 and anti-cyclin E (Bioss) all at 1:200 dilutions at 4°C overnight. HRP-conjugated goat anti-rabbit antibodies were used as a secondary antibody, at 1:5,000 dilution for 2 h at room temperature. The specific proteins were detected using enhanced chemiluminescent reagents (Pierce Biotechnology). Densitometry quantification was performed using the Image-Pro Plus v 6.0 software (Media Cybernetics, Inc., Bethesda, MD, USA).

Genomic DNA from the six treatment groups were digested with Hinf/RsaI enzyme at 37°C for 2 h. Electrophoresis of the digested genomic DNA (20 µg) was performed in 0.7% agarose gels (Yeasen, Shanghai, China) at 1 V/cm. Following electrophoresis, the gels were denatured and neutralized; and the DNA was transferred in 20X SSC onto nylon membrane filters. The membrane was prehybridization at 42°C for 1 h in 10 ml prehybridization solution, containing 3 m 20X SSC, 1 ml 50X Denhardt solution, 0.5 ml 10% SDS, and 5.5 ml H₂O (Klamar, Shanghai, China), followed by incubated at 42°C overnight following the addition of telomere probe (100 ng/ml). Finally, the membrane was washed twice in 2X SSC containing 0.1% SDS (Klamar) for 30 min, blocked for 15 min, and mixed by agitation with streptavidin-HRP (1:400; Boster, Wuhan, China) buffer at 37°C for 40 min. The membrane was then washed in PBS three times, followed by 10 min incubation with 3,3′-diaminobenzidine and capture of images. The intensity of the signals were determined using AlphaView System software (Alpha Innotech Corporation, St. San Leandro, CA, USA) and the mean lengths of the TRFs were calculated with L=Σ (ODi:l:Li)=Σ (ODi).

A total of 1×10⁶ cells were washed once in ice-cold wash buffer, resuspended and centrifuged at 800 x g for 5 min at 4°C. The precipitation was homogenized with 40 µl cold lysis buffer and lysad for 30 min in ice, followed by centrifugation at 1,500 rpm for 30 min at 4°C and collection of the supernatant (optical density 260/280 of RNA: 1.8–2.0). To the extension reaction, 50 µl of a solution, containing 5 µl 10XTRAP buffer, 1 µl dNTPs, 1 µl Taq-DNA polymerase, 1 µl TS primer, 2 µl telomerase extraction, 39 µl DEPC H₂O and 1 µl CX primer was added. Prior to qPCR, solution without CX primer was pre-incubated at 23°C for 30 min. The PCR for the TRAP assay was performed using the following program: 94°C for 5 min; 35 cycles at 94°C for 30 sec, 50°C for 30 sec, 72°C for 90 sec; 72°C for 10 min. SYBR Green dye (1 µl) was added to the PCR tube, mixed well, and incubated for 10 min at room temperature. PCR was conducted using a Bio-Rad sequence detection system (cfx96; Bio-Rad Laboratories, Inc., Hercules, CA, USA). The fluorescence intensity was determined using a fluorospectrophotometer (Bio-Rad Laboratories, Inc.), and the ratio of telomerase activity/fluorescence intensity:protein concentration was determined.

All values are expressed as the mean ± standard deviation. Analyses were performed using repeated measures of analysis of variance and an unpaired two-tailed Student's t-test. P<0.05 was considered to indicate a statistically significant difference.

To observe the effects of Rg1 on Sca-1⁺ cells in vitro, the Sca-1⁺ cells were treated with an achievable plasma concentration of 10 µmol/l Rg1. To enable direct comparison with the effect of Rg1 on aging cells, side by side investigations were performed using t-BHP, owing to the fact that 100 µmol/l t-BHP effectively induces Sca-1⁺ cells aging following 6 h in vitro co-culture (19). The results of the SA-β-Gal staining assay demonstrated that there was a marked increase following the cells treated with 100 µmol/l t-BHP, compared with the control group cells. Otherwise, no significantly difference in the number of positively-stained Sca-1⁺ were observed cells between the groups treated with or without 10 µmol/l Rg1. To determine whether Rg1 had protective effects on the aging Sca-1⁺ cells, the Sca-1⁺ cells were primarily cultured with 100 µmol/l t-BHP for 6 h, and then washed with sterile PBS and transferred into 10 µmol/l Rg1 medium for another 12 h co-culture. Following the SA-β-Gal staining assay, the numbers of positively-stained Sca-1⁺ cells were counted. As shown in Fig. 1, the higher rate of positive staining of the Sca-1⁺ cells caused by t-BHP exposure decreased following treatment with Rg1 (8.33±2.41, 7.04±2.53, 68.50±4.95 and 30.08±2.44 for the control, Rg1, t-BHP and Rg1+t-BHP groups, respectively).

To determine whether Rg1 functionally affected the ability of Sca-1⁺ cells to form colonies, methylcellulose colony-forming unit (CFU) assays were performed. The results revealed that Sca-1⁺ cells exposed to 10 µmol/l Rg1 exhibited a minor/moderate increase in CFUs. By contrast, the Sca-1⁺ cells exposed to 100 µmol/l t-BHP exhibited a significant decrease in CFUs. The decreased abilities of Sca-1⁺ cells to form colonies caused by t-BHP was apparently compensated for by treatment with 10 µmol/l Rg1 (15.04±1.78, 18.25±2.27, 3.34±1.58 and 9.34±2.23 CFUs in the control, Rg1, t-BHP and Rg1+t-BHP groups, respectively; Fig. 1). These data indicated that Rg1 had a protective effect on normal and on t-BHP-induced aged Sca-1⁺ cells in vitro.

