α₂M was obtained from Enzo Life Sciences (Farmingdale, NY, USA). hBMMSCs were obtained from Cyagen Biosciences (Guangzhou) Inc. (HUXMA-01001; Guangzhou, China). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin mixture and 0.25% trypsin-EDTA were purchased from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Osteogenic induction medium was purchased from Cyagen Biosciences (Guangzhou) Inc. (GUXMX-90021). Antibodies against Beclin1 (1:1,000; cat. no. 11306-1-AP), microtubule-associated protein 1A/1B (LC-3; 1:1,000; cat. no. 14600-1-AP), sex determining region Y (Sox2; 1:5,000; cat. no. 11064-1-AP), Nanog (1:2,000; cat. no. 14295-1-AP), manganese superoxide dismutase (MnSOD; 1:3,000; cat. no. 24127-1-AP) and GAPDH (1:10,000; cat. no. 60004-1-Ig) were purchased from ProteinTech Group (Chicago, IL, USA). The runt-related transcription factor 2 (RUNX2; 1:1,000; cat. no. 12556) antibody was purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA), the osteoglycin (OGN; 1:1,000; cat. no. sc-365228) antibody was obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA), and the goat anti-rabbit (1:10,000; cat. no. ab6721) and goat anti-mouse (1:10,000; cat. no. ab6789) horseradish peroxidase (HRP) conjugated secondary antibodies were obtained from Abcam (Cambridge, MA, USA).

hBMMSCs were cultured in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C and 5% CO₂ with saturated humidity. The culture medium was replaced every 3 days. When the cells reached 80% confluence, they were digested with trypsin and passaged at a 1:3 ratio. The fourth generation of cells was used for subsequent analysis.

A total of 1 mg α₂M was dissolved in 0.5 ml sterilized ddH₂O to prepare a solution of 2.0 mg/ml α₂M, which was stored at −20°C. X-ray irradiation was performed when hBMMSCs attained 70% confluence. hBMMSCs were irradiated at the Sun Yat-Sen University Medical Experimental Center (Guangzhou, China). An RS2000 X-ray irradiator (Radsource, LLC, Brentwood, TN, USA) was used at a voltage of 160 kV, a current of 25 mA, and a dose rate of 1.24 Gy/min. Cells were irradiated at room temperature for 1.61, 3.23, 6.45 and 9.68 min to obtain a dose of 2, 4, 8 and 12 Gy, respectively.

hBMMSCs were randomly divided into the following groups: Blank control, radiation control, 0.25 and 0.50 mg/ml α₂M, respectively. The blank control group was not administered any treatment, whereas the remaining three groups received X-ray irradiation. The α₂M treatment groups were supplemented with α₂M to the final concentrations of 0.25 and 0.50 mg/ml, respectively, in the complete medium (DMEM supplemented with 10% FBS and 1% penicillin-streptomycin) and osteogenic induction medium (DMEM supplemented with 10% FBS, 1% penicillin-streptomycin, 50 mg/l ascorbic acid, 10 mmol/l β-glycerophosphate and 1 µmol/l dexamethasone). Following irradiation, cells in complete medium were treated with α2M for 24 h, whereas cells in the osteogenic induction medium were treated with α2M for 3 days at 37°C.

hBMMSCs were seeded into 96-well plates at a density of 5×10³ cells/well, with five wells per group. Following adhesion to the well, the cells were exposed to various doses of X-ray radiation, to explore the relationship between dose and cell proliferation. After incubation for 1, 3, 5 and 7 days, 10 µl CCK-8 (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) solution was added to each well. Following incubation for 2 h, the absorbance was measured at a wavelength of 450 nm using a 96-well-plate reader (Thermo Fisher Scientific, Inc.).

hBMMSCs were seeded at 100 cells/well and cultured in 6-cm petri dishes. After 2 weeks culture in complete medium at 37°C and 5% CO₂ with saturated humidity, the cells were washed twice with PBS, fixed with 4% paraformaldehyde for 20 min at room temperature, washed twice with ddH2O, and stained with 0.1% crystal violet (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) for 15 min at room temperature. Images of the colonies were obtained using an Epson scanner (Seiko Epson Corp., Suwa, Japan). Then, images were collected for analysis.

