The role of SIRT1 in BMP2-induced chondrogenic differentiation and cartilage maintenance under oxidative stress

The C3H10T1/2 cells infected with Ad-BMP2, Ad-SIRT1, or Ad-GFP exogenously expressed BMP2 and SIRT1

In order to obtain high levels of transgene expression, we constructed recombinant adenoviruses expressing Ad-BMP2, Ad-SIRT1, and Ad-GFP. After 24 h of virus infection, the morphology of the C3H10T1/2 cells was observed under a fluorescence microscope, and the intensity of fluorescence infection showed that the C3H10T1/2 cells were successfully infected with adenovirus (Figure 1A). The results of real-time fluorescence quantitative PCR (Figure 1B, 1C) and western blotting showed that C3H10T1/2 cells transfected with the Ad-BMP2 and Ad-SIRT1 highly expressed BMP2 and SIRT1, but this did not occur in cells infected with Ad-GFP (Figure 1D, 1E). These data indicated that we successfully constructed recombinant adenoviruses of Ad-BMP2, Ad-SIRT1, and Ad-GFP and effectively infected the C3H10T1/2 cells.

Figure 1. The successful infection of the C3H10T1/2 cells with Ad-SIRT1, Ad-BMP2, and Ad-GFP. (A) C3H10T1/2 cells were infected with Ad-SIRT1, Ad-BMP2, or Ad-GFP in plate cultures. Bright light and fluorescence microscopy (100X) examination showed the efficiency of recombinant adenoviruses after 24 h of virus infection. (B, C) Total RNA was isolated 24 h after infection, and the mRNA expression of SIRT1 and BMP2 was measured using real-time PCR. (D, E) The SIRT1 and BMP2 protein levels were measured after 48 h of infection using the western blotting method. The data are denoted as the mean ± SD.*: p < 0.05 vs. Ad-GFP.

SIRT1 potentiated BMP2-induced chondrogenic differentiation of the C3H10T1/2 cells in vitro

Sox9, a key regulator of early chondrogenic differentiation expression, was detected by western blotting on the 5^th, 7^th, and 9^th day after the C3H10T1/2 cells were infected with Ad-GFP, Ad-SIRT1, Ad-BMP2, or Ad-SIRT1+Ad-BMP2. We found that the expression of Sox9 in the Ad-SIRT1+Ad-BMP2 group was significantly higher than that in the Ad-BMP2 group, and the expression levels were significantly higher in both groups compared with that in the control group (Figure 2, P < 0.05). We also detected the Sox9 downstream regulatory genes type II collagen (Col2a1) and aggrecan using the western blotting method. The results showed that SIRT1 could significantly promote the expression of Col2a1 and aggrecan in BMP2-induced chondrogenic differentiation of the C3H10T1/2 cells (Figure 2, P < 0.05). At the same time, we examined the levels of Sox9 and Col2a1 mRNA on the 5^th (Figure 3A, 3E, p < 0.05), 7^th (Figure 3B, 3F, p < 0.05), and 9^th (Figure 3C, 3G, p < 0.05) day using real-time PCR. PCR analysis showed that the expression of cartilage markers (Sox9 and Col2a1) in both the Ad-SIRT1+Ad-BMP2 group and Ad-BMP2 group was significantly higher than that in the control group, and the expression of cartilage makers in the Ad-SIRT1+Ad-BMP2 group was significantly higher than that in the Ad-BMP2 group. In addition, Alcian blue staining was used to observe the secretion of extracellular matrix after the 5^th (Figure 3D) and 7^th (Figure 3H) day of adenovirus infection. The results showed that cells in the Ad-BMP2+Ad-SIRT1 group secreted more extracellular matrix than those in the BMP2 group.

Figure 2. SIRT1 potentiated the BMP2-induced expression of chondrogenic differentiation makers. (A) The Sox9, Col2a1, and aggrecan protein levels in C3H10T1/2 cells infected with Ad-GFP, Ad-SIRT1, Ad-BMP2, or Ad-BMP2+Ad-SIRT1 for 5 days. (B) The Sox9, Col2a1, and aggrecan protein levels in C3H10T1/2 cells infected with Ad-GFP, Ad-SIRT1, Ad-BMP2, or Ad-BMP2+Ad-SIRT1 for 7 days. (C) The Sox9, Col2a1, and aggrecan protein levels in C3H10T1/2 cells infected with Ad-GFP, Ad-SIRT1, Ad-BMP2, or Ad-BMP2+Ad-SIRT1 for 9 days. All of the relative protein expression level results were compared to those of GAPDH using Quantity one. The data are denoted as the mean ± SD.*:p < 0.05 vs. Ad-GFP; #:p < 0.05, Ad-BMP2 group vs. Ad-BMP2+Ad-SIRT1 group.