It is known that the p16^INK4a/pRb signaling pathway is important in the process of replicative senescence in fibroblasts. To gain insight into the molecular basis of Rg1-mediated senescence alleviation in Sca-1⁺ cells, western blot analysis was performed to examine the expression patterns of the important senescence-asssociated proteins, P16 and Rb, in Sca-1⁺ cells exposed to different agents. The results demonstrated that the protein expression levels of P16 and Rb are markedly increased following t-BHP exposure in vitro. However, the higher protein expression levels of P16 and Rb in t-BHP-mediated Sca-1⁺ cells was decreased by 10 µmol/l Rg1 in vitro. Cell senescence is linked with specific changes in the expression of cell cycle regulators, contributing to cell proliferation arrest. To confrim the protective effects of Rg1, the present study further analyzed the expression levels of CDK2, CDK4 and cyclin E in the Sca-1⁺ cells. Rg1 was observed to significantly restore the higher protein expression levels of CDK2, CDK4 and cyclin E in the t-BHP-induced Sca-1⁺ cells. No other detectable differences were observed in the levels of protein expression between the Sca-1⁺ cells with or without Rg1 exposure in vitro (Fig. 2). These results implied that Rg1 substantially alleviated the cell cycle arrest, which was induced by t-BHP in vitro, and was associated with the regulation of p16^INK4α/pRb signaling.

To validate the protective effects of Rg1 on Sca-1⁺ hematopoietic cells in mice, a serial transplantation assaywas performed and Sca-1⁺ cells were collected from male mice and transplanted into lethally irradiated female mice to reconstitute the hematopoietic compartment. At 4 weeks post-transplantatation, the functional changes of the Sca-1⁺ cells were assessed using a colony forming units assay. These results indicated that the Rg1-treated Sca-1⁺ cells had higher colony-forming capacity, leading to an increased number of CFUs, compared with the control group of untreated cells (Fig. 3). The first indication of increased colony-forming capacity in the Rg1-treated Sca-1⁺ cells transplanted into the primary recipient mice was evident from the evaluated level of colony-forming capacity in Sca-1⁺ cells obtained from secondary recipients. At 8 weeks post-transplantation, the Rg1-treated Sca-1⁺ cells from secondary recipient mice exhibited higher colony-forming capacity compared with the cells in the control group. A similar tendency was also observed in the colony-forming capacity of the Sca-1⁺ cells from the third recipient mice.

To determine the aging profile of the Sca-1⁺ cells during the process of transplantation, BM Sca-1⁺ cells from the Rg1-treated or control group recipient mice were analyzed using an SA-β-Gal staining assay. The results revealed an increased number of positively stained cells within the transplant process, indicating that the regulation of replicative senescence was, at least in part, responsible for Sca-1⁺ cell aging during the process of serial transplantation. The beneficial effects of Rg1 on Sca-1⁺ cells aging was evidenced by the return of the aging cells close to normal levels following Rg1 treatment.

To gain insight into the basis for Rg1 on Sca-1⁺ cells in mice, Sca-1⁺ cells from the tibial and femoral bone marrow of the third recipient mice were collected and analyzed using western blotting to observe the age-associated changes in the protein expression levels of p16-Rb signaling pathways. The results demonstrated that there were decreases in the levels of P16 and Rb, but increases in the levels of CDK2, CDK4 and cyclin E in the Rg1-treated mice, compared with the control group (Fig. 4). These findings correlated with a significant change in the expression levels of P16, Rb, CDK2, CDK4 and cyclin E in the Sca-1⁺ cells in vitro assays. These results implied that p16^INK4a/pRb signaling was responsible for the regulation of Rg1 on Sca-1⁺ cells aging alleviation in mice.

Telomeres are formed by tandem repeats of the TTAGGG sequence, which has a base attrition loss during each cell division. Telomere shortening progressively occurs during cell replicative senescence. Abnormalities in the telomere length and telomerase activities of the recipient mice further promoted an examination of the senescence-associated p16-Rb signaling pathways (21). To analyze whether Rg1-mediated Sca-1⁺ cells aging alleviation is linked to telomere changes, the present study performed southern blotting to observe changes in telomere length in Sca-1⁺ cells from the third recipient mice. As shown in Fig. 5, the telomere lengths of the Sca-1⁺ cells from recipient mice were marginally decreased, compared with the control group cells, whereas a marked restoration in the telomere length of the Sca-1⁺ cells was identified following Rg1 treatment. This recovery was observed at similar time-points in three independent experiments. Telomeres are important in the aging of an organism and in the regulation of cell senescence, which protects chromosome telomeres from degradation (22). To further confirm that the protective effects of Rg1 on Sca-1⁺ cells aging is consistent with telomere length compensation, the present study compared the telomerase activities of the Rg1-treated ad control group Sca-1⁺ cells from third recipient mice. In agreement with the effects of Rg1 on telomere length restoration in the Sca-1⁺ cells, these results indicated that the telomerase activities of the Sca-1⁺ cells improved following Rg1 administration, compared with the control group cells in the third recipient mice, as is shown in Fig. 5.