Mouse pre-osteoblast cell line MC3T3-E1 was purchased from American Type Culture Collection. E2 was obtained from Sigma-Aldrich (cat. no. E4260; HPLC>98%; Merck KGaA). Transfection reagent Lipofectamine^® 3000 was purchased from Invitrogen (Thermo Fisher Scientific, Inc.). Cell Counting Kit-8 (CCK-8) was obtained from Nanjing Jiancheng Bioengineering Institute.

MC3T3-E1 cells (1×10⁶ cells/ml, 24-well plates) were cultured in a-minimum essential medium (α-MEM; Gibco; Thermo Fisher Scientific, Inc.) containing 10% FBS (Gibco; Thermo Fisher Scientific, Inc.), 1% antibiotics (100 µg/ml streptomycin and 100 U/ml penicillin) at 37°C in a humidified atmosphere with 5% CO₂. Lipofectamine^® 3000 was used following the manufacturer's protocol. Briefly, 2 µl Lipofectamine^® 3000 and 40 pmol of small interfering (si)RNA targeting Nrf2 (5′-GAGUAUGAGCUGGAAAAACUU-3′) or negative control siRNA (siNC; 5′-GACGAGCGGCACGUGCACAUU-3′) (Shanghai GenePharma Co., Ltd.) were mixed in 50 µl serum-free medium and incubated at room temperature for 15 min. The lipid compounds were diluted in 300 µl serum-free medium and 600 µl medium containing FBS, added to the cells and incubated at 37°C with 5% CO₂ for 24 h. Following transfection for 24 h, cells were harvested and used for subsequent experiments.

Different concentrations (0, 10, 50, 100, 200 and 300 mg/dl) of glucose were added to the medium and used to treat MC3T3-E1 cells to test viability and to determine the optimal glucose concentration for further experiments. The cells were incubated at 37°C with 5% CO₂ for 96 h. Then, the cell viability of the cells treated with the optimal glucose concentration for 1, 3, 5 and 7 days was determined. MC3T3-E1 cells were treated with 1, 10 nM, 0.1 and 1 µM E2 to test cell viability and to select an appropriate dose for further experiments (20). In subsequent experiments, cells were divided into the following groups: i) Control, cells were added to normal medium; ii) HG, cells treated with 200 mg/dl HG; iii) E2, cells treated with 0.1 µM E2; iv) siNrf2, cells transfected with siNrf2; v) NC, cells transfected with siNC; vi) HG + E2, cells co-treated with 200 mg/dl HG and 0.1 µM E2; vii) E2 + siNrf2, cells were co-treated with 0.1 µM E2 and siNrf2; viii) HG + E2 + siNC, cells co-treated with 200 mg/dl HG, 0.1 µM E2 and siNC; ix) HG + E2 + siNrf2, cells co-treated with 200 mg/dl HG, 0.1 µM E2 and siNrf2.

Cell viability was detected by CCK-8 assay according to the manufacturer's protocol. A total of 10 µl of CCK-8 solution was added to MC3T3-E1 cells (3×10³ cells/well) and the cells were incubated at 37°C for 2 h in the dark with 5% CO₂. Subsequently, the optical density of each well at 450 nm was determined by a microplate reader (Bio-Rad Laboratories, Inc.).