hBMMSCs were seeded into 6-well plates at a density of 4×10⁵ cells/well, and total RNA was extracted using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). RNA was reverse transcribed into cDNA using a PrimeScript™ RT Reagent Kit with gDNA Eraser (Takara Bio, Inc., Otsu, Japan), according to the manufacturer's instructions and amplified using a SYBR^® Premix Ex Taq™ II kit (Takara Bio, Inc.). The PCR cycling conditions were as follows: 95°C for 30 sec, followed by 40 cycles of 95°C for 5 sec and 60°C for 20 sec. The housekeeping gene GAPDH was used for normalization, and the relative expression was calculated by the 2^−ΔΔCq relative quantitation method (13). The primers used are listed in Table I. All primer sequences were determined via established GenBank sequences (https://www.ncbi.nlm.nih.gov/genbank/).

hBMMSCs were seeded into 6-well plates at a density of 4×10⁵ cells/well, and total protein was extracted using radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime Institute of Biotechnology, Haimen, China). Protein concentration was determined via a Bicinchoninic Acid (BCA) kit (Thermo Fisher Scientific, Inc.). A total of 30 µg protein was separated by 10% SDS-PAGE (Beyotime Institute of Biotechnology) at 120 V for ~1.5 h. Following the completion of electrophoresis, proteins were transferred to a polyvinylidiene difluoride membrane at a current of 280 mA for ~90 min. The membrane was blocked in 5% skim milk for 1 h at room temperature and then incubated at 4°C overnight with primary antibodies. The membrane was washed three times in tris-buffered saline with 0.1% Tween-20 (TBST) three times, followed by incubation with secondary antibodies at room temperature for 1 h. The membrane was washed three times with TBST, then treated with an Immobilon Western Chemiluminescent HRP Substrate (EMD Millipore, Billerica, MA, USA), according to the manufacturer's protocols. The grayscale values of the bands were analyzed by using ImageJ software v. 1.47 (National Institutes of Health, Bethesda, MA, USA) and the expression levels of the target protein were calculated by normalizing the gray value of the target protein to that of GAPDH.

When hBMMSCs attained 70% confluence, the complete medium was replaced with osteogenic induction differentiation medium, and cells were cultured at 37°C and 5% CO₂ with saturated humidity. The induction medium was replaced every 3 days. The gene expression of RUNX2 and OGN was detected following 1 and 2 weeks of culture in osteogenesis induction medium, respectively.

hBMMSCs were seeded into 12-well plates at a density of 4×10⁴ cells/well, and 200 µl of 1% Triton X-100 (Beyotime Institute of Biotechnology) was added to each well for lysis overnight at 4°C. Following the completion of lysis, 30 µl lysate was obtained for analysis. An ALP assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) was used according to the manufacturer's protocols to determine the absorbance at a wavelength of 520 nm, using a 96-well-plate reader (Thermo Fisher Scientific, Inc.). An additional 10 µl lysate was used to determine the protein concentration of each group of samples by using the BCA method. The activity was normalized to cellular protein and expressed as U/g.

hBMMSCs were seeded into 12-well plates at a density of 4×10⁴ cells/well and cultured in osteogenic induction medium for 3 weeks before 1 ml of 4% formaldehyde was added to each well at room temperature. After 30 min, the formaldehyde solution was removed, and cells were washed twice with PBS. Cells were then treated with 0.5 ml Alizarin red solution [Cyagen Biosciences (Guangzhou) Inc.] for 5 min at room temperature; following staining with Alizarin red dye, the cells were washed with PBS three times. Cells were analyzed under an optical microscope (Nikon Corporation, Tokyo, Japan; magnification, ×100) to observe staining. After washing with PBS, 10% cetylpyridinium chloride (Sigma-Aldrich; Merck KGaA) was added, and cells were lysed for 1 h at room temperature. The lysate was transferred to a 96-well plate, and the absorbance was measured at 570 nm using a 96-well-plate reader (Thermo Fisher Scientific, Inc.).

hBMMSCs were cultured in 6-well plates at a density of 4×10⁵ cells/well. Apoptosis was determined by flow cytometry using an Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining kit (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China). Cells were collected and stained in working solution (500 µl binding buffer with 5 µl Annexin V-FITC and 5 µl PI) for 15 min at room temperature in the dark. Apoptotic cells were analyzed using a BD FACS Canto II flow cytometer running BD FACSDiva software version 4.1 (Franklin Lakes, NJ, USA). Annexin V-FITC single positive and Annexin V-FITC/PI double positive cells were considered apoptotic cells. The ratio of apoptotic cells to total cells was used to calculate the cell apoptosis rate.

