Reagents
Penicillin, streptomycin and HRP-conjugated goat anti-rabbit IgG (H+L) were obtained from Zhongshan Golden Bridge Biotechnology (Beijing, China), the senescent cell histochemical staining kit was obtained from Beyotime Institute of Biotechnology (Haimen, China), and the cell counting kit-8 (CCK-8) assay was obtained from Tongren Institute of Chemistry (Japan). VEGF and bFGF enzyme-linked immunosorbent assay (ELISA) kits were purchased from Wuhan Yunkelong Technology Co., Ltd. (SEA143Ra and SEA551Ra, Hubei, China). SIRT1 and FoxO3a were both from Cell Signaling Technology (#9475 and #2497, Danvers, MA, USA), LC3 was from Abcam ( ab63817, Cambridge, MA, USA), Beclin1 was from Affinity Biosciences (AF5128, OH, USA), p21 and p16 were obtained from Hua Biotechnology Co., Ltd., (ER1914-57 and ET1602-9, Zhejiang, China).
Experimental diabetes model
All animals were housed in an accredited facility with appropriate temperature and humidity. The rats were fed standard chow. The experimental protocols were approved by Xuzhou Medical University. To establish the DM animal model, the rats were fed with high-fat diet for 8 weeks, intraperitoneally injected with streptozotocin (STZ) (25 mg/kg/d in 0.1 mM sodium citrate buffer; Sigma, St. Louis, MO, USA) and following high-fat diet for 4 weeks. Blood samples were then collected to determine the blood glucose (BG) levels using an automatic BG monitor (Lifescan Inc., Milpitas, CA, USA). Animals with fasting glycaemia levels higher than 300 mg/dl and presenting signs of polydipsia and polyuria were considered diabetic and were selected for subsequent study [17].
Cell culture and treatments
MSCs were isolated from the bone marrow of Sprague-Dawley (SD) rats (weighing 60–80 g), as previously described. Briefly, the femurs and tibias were removed from the SD rats, and the bone marrow was washed out using 10 ml of Dulbecco's modified Eagle’s medium/normal glucose (DMEM/L; HyClone, Waltham, MA, USA) supplemented with 1% penicillin/streptomycin (Beyotime Institute of Biotechnology, Haimen, China). The cells were centrifuged at 300 ×g for 5 min. The resulting cell pellets were resuspended in 6 ml of DMEM/L supplemented with 10% foetal bovine serum (HyClone) and 1% penicillin/streptomycin and plated in a 25 cm2 plastic flask at 37°C in a humidified atmosphere containing 5% CO2 to allow the MSCs to adhere. After culturing the cells for 3 days, the medium was changed, and the nonadherent cells were removed. The medium was replaced every 2 days. Upon reaching 80–90% confluence, the adherent cells were detached from the dishes using 0.25% trypsin (Beyotime Institute of Biotechnology) and expanded at a dilution of 1:2 or 1:3. MSCs were characterized by flow cytometric analysis for the expression of the typical markers, CD90, CD29, and CD44 (All from BD Biosciences, Franklin Lakes, NJ, USA), and the absence of the hematopoietic markers CD45 (eBioscience, San Diego, CA, USA) and CD34 (Santa Cruz Biotechnology, Inc., Dallas, TX, USA), as previously reported [18]. MSCs from DM rat were culture under high glucose medium (25 M glucose), while MSCs from normal rat were culture under normal glucose medium (5.5 M glucose). MSCs at passage 3 were used in the experiment. For the other MSCs used in our following study, they were obtained from normal rats, for normal glucose group, the cells were cultured with 5.5 M glucose, while the high glucose group was cultured with 25 M glucose. Cells were cultured for as long as 28 days.
Cell transfection
Before transfection, MSCs were replayed into six-well plates at a density of 2 × 105 cells/well and incubated overnight. For over-expression or inhibition of miR-34a, cells were transfected with 20 nM of miR-34a mimic or miR-34a inhibitor (both from GenePharma Co., Ltd, Shanghai, China), respectively. For SIRT1 inhibition, 100 nM Akt siRNA (GenePharma Co., Ltd, Shanghai, China) was transfected into cells. All miRNAs and siRNA were transfected into MSCs using a commercial transfection reagent (X-treme siRNA transfection reagent; Roche Applied Science, Penzberg, Germany) according to the manufacturer’s protocol. Forty-eight or seventy-two hours after transfection, the cells were harvested for further analysis.
Senescence-associated β-galactosidase staining
MSCs senescence was determined by in situ staining for senescence-associated β-galactosidase (SA-β-gal) using a senescent cell histochemical staining kit (Beyotime Institute of Biotechnology, Haimen, China). Briefly, after treatment, MSCs were first fixed for 30 min at room temperature in fixation buffer. The cells were washed with PBS and incubated with β-galactosidase staining solution for 16 h at 37°C without CO2. The reaction was stopped by the addition of PBS. Statistical analysis was performed by counting 500 cells for each sample.