Treated MC3T3-E1 cells (2×10⁴ cells/well in 6-well plates) were washed twice with PBS, lysed with RIPA buffer (Cell Signaling Technology, Inc.) for 2 h on ice, and centrifuged at 12,000 × g for 30 min at 4°C. Subsequently, supernatant was collected. The nuclear and cytoplasmic extracts were prepared using an NE-PER Nuclear and Cytoplasmic Extraction Reagents kit (Pierce; Thermo Fisher Scientific, Inc.) according to the manufacturer's directions. The protein concentration was determined using the BCA protein kit (Bio-Rad Laboratories, Inc.) and adjusted to a concentration of 5 µg/µl using 1X loading buffer and diethyl pyrocarbonate (DEPC)-treated water. Proteins (at least 30 µg/lane) were separated by 10% SDS-PAGE, transferred to a PVDF membrane (Bio-Rad Laboratories, Inc.), blocked with 5% non-fat milk at room temperature for 2 h and washed with PBS three times for 5 min. The membranes were then incubated overnight at 4°C with the corresponding primary antibody, washed with PBS three times for 15 min, incubated with horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit immunoglobulin G secondary antibody (1:2,000; cat. nos. sc-2005 and sc-2004; Santa Cruz Biotechnology, Inc.) for 2 h at room temperature, washed with PBS three times for 15 min, and washed with PBS + 0.1% Tween-20 for 15 min. Development was carried out using the EZ-ECL developer kit (Biological Industries), densitometric analysis was performed using ImageJ 5.0 (National Institutes of Health). The antibodies used in the present study were as follows: Rabbit anti-β-actin (1:1,000; cat. no. LS-B1625; LifeSpan BioSciences, Inc.), mouse anti-Lamin B1 (1:1,000; cat. no. LS-B6062; LifeSpan BioSciences, Inc.), rabbit anti-Nrf2 (1:1,000; cat. no. ab62352; Abcam) and mouse anti-HO1 (1:1,000; cat. no. ab13248; Abcam). The detection method for nuclear Nrf2 protein was the same as for the other genes mentioned above, and the Lamin B1 was used an internal reference.

Total RNA was extracted from treated MC3T3-E1 cells (2×10⁵ cells) using TRIzol reagent (1 ml/well; Invitrogen; Thermo Fisher Scientific, Inc.). The lysate was transferred to 1.5 ml Eppendorf tubes and kept at room temperature for 5 min; subsequently 200 µl chloroform was added to each tube and inverted for 15 sec. Following emulsification, the tubes were left in room temperature for 5 min, centrifuged at 12,000 × g at 4°C for 15 mins, and the upper aqueous phase was pipetted into new 1.5 ml tubes with an equal volume of isopropanol (~400 µl); the tubes were kept at room temperature for 10 min. Following another centrifugation at 12,000 × g at 4°C for 15 min, the supernatant was discarded and 1 ml of pre-cooled 75% ice ethanol was added. The tubes were centrifuged at 7,500 × g at 4°C for 10 min, and the supernatant was discarded. DEPC-treated water (20 µl) was added to the tubes to dissolve the RNA. The purity and concentration of RNA was tested using the NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies; Thermo Fisher Scientific, Inc.) at 260/280 nm. RevertAid First Strand cDNA Synthesis kit (Thermo Fisher Scientific, Inc.) was used according to the manufacturer's protocol to reverse transcribe 1 µg total RNA to cDNA (42°C for 60 min followed by 70°C for 5 min). SYBR Green PCR Master Mix (Roche Diagnostics) was used to perform qPCR using ABI 7500 Real-Time PCR Detection System (Thermo Fisher Scientific, Inc.). The PCR cycle was as follows: Initial denaturation at 95°C for 10 min, followed by 40 cycles of 94°C for 15 sec and 60°C for 1 min; final extension at 60°C for 1 min. Relative mRNA quantity was determined using the 2^−ΔΔCq method (21) and normalized to the internal reference gene GAPDH. The primer sequences used for RT-qPCR analysis are presented in Table I.

Treated MC3T3-E1 cells (1.3×10⁵ cells/well) were seeded in six-well plates. Supernatant was collected in a 15 ml centrifuge tube, and the culture flask was washed once with 2 ml PBS. The cells were digested with 1 ml trypsin without EDTA and gently shaken. The mixture was kept at room temperature for 1 min, and α-MEM containing 10% FBS was added to terminate the digestion. The cells were centrifuged at 1,000 × g for 3 min and the supernatant was removed. The cells were washed twice with pre-cooled PBS and resuspended in 1X Annexin V binding buffer. Annexin-V-FITC Cell Apoptosis Detection kit (cat. no. K201-100; BioVision, Inc.) was used according to the manufacturer's protocol. Briefly, cells were collected and stained with Annexin V-FITC and propidium iodide (PI) at room temperature for 15 min and counted by flow cytometry using BD FACSCalibur™ with FlowJo software (version 10.0; BD Biosciences). Flow cytometry scatter diagrams demonstrate living cells in the lower left quadrant, necrotic cells in the upper left quadrant, advanced apoptotic cells in the upper right quadrant and early apoptotic cells in the lower right quadrant. The apoptosis rate of total apoptotic cells (early + advanced) was calculated.

