Sprague-Dawley (SD) rats (weight, 10.0–15.0 g; age, 7 days; male, n=30; female, n=30; total n=60) were obtained from Zhejiang Medical Academy of Science (Hangzhou, China; permit number SCXK (Zhejiang) 2008–0033) and housed separately at 20–25°C under a 12-h light/dark cycle and fed ad libitum with a normal diet. The investigation was permitted by the Law of the People's Republic of China on the Protection of Wildlife and the protocol was approved by the Ethical Committee of the Zhejiang University City College (Hangzhou, China). SD rats were sacrificed by cervical dislocation sacrificed by cervical dislocation and immersed in 75% alcohol and disinfectant for 3 min. The rats were subsequently transferred to a new dish and the whole skin was removed from the hind limbs and forelimbs. The femurs and tibias were removed from the SD rats and bone marrow was flushed out using 10 ml PBS with 100 U/ml heparin in a syringe. The cells were centrifuged at 110 × g at room temperature for 8 min. The cell pellet was resuspended in 2.5 ml Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Hangzhou Sijiqing Biological Engineering Materials Co., Ltd., Hangzhou, China) and plated in a 25 cm² plastic flask (Corning Incorporated, Corning, NY, USA) to allow the MSCs to adhere at 37°C with 5% CO₂ in a humidified atmosphere. Following a 3 day period, the medium was replaced and the nonadherent cells were discarded. The medium was completely replaced every 3 days thereafter. The cells became ~80% confluent at 7–10 days following seeding. The adherent cells were released from the dishes with 0.25% trypsin (Hyclone; GE Healthcare Life Sciences, Logan, UT, USA) and seeded into fresh culture flasks. All the experiments described below were performed using MSCs from the third to the fifth passages.

In the control groups, MSCs were cultured for 14 days in DMEM supplemented with 10% FBS. In the glucose treatment groups: MSCs were incubated in the culture medium containing 5.5, 11.0 or 22.0 mM glucose for 14 days. In the high glucose control group, MSCs were treated with 22.0 mM glucose for 14 days to induce cellular senescence. In the MK-2206 treatment group, the cells were incubated in the culture medium containing 0.1, 1.0 or 10.0 nM MK-2206 and 22.0 mM glucose for 14 days. The medium was completely replaced every 3 days. The cells of all of the above groups were cultured in a humidified incubator at 37°C and 5% CO₂.

SA-β-gal staining was performed using a Senescence-associated β-Galactosidase Staining kit (Beyotime Institute of Biotechnology, Haimen, China) following the manufacturer's protocol. The treatment methods for the MSCs in each group were the same as the aforementioned. The cells were fixed in 4% (v/v) formaldehyde for 5 min and were then stained with SA-β-gal staining solution at pH 6.0 for 12 h. The SA-β-gal-positive cells exhibited a blue coloration. The number of positive cells were counted under a phase-contrast microscope. The experiment was repeated five times in each group.

Briefly, cells were fixed with 10% trichloroacetic acid solution for 1 h, wells were rinsed five times with tap water and then cells were stained with 0.4% SRB solution (100 µl per well) for 20 min at room temperature. The wells were then rinsed with 1% acetic acid to remove unbound dye and were left to air dry. The SRB dye was then solubilized by placing 100 µl unbuffered Tris-based solution in each well, and the absorbance was measured at a wavelength of 515 nm using a multiscan spectrum. The experiment was repeated five times in each group.