Figure 3. The C3H10T1/2 cells were cultured in 6-well plates after being infected with Ad-GFP, Ad-SIRT1, Ad-BMP2, or Ad-BMP2+Ad-SIRT1. (A–C) The Sox9 mRNA levels of C3H10T1/2 cells were measured after 5, 7 and 9 days using real-time PCR. (E–G) The Col2a1 mRNA levels of C3H10T1/2 cells were measured after 5, 7 and 9 days using real-time PCR. (D, H) Alcian blue staining was used to observe the secretion of extracellular matrix after the 5 and 7 days of Ad-GFP, Ad-SIRT1, Ad-BMP2, and Ad-BMP2+Ad-SIRT1 infection. The dyed 12-well plates were photographed by a camera attached to a light microscope (100X). All of the relative mRNA expression level results were compared to those of GAPDH using Quantity one. The data are denoted as the mean ± SD.*:p < 0.05 vs. Ad-GFP; #:p < 0.05, Ad-BMP2 vs. Ad-BMP2+Ad-SIRT1.

SIRT1 potentiated BMP2-chondrogenic differentiation of C3H10T1/2 cells in vivo

In order to verify the effect of SIRT1 on BMP2-induced chondrogenic differentiation of stem cells, we also carried out stem cell transplantation experiments. After 2 and 4 weeks of injection with C3H10T1/2 cells, the animals were euthanized, and the bony mass was removed. The C3H10T1/2 cells infected with either Ad-SIRT1 or Ad-GFP alone did not yield any detectable masses (Figure 4A). We measured the bone tissue size of the Ad-BMP2 and Ad-BMP2+Ad-SIRT1 group, and there was no significant difference between them (Figure 4B). Hematoxylin and eosin, Alcian blue, and Masson trichrome staining were performed on the extracted bony mass (Figure 4C). The staining showed that the cartilage morphology including chondrocytes and chondrocyte lacunae in the Ad-SIRT1+Ad-BMP2 group was more obvious than that in the Ad-BMP2 group after 2 weeks of injection. At the fourth week, the cartilage lacuna and differentiation of bone tissue were more obvious compared in two weeks. Simultaneously, in the Ad-SIRT1+Ad-BMP2, the bone tissue was more mature and differentiated into osteoblasts compared with group infected with BMP2 in four weeks.

Figure 4. SIRT1 potentiated BMP2-induced chondrogenesis in vivo. (A) Macrographic images of the ectopic bone mass. The ectopic osseous masses were retrieved after 2 and 4 weeks. (B) The bone mass volumes were evaluated. (C) The bone masses retrieved from the Ad-BMP2 and Ad-BMP2+Ad-SIRT1 group were fixed and decalcified, and hematoxylin and eosin, Alcian blue, and Masson trichrome staining was performed on the extracted bony mass (200X). NS: p > 0.05.

SIRT1 alleviated the apoptosis of BMP2-induced chondrogenic differentiation stem cells under oxidative stress

CCK8 was used to detect apoptotic cells treated with different concentrations (0, 50, 100, 200, and 400 μM) of H₂O₂ (Figure 5A). With the increase in H₂O₂ concentration, the decrease of cell absorbance at 450 nm indicated an increase in cell apoptosis. We selected the appropriate concentration (200 μM) according to the results of the CCK8 assay for subsequent experiments. We used flow cytometry to detect apoptosis of Ad-BMP2, Ad-BMP2+H₂O₂, and Ad-BMP2+Ad-SIRT1+H₂O₂ cells (Figure 5B). The results showed that H₂O₂ could promote cell apoptosis, but the apoptotic rate of the Ad-BMP2+Ad-SIRT1+H₂O₂ group was significantly lower than that of the Ad-BMP2+H₂O₂ group. In addition, we detected apoptosis-related proteins (Bcl-2, Bax, Cleaved-PARP-1, Cleaved-Caspase3) using the western blotting method (Figure 5C, 5D). The results showed that the expression of Bax, Cleaved-PARP-1, and Cleaved-Caspase3 proteins increased and the expression of Bcl-2 protein decreased in the Ad-BMP2+H₂O₂ group compared with the Ad-BMP2 group, and the Ad-BMP2+Ad-SIRT1+H₂O₂ group exhibited opposite results.