Cell proliferation assay
Cell proliferation was assessed with the CCK-8 assay (Tongren Institute of Chemistry, Japan). For the CCK-8 assay, cells were plated onto 96-well plates (3×103 cells/well). Assays were performed from 1 to 4 days after plating with the addition of 100 μl of fresh medium and 10 μl of CCK-8 solution for another 2 h at 37°C. The optical density (OD) at 450 nm was measured. The assay was repeated three times.
Cytokine measurement via ELISA
Using 500 μl of supernatant containing 5×105 cells, the VEGF and bFGF concentrations were assessed by a standard sandwich ELISA (Wuhan Yunkelong Technology Co., Ltd., Hubei, China) according to the manufacturer’s instructions. Briefly, 100ul supernatant were transferred to the plates for 1h at 37 °C. After washing 3 times, 100ul reacting agent was added and was incubated for 30 min at 37 °C. After washing 5 times, the reaction was stopped by adding 90ul chromogenic agent. The VEGF and bFGF concentrations are expressed in nanograms per millilitre and were calculated from calibration curves constructed from serial dilutions of human recombinant standards. The sensitivity of the VEGF and bFGF assays was 2 pg/ml.
Western blotting
After the designated treatments, the cells were lysed in RIPA buffer. The protein concentration was determined with a bicinchoninic acid (BCA) kit (Beyotime, Haiman, China) according to the manufacturer’s instructions. For western blot analysis, 50-80 µg of denatured protein was separated on SDS-PAGE gels and transferred onto PVDF membranes. The membranes were blocked with 5% skim milk in TBS containing 0.5% Tween 20 (TBST) for 1 h at room temperature (RT) on a shaking table. The membranes were then incubated overnight at 4°C with primary antibodies diluted in TBST. The membranes were subjected to three 10-min washes with TBST (TBS containing 0.5% Tween 20) and then reprobed with HRP-conjugated secondary antibodies at RT for 1 h. The membranes were then washed three times with TBST as described previously and visualized with an ECL detection system and Beyo ECL Plus reagent (Beyotime, Haimen, China), and the images were analyzed using Image Lab software (version 4.1, Bio-Rad). The following antibody dilutions were used: antibodies against p21 (1:1000), p16 (308) (1:1000), SIRT1 (473) (1:1000), FoxO3a (1:1000), LC3 (1:750) and Beclin1(1:1000), and HRP-conjugated secondary rabbit antibodies (1:5000).
Transmission Electron Microscopy (TEM)
MSCs with different treatment were fixed for 4 h at 4°C in 4% glutaraldehyde (v/v) buffer, washed with 0.01 mol/L phosphate buffer solution (PBS, pH 7.0), fixed again for 2h at 4°C in aqueous 1% (w/v) osmium tetroxide, and then embedded in Epon. Observation and photography were carried out by an electron microscope (JEM-2000EX TEM, JEOL, Tokyo, Japan).
MI model and MSCs transplantation
To induce an acute MI model, the left anterior descending coronary artery (LAD) was ligated as previously described [19]. After LAD ligation (besides the sham group), the rats randomly received one of the following treatments: (a) sham; (b) MI; (c) 3×105 MSCs (NG); (d) 3×105 MSCs (HG); (e) 3×105 MSCs (HG +miR-34a I). The group of rats that underwent thoracotomy without LAD ligation served as the sham group. All MSCs were suspended in 100 μl of PBS and injected intramuscularly at four sites around the border zone of the infarcted heart. Cardiac function in each rat was assessed by transthoracic echocardiography (Ultramark 9; Soma Technology) at baseline (before MI), and at 1 week or 3 weeks following MI. Left ventricle ejection fraction (LVEF) and left ventricular fraction shortening (LVFS) were calculated as previously described [19].
Masson's staining
After echocardiography assessment at 3 weeks post-MI, all rats were sacrificed, and the heart tissues were harvested, embedded and sectioned. The infarction sizes of the rat hearts, as evidenced by fibrosis, were examined by a Masson's staining kit according to the manufacturer's protocol (HT15, Sigma). The percent infarct size was calculated as the ratio of the fibrosis area to the total LV area ×100%.
TTC staining
2,3,5-Triphenyltetrazolium chloride (TTC) staining was performed after the heart was removed, rinsed and weighed. Next, the heart was frozen at -20°C for 20 min and cut into 5 sections (approximately 1-2 mm in thickness). Thereafter, the sections were subjected to a water bath containing 1% TTC phosphate buffer (pH 7.4) at 37°C for 15 min (protected from the light and shaken) and fixed with 10% formalin. Finally, a fluorescence microscope was utilized for to capture photographs.
Data analysis and statisticsThe data are expressed as the means ± SD of at least three independent experiments. When only two value sets were compared, the statistical analysis was performed with Student’s t-tests. The significance of differences between three or more experimental groups was determined by one-way analysis of variance. Values of P < 0.05, P < 0.01, and P < 0.001 were considered statistically significant, and these values are indicated by *, ** or ***, respectively.