2.1 Cell culture and differentiation
MC3T3-E1 cells were cultured in α-MEM with 10% fetal bovine serum (FBS, Gibco, USA) and 1% penicillin–streptomycin (Beyotime, China) at 37℃ in atmosphere containing 5% CO2. The cells were passaged every 2 days once they grew to approximately 80% confluence. Osteogenic differentiation was induced with a medium containing 10% FBS, 1% penicillin–streptomycin, 10 mM β-glycerophosphate (Solarbio, USA), 50 µM ascorbic acid (Solarbio, USA), and 100 nM dexamethasone (Solarbio, USA). In order to examine the effect of melatonin (MLT, Beyotime, China) on osteoblasts treated with H2O2, cells were incubated in α-MEM alone (control), H2O2 (pretreated with 400 µM H2O2 for 4 h and then incubated with α-MEM for 24 h), H2O2 with MLT (pretreated with 400 µM H2O2 for 4 h then incubated with 100 µM melatonin for 24 h), H2O2 with EX527 and MLT (EX527 is a SIRT1 inhibitor, cells were pretreated with 400 µM H2O2 for 4 h, then incubated with 10 µM EX527 for 2 h and 100 µM melatonin for 24 h).
2.2 Alkaline phosphatase (ALP) staining and activity assay
To evaluate the impact of melatonin on the osteogenic differentiation of MC3T3-E1 cells under the influence of H2O2, ALP staining and activity assays were conducted. Seven days after osteogenic induction, 4% paraformaldehyde (Beyotime, China) was added to fix the cells for 20 min and then stained with an BCIP/NBT working solution (Beyotime, China). The stained cells were then photographed using a microscope. To assess ALP activity, the Alkaline Phosphatase Assay Kit (Nanjing Jiancheng Bioengineering Institute, China) and BCA Protein Assay Kit (Beyotime, China) were used in accordance with the manufacturer’s instructions.
2.3 Alizarin Red S (ARS) staining and mineralization assay
Twenty-one days after osteogenic induction, 4% paraformaldehyde was added to fix the cells for 20 min and then stained with 0.2% ARS (Solarbio, China). To quantify mineralization, the stained cells were incubated with 10% cetylpyridinium chloride (Solarbio, USA) for 1 h and the absorbance was measured by a microplate reader (Bio Tek Instruments, Inc; USA) at 570 nm.
2.4 Evaluation of intracellular ROS levels
The cells were washed with PBS and then stained with 2’,7’-dichlorodihydro-fluorescein-diacetate (DCFH-DA) to detect ROS levels using the Reactive Oxygen Species Assay Kit (KeyGEN BioTECH, China). Images were recorded using a fluorescence microscope (Nikon eclipse Ti-U; USA). Meanwhile, the mean fluorescence intensity was measured using flow cytometry (FC500MPL, Beckman Coulter, USA) to evaluate the levels of ROS.
2.5 Assays for malondialdehyde (MDA) levels and superoxide dismutase (SOD) activity
MDA and SOD, both biomarkers of oxidative stress, were detected using the Cell Malondialdehyde assay kit and Superoxide Dismutase assay kit (Nanjing Jiancheng Bioengineering Institute, China), respectively, in accordance with the manufacturer’s instructions. The protein concentration of each sample was measured using a BCA Protein Assay Kit. MDA concentrations were manifested as nmol/mg protein and SOD activity as U/mg protein.
2.6 Detection of mitochondrial membrane potential (MMP)
JC-1 is a sensitive indicator of MMP. When MMP is high, the JC-1 fluorescent probe emits red fluorescence, but when it is low, this probe emits green fluorescence. Low MMP implies cell apoptosis. JC-1 aggregates and JC-1 monomers were detected in treated cells and photographed using a fluorescence microscope. The acquired images were further assessed using Image J software (version 1.52v, NIH, USA).
2.7 Analysis of apoptosis
The Annexin V-FITC/PI Apoptosis Detection Kit (KeyGEN BioTECH, China) was used to detect the effect of different treatments on apoptosis. Stain cells with Annexin V and PI and then subjected to flow cytometry. Positive Annexin V staining and negative PI staining indicated viable apoptotic cells, whereas double positive staining indicated non-viable apoptotic cells. The sum of the two counts was used to calculate the apoptosis rate.