To assay the expression of phosphorylated (p)-16^INK4a, p53, p21, p-mTOR, p-Akt, p-glycogen synthases kinase (GSK)-3β and β-catenin, the total cellular protein was extracted from MSCs from different treatment groups. The cells were first washed in cold-buffered PBS and lysed in radioimmunoprecipitation assay buffer (150 mM NaCl, 1% Triton X-100, 0.5% NaDOD, 0.1% SDS and 50 mM Tris, pH 8.0). Following centrifugation (9660 × g for 5 min) at 4°C, the protein supernatant was transferred into different tubes. The protein concentration of the samples was determined using a bicinchoninic acid protein assay (Beyotime Institute of Biotechnology). A 40 µg sample of the total protein was resolved using 8–12% SDS-PAGE and transferred onto polyvinylidene difluoride (EMD Millipore, Billerica, MA, USA) membranes. The membranes were blocked with 5% nonfat milk at room temperature for 1 h in Tris-buffered saline containing Tween-20 (TBST) and incubated overnight at 4°C with the following Primary antibodies: rabbit anti-p16^INK4a (1:1,000; cat. no. sc-1661; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), mouse anti-p53 (1:2,000; cat. no. 554147; BD Biosciences, Franklin Lakes, NJ, USA), mouse anti-p21 (1:1,000; cat. no. sc-6246; Santa Cruz Biotechnology, Inc.), rabbit anti-p-mTOR (1:2,000; Ser2448; cat. no. 2971S; Cell Signaling Technology, Inc., Danvers, MA, USA), rabbit anti-p-Akt (1:2,000; Ser473; cat. no. 4058S; Cell Signaling Technology, Inc.), rabbit anti-p-GSK-3β (1:2,000; Ser9; cat. no. 5558; Cell Signaling Technology, Inc.), mouse anti-β-catenin (1:1,000; cat. no. sc-7963; Santa Cruz Biotechnology, Inc.) and mouse anti-β-actin (1:5,000; cat. no. 612657; BD Biosciences), were incubated overnight with the membranes at 4°C. Membranes were incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit or anti-mouse secondary antibodies (1:2,000; cat. nos. 7074 and 7076; Cell Signaling Technology, Inc.) for 1 h at room temperature, and proteins were detected by enhanced chemiluminescence (cat. no. RPN2106; GE Healthcare Life Sciences, Little Chalfont, UK). β-Actin was used as the internal control to normalize the loading materials.

All data are presented as the mean ± standard deviation. Statistical significance was determined using one-way analysis of variance to compare data from different experimental groups, followed by the Student-Newman-Keuls multiple comparison test. SPSS software, version 11.5 (SPSS, Inc., Chicago, IL, USA) was used to process data. P<0.05 was considered to indicate a statistically significant difference.

SA-β-gal staining was used to observe the effect of HG on MSCs aging. The results demonstrated that SA-β-gal-positive cells appeared larger and of a flatter shape and notably, few SA-β-gal-positive cells were observed to be present in the control group. However, in the 11.0 and 22.0 mM glucose groups, the number of SA-β-gal-positive cells increased (Fig. 1A). The cell counting indicated that the number of SA-β-gal-positive cells in the 11.0 mM and 22.0 mM glucose group (33.2±7.3 and 48.2±8.6/100 cells, respectively) were significantly increased compared with the control group (8.6±1.7; P<0.01; Fig. 1B), which suggested that HG promoted MSCs senescence.

To investigate the effects of HG on the expression of senescence-associated proteins, the present study examined p16^INK4a, p53 and p21 expression levels via western blot analysis. The results demonstrated that, following cell culture in 11.0 or 22.0 mM glucose for 14 days, the expression levels of p16^INK4a, p53 and p21 were increased compared with the control group (Fig. 2). The results suggested that p53/p21 and p16^INK4a acted as potential mediators in MSC senescence, induced by HG.

The proliferation rate of MSCs in each group was examined via the SRB assay. Following MSC incubation with 11.0 or 22.0 mM glucose for 14 days, the absorbance value in the 11.0 mM and 22.0 mM glucose groups was decreased compared with the control (1.10±0.10 or 0.85±0.06 vs. 1.38±0.10; P<0.05 or P<0.01; Fig. 3). These data suggested that HG inhibited the proliferation of MSCs.

To investigate the mechanisms of MSC senescence induced by HG, the expression levels of p-mTOR, p-Akt, p-GSK-3β and β-catenin were examined. Western blot analyses indicated that the p-mTOR and p-Akt expression levels were increased in the 11.0 mM or 22.0 mM glucose groups compared with the control group and HG did not affect the expression of p-GSK-3β and β-catenin (Fig. 4). These results indicated that HG activated the Akt/m-TOR signaling pathway in MSCs.

To further define the role of Akt/mTOR signaling in MSC senescence promoted by HG, MK-2206, an Akt-specific small molecule inhibitor, was used to inhibit the Akt/mTOR signaling. SA-β-gal staining demonstrated that the number of SA-β-gal-positive cells in the 0.1, 1.0 and 10.0 nM MK-2206 groups decreased in a dose-dependent manner, compared with the HG control group (Fig. 5A). The cell count revealed that the number of SA-β-gal-positive cells in the 1.0 and 10.0 nM MK-2206 groups (27.1±4.0 and 13.2±3.7/100 cells) was significantly decreased compared with the glucose control group (44.3±7.7/100 cells; P<0.05 or P<0.01; Fig. 5B). Western blot analyses indicated that MK-2206 inhibited the expression of p-Akt, and furthermore the enhancing effects of HG on p53 and p16^INK4a expression levels were reversed by incubation with MK-2206 (Fig. 5C). These data indicated that the Akt/mTOR signaling pathway was important in the MSC senescence induced by HG.