The positive effects of Ginsenoside Rg1 upon the hematopoietic microenvironment in a D-Galactose-induced aged rat model

Three month old male Sprague-Dawley rats were purchased from the Medical and Laboratory Animal Center of Chongqing (Chongqing, China) and housed in a temperature and light-controlled room with free access to water and food. All treatments were performed under sodium pentobarbital anesthesia, and all efforts were made to minimize animal suffering. The Committee on Ethics of Animal Experimentation at Chongqing Medical University (Chongqing, China) approved the protocols of this study prior to its implementation (Approval no.: 2013031).

Twenty rats were randomly divided into four groups (control group, D-gal-administration group, Rg1-treatment group, and D-gal-administration + Rg1-treatment group). In the D-gal-administration group, D-gal (120 mg/kg · d) was injected subcutaneously daily into rats for 42 days. In the D-gal-administration + Rg1-treatment group, ginsenoside Rg1 (20 mg/kg · d) was injected peritoneally daily concomitantly for 28 days from day 15 of D-gal injection. All control animals were given saline in the same volume subcutaneously and peritoneally, respectively. In the Rg1-treatment group, saline at the same volume with D-gal injection was injected subcutaneously for 42 days, and Rg1 (20 mg/kg · d) was injected peritoneally for 28 days from day 15 of saline injection.

Ginsenoside Rg1 (RSZD-121106, Purity = 98.3%) was purchased from Xi’an Haoxuan Biological Technology Co., Ltd (Xi’an, China), dissolved in ddH2O at the concentration of 20 mg/ml, and sterilized by ultrafiltration. The SOD kit and MDA kit were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The IL-2 kit, IL-6 kit, and goat anti-rabbit secondary antibody were purchased from Wuhan Boster Bio-engineering Co., Ltd. (Wuhan, China). The TNF-α Kit was obtained from Uscn Life Science Inc. (Wuhan, China). The BCA kit and SA-β-gal Staining kit were purchased from Beyotime Institute of Biotechnology (Shanghai, China). The anti-β-tubulin III antibody was obtained from Sigma Co. LLC. The anti-GFAP antibody was purchased from Wuhan Sanying Biotechnology Inc. (Wuhan, China).

The bone marrow cells were collected according to Raghavendran’s method [5]. The femoral bones were separated out of the body, briefly soaked in 75% alcohol, and flushed three times in phosphate buffer solution (PBS) containing antibiotics (Penicillin–Streptomycin) under sterile conditions. The epiphyses of each bone were cut off. Bone marrow cell suspensions were prepared by flushing the diaphysis with PBS through syringe needles for three to five times. Then, full bone marrow cells were implanted in 25-cm² tissue flasks containing 5 ml DMEM supplemented with 10% FBS and 1% penicillin-streptomycin. The cells were incubated in a 37°C, 5% CO₂-humidified chamber. After 72 h of incubation, the supernatant and unattached cells were removed. The adherent cells were fed with fresh medium and incubated for an additional two days. The medium was pipetted off and discarded, the cells were trypsin-digested and detached, and then passaged to a new flask. The cultures were fed every three days by replacement of the medium. After three passages, BMSCs achieved 80% confluence. The confluent cell layers were washed twice with PBS. Adherent BMSCs were detached with 0.25% trypsin with EDTA and collected for further assays.

SA-β-gal is one of the most widely used biomarkers for aging cells [7,8]. The BMSCs were collected at passage three, and the SA-β-gal staining was performed according to the manufacturer’s instructions. Briefly, 5 × 10⁴ purified BMSCs from each animal were inoculated onto a six-well plate (with one slide per each well) and incubated for three days. Slides were washed twice with PBS, fixed in Fixative Solution for 10 min at room temperature, and stained with Staining Solution for 12 h at 37°C without CO₂. Approximately 1 × 10⁴ cells were separated on each slide, and 400 cells were analyzed for each animal. The percentage of SA-β-gal-positive cells was calculated by counting the number of blue cells under bright field illumination and then calculating the ratio of SA-β-gal staining positive cells (%) in each group.

BMSCs were collected at a concentration of 5 × 10⁵ cells/ml and fixed with 70% cold ethanol at 4°C overnight. After centrifugation at 1000 rpm for 5 min, cells were incubated with propidium iodide (PI) at 4°C for 30 min in the dark and were subjected to flow cytometry using a FAC Scan Flow cytometer (BectonDickinson, New Jersey, USA) to determine the percentage of cells in each cell cycle phase.

For the quantitative determination of cellular proliferation and viability, we performed the CCK-8 assay. This assay was performed after BMSCs were collected at passage three. The cells were washed, counted, and seeded at a density of 4 × 10⁵ cells/ml per well in 96-well plates. At 24, 48, 72, 96, and 120 h after BMSC seeding, CCK-8 solution was added 4 h prior to the end of incubation. Cell viability was measured with a spectrophotometer at an absorbance of 450 nm.

The supernatant of the cell culture on passage three was collected, and levels of three inflammatory cytokines (IL-2, IL-6, and TNF-α) as well as SCF in each group were measured by ELISA according to the kit manufacturer’s instructions.

At passage three, BMSCs were collected and lysed in an ice bath for 30 min. After centrifugation (12000 rpm, 4°C, 30 min), the supernatant was collected. SOD activity -- determined using the commercially-available WST-1 assay kit -- and MDA content were detected by chemical colorimetric analysis according to the kit manufacturer’s instructions. The SOD activity assay is based on its ability to inhibit the oxidation of hydroxylamine by O₂- produced by the xanthine-xanthineoxiase system. The results of the SOD activity assay were expressed as ratios of SOD suppression.