Figure 5. SIRT1 alleviated the apoptosis of BMP2-induced chondrogenic differentiation stem cells under oxidative stress. (A) Cell Counting Kit-8 assay to detect cell proliferation under different concentrations (0, 50, 100, 200, or 400 μM) of H₂O₂. (B) The cell apoptotic rates were measured using a flow cytometry assay under 200 μM of H₂O₂. (C, D) H₂O₂ increased the expression of Cleaved-Caspase3, Bax, and Cleaved-PARP-1 and reduce the expression of Bcl-2 compare to the control group. The Ad-BMP2+Ad-SIRT1+H₂O₂ group exhibited opposite results. The data are denoted as the mean ± SD.*: p < 0.05 Ad-BMP2+H₂O₂ vs. Ad-BMP2; #: p < 0.05, Ad-BMP2 + H₂O₂ vs. Ad-BMP2+Ad-SIRT1+H₂O₂.

SIRT1 inhibited the decomposition of extracellular matrix in BMP2-induced-C3H10T1/2 cells under oxidative stress

We detected the secretion of extracellular matrix (Col2a1, aggrecan) in BMP2-induced C3H10T1/2 cells treated with 200 μM of H₂O₂ by an immunofluorescence assay (Figure 6A, 6B). The results showed that there were less Col2a1 and aggrecan proteins in the Ad-BMP2+H₂O₂ group than in the Ad-BMP2 group, while there were more proteins expressed in the Ad-SIRT1+Ad-BMP2+H₂O₂ group than in the Ad-BMP2+H₂O₂ group. At the same time, we detected the synthesis and decomposition of extracellular matrix proteins (Sox9, Col2a1, aggrecan, MMP2) by the western blotting method (Figure 6C, 6D). The results showed that the expression of Sox9, Col2a1, and aggrecan proteins decreased and the expression of MMP2 increased in the Ad-BMP2+H₂O₂ group compared with the Ad-BMP2 group, and Ad-BMP2+Ad-SIRT1+H₂O₂ group showed opposite results. We also detected the acetylation level of p65 (Figure 6E, 6F) and found that the acetylation level of p65 increased after adding H₂O₂ stimulation, while the acetylation level of p65 in the Ad-SIRT1+BMP2+H₂O₂ group was significantly lower than that of the Ad-BMP2+H₂O₂ group. These results suggested that SIRT1 not only alleviated apoptosis but also inhibited the decomposition of extracellular matrix in BMP2-induced chondrogenesis in C3H10T1/2 cells under oxidative stress.

Figure 6. SIRT1 inhibited the decomposition of extracellular matrix in BMP2-induced-C3H10T1/2 cells under oxidative stress. (A, B) Immunofluorescence analysis of the expression of Col2a1 and aggrecan among the Ad-BMP2, Ad-BMP2+H₂O₂, and Ad-BMP2+Ad-SIRT1+H₂O₂ groups (200X). The Col2a1 and aggrecan proteins were labeled in red, and the nuclei were labeled in blue by DAPI staining. (C, D) The expression of Sox9, Col2a1, and aggrecan proteins decreased in the Ad-BMP2+H₂O₂ group compared with the Ad-BMP2 group, while the expression of Sox9, Col2a1, and aggrecan proteins increased in the Ad-SIRT1+Ad-BMP2+H₂O₂ group compared with that of the Ad-BMP2+H₂O₂ group. The expression of MMP2 increased in the Ad-BMP2+H₂O₂ group compared with that in the Ad-BMP2 group, while the expression of MMP2 in the SIRT1+BMP2+H₂O₂ group was significantly lower than that of the Ad-BMP2 group. (E, F) The expression of Ac-p65 and p65 among Ad-BMP2, Ad-BMP2+H₂O₂, and Ad-BMP2+Ad-SIRT1+H₂O₂ groups. The data are denoted as the mean ± SD. *: p < 0.05, Ad-BMP2+H₂O₂ vs. Ad-BMP2; #: p < 0.05, Ad-BMP2+Ad-SIRT1+H₂O₂ vs. Ad-BMP2+H₂O₂.

