Table 1.
Primers Used for qPCR.
Fig 1.
Age-related changes in cell morphology.
Morphological changes were observed in MSCs using phase-contrast microscope. (A) No significant morphological differences were observed in primary MSCs between young and old rats. (B) MSCs at passage 3 in the young group maintained a long and fusiform shape, while cells in the old group appeared flattened and enlarged and had lost their stereoscopic perception. The cell areas were clearly larger (C) and the cell aspect ratios were markedly lower (D) in the old group than in the young group. Statistically analyzed values are shown and indicate the mean ± SD (*p< 0.05, **p< 0.01).
Fig 2.
Age-induced variations in biological characteristics.
Cell growth curves and population doubling times were used to detect changes in proliferation in both groups. In the MSCs obtained from the old rats, growth slowed (A), and population doubling times were extended (B). The cell cycle was analyzed using flow cytometry. Most of the MSCs in the old group were arrested in G0/G1 phase at the expense of S phase (C). The S-phase fraction (D) and proliferation index (E) were lower in the old group than in the young group. Values indicate the mean ± SD (*p< 0.05, **p< 0.01).
Fig 3.
Cell senescence in MSCs obtained from old rats.
(A) β-galactosidase staining. The ratio of SA-β-gal-positive cells was higher in MSCs obtained from old rats than in those obtained from young rats. As indicated by the arrows, senescent cells were stained a blue color. (B) Cell apoptosis was measured using flow cytometry. The results demonstrated that there was no significant difference in the rate of apoptosis between the young and old groups. The values shown indicate the mean ± SD (* p< 0.05, ** p< 0.01, N p> 0.05).
Fig 4.
Cellular senescence is increased in MSCs obtained from old rats.
(A) Intracellular ROS levels were determined using H2DCFDA staining and flow cytometry. The DCFH-fluorescent intensity was much higher in the old group than in the young group. (B) DNA damage was detected using comet assays. No clear comet tails were observed in the MSCs obtained from young rats, while almost every cell in the old group had a long and apparent comet tail. DNA damage was quantified by measuring olive tail moments (OTMs). The length of each OTM increased with the ages of the rats. (C) Telomerase activity was analyzed using a TeloTAGGG Telomerase PCR ELISA Plus Kit. Telomerase activity was clearly lower in the MSCs obtained from old rats than in the MSCs obtained from young rats. (D) Real-time qPCR analyses of the expression of the senescence-related genes pl6INK4A and p21WAF1/CIP1. Actin was used as the reference gene. MSCs obtained from old rats expressed higher levels of the senescence markers pl6INK4A and p21WAF1/CIP1 than were observed in the young group. The values shown indicate the mean ± SD (* p< 0.05, ** p< 0.01, N p> 0.05).
Fig 5.
Nampt expression in MSCs obtained from young and old rats.
Nampt protein levels were evaluated using Western blot analysis (A) and immunofluorescence (C). mRNA levels were detected using RT-qPCR (B). Nampt expression at both the protein and gene level were reduced in an age-dependent manner. Actin was used as the internal standard. The values shown indicate the mean ± SD (* p < 0.05, ** p< 0.01).
Fig 6.
Sirt1 expression and activity and the intracellular levels of NAD+ in MSCs.
Sirt1 protein levels were measured using Western blot analysis (A) and immunofluorescence staining (C), and mRNA levels were tested using real-time qPCR (B). Sirt1 activity was evaluated using SIRT1 Assay Kits (D). Intracellular NAD+ levels were detected using a NAD/NADH Quantitation Colorimetric Kit (E). Sirt1 expression and activity were dramatically lower in the old group than in the young group, and this decrease was associated with a reduction in intracellular NAD concentrations. The values shown indicate the mean ± SD (* p < 0.05, ** p< 0.01).
Fig 7.
Inhibiting Nampt using FK866 increased cell senescence in MSCs obtained from the young group.
Cells were treated with 10 nM FK866 for 72 h. (A) Unlike the controls, which displayed normal morphologies, the MSCs treated with FK866 were flattened and enlarged and displayed senescence-like morphological features. (B-C) SA-β-gal activity was then measured. The percentage of β-Gal-positive cells and their staining intensity were significantly higher in the FK866 group than in the control group. (D) pl6INK4A and p21WAF1/CIP1 expression levels were evaluated using RT-qPCR. When Nampt was inhibited using FK866 in MSCs obtained from young rats, the expression levels of both pl6INK4A and p21WAF1/CIP1 were upregulated. Furthermore, both intracellular NAD+ levels (E) and Sirt1 activity (F) were decreased in the FK866 treatment group compared to those in the control group. The values shown indicate the means ± SD (* p< 0.05, **p< 0.01).
Fig 8.
Nampt overexpression attenuated cell senescence in MSCs obtained from the old group.
Cells were transduced with the lentivirus system. (A) Fluorescence images showed that Nampt was successfully overexpressed in MSCs from old rats. LV-Nampt: lentivirus encoding Nampt; LV-Vector: lentivirus encoding enhanced green fluorescent protein (EGFP). (B) Nampt protein levels were confirmed using Western blot analysis. (C) Nampt mRNA levels were ascertained using RT-qPCR. (D) Nampt overexpression significantly decreased SA-β-gal activity in MSCs from old rats.