The thiobarbituric acid reaction (TBAR) method was used to determine the MDA, which can be measured at a wavelength of 532 nm by reacting TBA to form a stable chromophore. MDA content was expressed as nmol per milligram of BMSC protein. ROS production in BMSCs was measured using a DCFH-DA fluorescence flow cytometric assay. At passage three, the fluorogenic substrate solution was added to the medium and incubated at 37°C and 5% CO₂ for 45 min. After being washed with cold PBS, the cells were quickly analyzed by FACS Calibur (BectonDickinson, USA).

BMSCs in each group were collected at passage three. Total protein was extracted, and the concentrations were measured by a BCA procedure. Samples containing 50 μg protein were separated on SDS-PAGE and transferred to PVDF membranes. Membranes were incubated overnight at 4°C with anti-p16, anti-21, and anti-53 antibodies (all diluted 1:500). The secondary antibody was diluted 1:5000 in TBST. The membranes were visualized using the enhanced chemiluminescence (ECL) detection system (Pierce, USA). β-actin was used as an internal control. Integral optical density (IOD) was quantified using Image Pro Plus (Media Cybernetics).

SPSS v17.0 was used for all statistical analyses. One-way ANOVA was used for comparing mean values across groups, and multiple comparisons were made using the LSD test. Differences were considered significant at P < 0.05.

The intensity of SA-β-gal staining was evaluated by means of the percentage of SA-β-gal positive cells (%) (Figure 1). The percentages of SA-β-gal positive cells were not significantly different between the control group and the Rg1-treatment group (Figure 1). As expected, the D-gal-administration group induced a significant increase in the percentage of SA-β-gal positive cells compared to that of the control group (p < 0.05; Figure 1). However, in the D-gal-administration + Rg1-treatment group, the percentage of SA-β-gal positive cells was significantly reduced (p < 0.05; Figure 1). These findings suggest that Rg1 can protect BMSCs against senescence.

When compared to the D-gal-administration group, the BMSCs in both the Rg1-treatment and D-gal-administration + Rg1-treatment groups displayed G1-phase shortening, which is the reverse of the effect observed in the control group (p < 0.05; Figure 2). BMSCs from the D-gal-administration group displayed G1-phase arrest, and the percentages of cells in S phase were significantly decreased compared to that of the control group (p < 0.05; Figure 2). However, the percentages of cells in S phase in both the Rg1-treatment and D-gal-administration + Rg1-treatment groups were significantly elevated (p < 0.05; Figure 2). There were no significant differences among the four groups regarding cell percentages in M phase (Figure 2).

As expected, D-gal administration led to significant inhibition of BMSC proliferation compared to controls (p < 0.05; Figure 3). However, after Rg1 treatment, BMSC proliferation in the D-gal-administration + Rg1-treatment group was elevated significantly compared to the D-gal-administration group (p < 0.05; Figure 3).

The levels of IL-2, IL-6 and TNF-α significantly increased, and SCF significantly decreased, in the BMSCs from the D-gal-administration group when compared with the control group (p < 0.05; Figure 4). However, the levels of the three inflammatory cytokines were significantly reduced in the D-gal-administration + Rg1-treatment group relative to the D-gal-administration group (p < 0.05; Figure 4). Conversely, the level of SCF was significantly elevated in the D-gal-administration + Rg1-treatment group compared to the D-gal-administration group. Simultaneously, Rg1 treatment did not significantly reduce the levels of IL-2, IL-6 and TNF-α in the Rg1-treatment group compared with controls, but the level of SCF was significantly promoted in the Rg1-treatment group compared with controls (p < 0.05; Figure 4). These findings indicate that inflammation in D-gal-induced aged BMSCs is alleviated, while SCF expression is promoted, by Rg1 treatment.

The oxidative stress produced by reactive oxygen species (ROS) is one of the main causes of cell senescence. Superoxide dismutase (SOD) participates in the removal of ROS from the cellular environment, and malondialdehyde (MDA) is an end-product of ROS-induced peroxidation that is widely used as a marker of oxidative stress. We evaluated cellular ROS levels, SOD activity, and MDA content in the BMSCs to determine whether the anti-aging effects of Rg1 are mediated by alleviating oxidative stress. In the DCFH-DA assay, the cellular ROS levels were elevated significantly in the D-gal-administration group when compared with controls with significant inhibitory effects of Rg1 observed in the D-gal-administration + Rg1-treatment group (p < 0.05; Figure 5). In the Rg1-treatment group, the level of ROS production was not inhibited significantly compared to controls (Figure 5). When compared to the control group, the ratio of SOD suppression decreased significantly and the MDA content increased significantly in BMSCs in the D-gal-administration group (p < 0.05; Figure 5). Meanwhile, Rg1 significantly rescued the reduction in SOD activity, and partially rescued the increase in MDA content, in the D-gal-administration + Rg1-treatment group (p < 0.05; Figure 5). Interestingly, the Rg1-treatment group also significantly increased SOD activity and significantly reduced MDA content (p < 0.05; Figure 5), showing Rg1 exerts anti-oxidant effects by enhancing the activity of endogenous anti-oxidative defense enzymes.

The p16^INK4a-retinoblastoma (Rb) pathway and the p19^Arf-Mdm2-p53-p21^Cip1/Waf1 pathway are two important signal transduction pathways involved in cell aging and cell cycle arrest. According to Western blotting analysis, the levels of p16^INK4a, p21^Cip1/Waf1, and p53 were significantly higher in the D-gal-administration group than in the control group (p < 0.05; Figure 6). With Rg1 treatment, the levels of these three proteins declined significantly (p < 0.05; Figure 6).