2.8 Western blotting
Treated cells were lysed on ice using RIPA buffer (Beyotime, China) containing a protease inhibitor (Beyotime, China), and the protein concentration was measured using the BCA Protein Assay Kit. The proteins were separated by SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with bovine serum albumin for 2 h and then incubated with the following primary antibodies overnight at 4℃: BMP2 (1:1000, AF5163, Affinity, USA), RUNX2 (1:1000, AF5186, Affinity, USA), OPN (1:1000, AF0227, Affinity, USA), Bcl2 (1:1000, AF6139, Affinity, USA), Bax (1:1000, AF0120, Affinity, USA), cleaved caspase3 (1:1000, AF7022, Affinity, USA), caspase3 (1:1000,AF6311,Affinity,USA), SIRT1 (1:1000, DF6033, Affinity, USA), p66SHC (1:1000,AF6245,Affinity,USA) and β-actin (1:10000, AF7018, Affinity, USA). The following day, the membranes were incubated with HRP-labeled secondary antibodies (1:5000, S0001, Affinity, USA) for 2 h. Then, the bands were developed using an enhanced chemiluminescence kit (KF005, Affinity, USA) and quantified by Image J software.
2.9 Experimental animals
All animal experiments were approved by the Ethical Committee of Yijishan Hospital. Forty eight-week-old female Sprague Dawley rats weighing 220–240 g were housed in the Central laboratory of Yijishan Hospital for groups of four. The temperature was controlled at 22±2℃, the humidity was controlled at 50%, the light cycle was controlled at 12 h light/dark and all the rats had free access to food and water.
2.10 Surgery and treatment
The standard method was used for bilateral ovariectomy (n=25) and sham surgery (n=15). After being ovariectomized (OVX), rats were fed normally for three months until the osteoporosis model was established. Then, five rats each from the OVX and sham group were sacrificed. Micro-computed tomography (micro-CT, SCANCO µCT-100, Switzerland) was used to measure the bone mineral density (BMD) of the left femur in each rat, and the right femur was used for hematoxylin-eosin (HE) staining to verify the successful establishment of the osteoporosis model. Once the osteoporosis model was established, all animals underwent surgery to induce bilateral femur defects according to the standard protocol. After the operation, the rats were segregated into three groups: control group (n=10, sham, bone defect surgery, and intraperitoneal injection of the same amount of saline from 4 to 6 pm daily), OVX group (n=10, ovariectomy, bone defect surgery, and intraperitoneal injection of the same amount of saline from 4 to 6 pm daily), and melatonin (MLT) group (n=10, ovariectomy, bone defect surgery, and intraperitoneal injection of 50 mg/kg melatonin from 4 to 6 pm daily). All rats were sacrificed two months later, the left femur was examined using micro-CT, and the right femur was subjected to HE staining, Masson’s trichrome staining, and immunohistochemical staining after decalcification.
2.11 Micro-CT analysis
To analyze the microstructure of the femoral metaphysis, micro-CT was used and the microarchitecture of the defect zone was evaluated. The following parameters were used: scanning voltage, 70 kV; current, 200 µA; exposure time, 300 ms; and thickness, 15 µm. To assess bone regeneration, a 2.0-mm-diameter circular area containing the bone defect zone was selected as the volume of interest. Meanwhile, BMD, bone volume fraction (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th) and trabecular separation (Tb.Sp) were measured using built-in software after 3D reconstruction.
2.12 Histological and immunohistochemical Analysis
Histological and immunohistochemical analysis were conducted after two months of drug treatment. After fixation, decalcification, and paraffin embedding, the femur was incised into 5 µm slices and stained with HE and Masson’s trichrome stains for morphologic assessment. New bone formation after bone injuries was examined using a light microscope (Nikon eclipse Ti-U; USA). After Masson’s trichrome staining, new bone tissue and collagen fibers were stained blue, and mature bone and muscle fibers were stained red. The new bone formation in defect area was measured by Image J software (version 1.52v, NIH, USA).
The expression levels of osteocalcin (OCN) and type 1 collagen (COL1) in the femur were detected using immunohistochemistry. Briefly, slices were deparaffinized with xylene, immersed in 3% H2O2 to remove endogenous catalase, blocked against non-specific antibodies, and then incubated with specific primary antibodies (OCN, 1:200, DF12303, Affinity; COL1, 1:100, AF7001, Affinity) overnight at 4℃. The next day, the slices were incubated with the secondary antibody and subjected to DAB staining, then counterstained with hematoxylin, dehydrated, and mounted. Antigen-expressing positive cells were stained brown. Photographs were obtained under a light microscope. The average optical density of OCN and COL1 were measured by Image J software.
2.13 Statistical analysis
All values were expressed as mean ± standard deviation (SD), and all statistical analysis were performed using GraphPad Prism 8 (USA). One-way analysis of variance (ANOVA) was used for comparisons among multiple groups, and independent samples t-tests were used for comparisons between two groups. P༜0.05 was considered statistically significant.