hBMMSCs were incubated in 12-well plates at 4×10⁴ cells/well, and 200 µl RIPA lysis buffer was added to each well. Following lysis, 40 µl lysate was obtained for analysis via a Superoxide Dismutase Assay kit (Nanjing Jiancheng Bioengineering Institute) according to the manufacturer's protocols. The absorbance values were measured at a wavelength of 450 nm using a 96-well-plate reader (Thermo Fisher Scientific, Inc.). The BCA method was applied using 10 µl lysate to determine the protein concentration for each group of samples, in which the protein per milligram of SOD activity was determined.

hBMMSCs were cultured in 6-well plates at 4×10⁵ cells/well in complete medium. 2′,7′-Dichlorodihydrofluorescein diacetate (Nanjing Jiancheng Bioengineering Institute) was diluted 1:1,000 with serum-free cell culture medium and incubated in the cell incubator for 20 min at 37°C. The cells were washed three times in serum-free cell culture medium, and analyzed under a Leica TCS-SP8 confocal microscope (Leica Microsystems GmbH, Wetzlar, Germany; magnification, ×100). An excitation wavelength of 502 nm was applied and images of the cells were collected.

All experiments were performed at least three times. The results were analyzed using SPSS software, version 20.0 (IBM Corp., Armonk, NY, USA) and are expressed as the mean ± standard deviation. The data were analyzed by one-way analysis of variance, followed by a Bonferroni post-hoc test for multiple comparisons. P<0.05 was considered to indicate a statistically significant difference.

hBMMSC proliferation decreased with increasing doses of radiation; the hBMMSCs treated with 8 and 12 Gy radiation exhibited significantly lower proliferation rates than the 0 Gy-treated group. Cell proliferation was too low in the 12 Gy group after 5 and 7 days of treatment; thus, 8 Gy was selected for follow-up experiments (Fig. 1A). The colony formation experiment demonstrated that hBMMSCs irradiated with 8 Gy X-rays and cultured with 0.50 mg/ml α₂M exhibited a significantly increased colony formation rate compared with in the irradiated group; however, the colony formation rate of the irradiated group was significantly decreased compared with in the control (Fig. 1B and C). RT-qPCR revealed that α₂M significantly downregulated the mRNA expression levels of Beclin1 and LC-3 in hBMMSCs in a dose-dependent manner compared with the irradiation group. Treatment with 0.50 mg/ml α₂M appeared to markedly downregulate the mRNA expression levels of Beclin and LC-3 compared with the 0.25 mg/ml α₂M-treated group; however, no notable differences were observed in the protein expression levels of LC-3 between the two α₂M treatment groups (Fig. 1D-G).

RT-qPCR revealed that the mRNA expression levels of Sox2 and Nanog were significantly upregulated at doses of 0.50 mg/ml α₂M, compared with in the radiation group; however, at doses of 0.25 mg/ml α₂M, Nanog mRNA expression was only markedly upregulated whereas Sox2 was significantly upregulated. Protein expression levels of Nanog and Sox2 were also significantly upregulated at doses of 0.50 mg/ml α₂M, compared with in the radiation group however, at doses of 0.25 mg/ml α₂M, Sox2 protein levels were markedly upregulated (Fig. 2A-D).

The mRNA and protein expression levels of RUNX2 and OGN were significantly increased with the addition of α2M following X-ray irradiation compared with in the radiation group; however, treatment with 0.25 mg/ml only markedly increased the protein expression level of RUNX2 (Fig. 3A-D). In addition, ALP activity significantly increased in response to α₂M treatment compared with in the radiation group (Fig. 3E). Microscopic analysis revealed the formation of calcium nodules and the degree of staining increased (Fig. 3F and G).

hBMMSCs were irradiated with X-rays and then treated with α₂M. The rate of apoptosis was significantly decreased in response to treatment with 0.50 mg/ml α₂M compared with in the irradiated group (Fig. 4A and B). Additionally, treatment with 0.50 mg/ml α₂M significantly increased SOD activity and the protein expression levels of MnSOD compared with in the radiation group (Fig. 4C-E). The levels of ROS were notably decreased following treatment with α₂M compared with in the radiation group (Fig. 4F).