HEK-293 cells and the C3H10T1/2 cell line culture

The HEK-293 cells and mesenchymal stem cell line (C3H10T1/2) used in this study were purchased from the American Type Culture Collection (ATCC; USA). They were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Hyclone, USA) supplemented with 10% fetal bovine serum (FBS, Biological Industries, Israel), 100 U/mL streptomycin, and 1% penicillin (Beyotime, China). The cells were incubated in an atmosphere containing 5% CO₂ at 37°C.

Recombinant adenoviruses expressing GFP, BMP2, and SIRT1 and cell transfection

The human SIRT1 was polymerase-chain-reaction (PCR)-amplified, cloned into an adenoviral shuttle vector, and subsequently used to generate recombinant adenoviruses in the HEK-293 cells. The primers used to amplify the coding regions included SIRT1 F:5’-ACAATTTAAATATGATTGGCACAGATCCTC-3’and R:5’-ATTAGCGGCCGCTGAT TTGTTTGATGGA-3’. SwaI and NotI enzymes were used to clone the BMP2 coding regions into the shuttle vector. The resulting adenoviruses were denoted as Ad-SIRT1 and Ad-GFP. The green fluorescent protein (GFP) was used to monitor infection efficiency via fluorescence microscopy (Nikon TE200-U, Japan). Ad-SIRT1, which expressed the SIRT1 protein, also expressed GFP. Ad-GFP, which only expressed GFP, was used as the control. The Ad-BMP2 was denoted by TC He, and it has been used in many studies [12, 14, 28].

Alcian blue staining

The C3H10T1/2 cells were seeded in a 12-well plate; infected with Ad-GFP, Ad-SIRT1, Ad-BMP2, or Ad-BMP2+Ad-SIRT1; and subsequently were stained with Alcian blue on the 5^th and 7^th day to detect the secretion of extracellular matrix. The plates were washed with phosphate buffered saline (PBS), fixed in 4% paraformaldehyde for 30 min, and washed again with PBS. The cells were stained with Alcian blue solution (Solarbio, China) according to the instructions and then photographed.

Cell proliferation assay

The cell counting kit-8 assay (CCK8, MedChemexpress, USA) was used to investigate cell proliferation. The C3H10T1/2 cells were seeded in 96-well plates at a density of 2000 cells/well, following treatment with H₂O₂ (0, 50, 100, 200, 400 μM). CCK8 reagent was added into wells, and cell proliferation was detected at 0, 24, 48, and 72 h by measuring the absorbance at 450 nm with a microplate reader.

Apoptosis assay

Flow cytometry was used to detect cell apoptosis. Cells were processed in 6-well plates for 48 h. Cells were digested with 0.25% EDTA-free trypsin, washed twice with PBS, and collected in 1.5 ml EP tubes. Annexin V APC-A (Threebond, China)/DAPI (Beyotime, China) was added according to the instructions, and the apoptosis was examined by a flow cytometer. The stained cells were subjected to flow cytometry to identify live cells (Annexin V APC-A-/DAPI PB 450-A-), early apoptotic cells (Annexin V APC-A+/DAPI PB 450-A-), late apoptotic or secondary apoptotic cells (Annexin V APC-A+/DAPI PB 450-A+), and necrotic cells (Annexin V APC-A-/DAPI PB 450-A+).

Immunofluorescence assay

The C3H10T1/2 cells were seeded in a 6-well plate with glass coverslips and infected with Ad-BMP2, Ad-BMP2+H₂O₂, or Ad-BMP2+Ad-SIRT1+H₂O₂ for 3 days. The cells were washed with PBS, fixed with 4% paraformaldehyde for 30 min at room temperature, and again washed with PBS. The cells were permeabilized with 0.1% Triton X-100 (Beyotime, China) at room temperature for 20 min, blocked with 1% bovine serum albumin (Beyotime, China) for 1 h, and incubated with primary antibody (anti-Col2a1, 1:200; anti-Aggrecan, 1:50) at 4°C overnight. Subsequently, the cells were incubated with Alexa Fluor®594-conjugated secondary antibody for 1 h in the dark at 37°C and washed with PBS. Finally, the nuclei were stained with DAPI (Beyotime, China) for 5 min in the dark.

Subcutaneous stem cell implantation

The C3H10T1/2 cells were infected with Ad-GFP, Ad-SIRT1, Ad-BMP2, or Ad-BMP2+Ad-SIRT1 at a density of 70%. Twenty four hours after infection, the cells were digested, collected, and resuspended with PBS to 5 × 10⁷ cells/ml. Each athymic nude mouse (groups of 4 to 6 week-old males, Beijing HFK Bioscience Corporation, China) was injected with 100 μl PBS (about 5 × 10⁶ cells), subcutaneously. After two or four weeks of subcutaneous implantation, nude mice were euthanized, and bone mass was removed. All of the experiments involving animals were performed according to institutional animal guidelines (Ethics Committee of Chongqing Medical University).

Hematoxylin and eosin, alcian blue, and Masson trichrome staining

The retrieved tissues were fixed in 4% paraformaldehyde, decalcified, and embedded in paraffin. Hematoxylin and eosin, Alcian blue, and Masson’s trichrome staining were performed as described [29].

Real-time PCR

Total RNA was extracted with TRIzol® reagent (Takara, China), following the manufacturer's protocols. Then, the total RNA was reverse transcribed into cDNA using the PrimeScript™ RT reagent kit (Takara, China). Finally, the cDNA was amplified using SYBR Green Super Mixture, and PCR was conducted with a CFX-Connect Real-Time PCR system (Bio-Rad). The amplification conditions were as follows: 95°C for 30 sec, followed by 40 cycles of 95°C for 5 sec and 60°C for 1 min, and the melt curve at 60°C to 95°C, with an increment of 0.5°C for 5 sec. The primer sequences were as follows:

Sirt1-F: 5’-TGATTGGCACCGATCCTCG-3’, Sirt1-R: 5’-CCACAGCGTCATATCATCCAG-3’; BMP2-F: 5’-GGGACCCGCTGTCTTCTAGT-3’, BMP2-R: 5’-TCAACTCAAATTCGCTGAGGAC-3’; Col2A1-F: 5’-GGGTCACAGAGGTTACCCAG-3’, Col2A1-R: 5’-ACCAGGGGAACCACTCTCAC-3’; Sox9-F: 5’-AGTACCCGCATCTGCACAAC-3’, Sox9-R: 5’-ACGAAGGGTCTCTTCTCGCT-3’; GAPDH-F: 5’-AGGTCGGTGTGAACGGATTTG-3’, GAPDH-R: 5’-GGGGTCGTTGATGGCAACA-3’.

Western blotting analysis

Proteins were extracted with lysis buffer and phenylmethanesulfonyl fluoride (Beyotime, China). A bicinchoninic acid kit (Beyotime, China) was used to detect the protein concentration. Total cell proteins were separated by SDS-PAGE (Beyotime, China) and transferred to a polyvinylidene fluoride membrane. The membranes were blocked in 5% skimmed milk for 1 h at room temperature and incubated in specific antibodies at 4°C overnight. The blots were washed three times with TBST and incubated in secondary antibodies for 1 h at room temperature. The Immobilon Western Chemiluminescent Kit (NCM Biotech, China) was used to detect protein levels. The primary antibodies used included: anti-aggrecan (Santa Cruz Biotechnology, USA; 1:500), anti-Sox9 (Abcam, USA; 1:1000), anti-Bcl-2 (Abcam, USA; 1:2000), anti-Bax (Abcam, USA; 1:2000), anti-SIRT1 (Abcam, USA; 1:1000), anti-GAPDH (Abcam, USA;1:10000), anti-NF-κB p65 (Abcam, USA; 1:1000), anti-Acetyl-NF-κBp65 (Lys310) (Affinity Biosciences, USA; 1:500), anti-BMP2 (Immunoway, USA; 1:500), anti-Col2a1 (Immunoway, USA; 1:500), anti-MMP2 (Immunoway, USA; 1:500), anti-Cleaved-Caspase3 (Immunoway, USA; 1:1000), and anti-Cleaved-PARP-1 (Immunoway, USA; 1:500).

Statistical analysis

Experiments were repeated three times, and the data are expressed as the mean ± standard deviation (SD). Any statistically significant differences between groups were evaluated using a one-way variance analysis (SPSS cooperation, SPSS 17.0). P < 0.05 was considered to indicate a statistically significant difference.