All data were analyzed using GraphPad Prism 6 software (GraphPad Software, Inc.). All data are expressed as mean ± SEM. Comparisons of multiple groups were performed by analysis of variance between groups followed by Dunnett's test. P<0.05 was considered to indicate a statistically significant difference.

CCK-8 assay was used to detect cell viability of MC3T3-E1 cells treated with various concentrations of glucose for 96 h, which revealed that high concentrations of glucose decreased cell viability compared with cells cultured in medium with normal glucose levels (Fig. 1A). Therefore, 200 mg/dl glucose was selected for subsequent experiments. In addition, cell viability was inhibited by 200 mg/dl glucose on treatment days 3, 5 and 7 compared with control (0 mg/dl) (Fig. 1B).

Cell viability was tested in MC3T3-E1 cells by CCK-8 assay using a range of concentrations of E2. Cell viability was significantly increased by E2 at 0.1 µM (Fig. 2A); thus, 0.1 µM E2 was used for subsequent experiments. To investigate the effect of E2 on HG-induced osteoblast injury, cell viability, osteoblast differentiation markers osteocalcin and Runx2, and Nrf2 and HO1 expression levels were determined following co-incubation with E2 and HG. The results demonstrated that compared with the HG-only group, cell viability was increased (Fig. 2B), osteocalcin and Runx2 mRNA expression levels were increased (Fig. 2C) in the HG + E2 group. The mRNA expression level of HO1 in the HG + E2 group was significantly higher than that in HG-only group, and there was no significant difference in Nrf2 mRNA expression in control group, HG group, HG+E2 group and E2 group (Fig. 2D). The protein levels of nuclear Nrf2 and downstream target effector HO1 were increased (Fig. 2E-G) in the HG + E2 group compared with the HG-only group.

Markers of osteoblast differentiation were examined to detect changes in cell differentiation following HG, E2 and siNrf2 treatment. Nrf2 was effectively downregulated by siRNA transfection (Fig. 3A). Following Nrf2 silencing, mRNA expression levels of osteocalcin, Runx2, Nrf2 and HO1 were significantly reduced, which was reversed by E2; HG treatment significantly reduced the mRNA expression of osteocalcin, Runx2 and HO1; E2 treatment alleviated the inhibitory effect of HG on the mRNA expression of osteocalcin, Runx2, Nrf2 and HO1, while Nrf2 silencing reversed this protective effect of E2, excepting the expression of Runx2 (Fig. 3B-E).

Western blotting was used to identify the protein expression levels of Nrf2 and HO1. siNrf2 reduced the protein expression of Nrf2 and HO1 and the Nrf2 and HO1 protein levels in E2 + siNrf2 group were significantly lower than those in E2 group; E2 treatment alleviated the inhibitory effect of HG on the protein expression of Nrf2 and HO1, while Nrf2 silencing reversed this protective effect of E2 (Fig. 4).

MC3T3-E1 cell viability and apoptotic rates were examined by CCK-8 assay and flow cytometry, respectively. SiNrf2 treatment alone, E2 treatment alone, and siNrf2 and E2 co-treatment had no significant effect on cell viability, although the cell viability was inhibited in the HG group; E2 treatment effectively enhanced cell viability in HG-treated cells, which was inhibited by cotreatment with siNrf2 (Fig. 5A). Additionally, SiNrf2 treatment alone, E2 treatment alone, and siNrf2 and E2 co-treatment had no significant effect on cell apoptosis, although the apoptosis rate was promoted in the HG group. The apoptotic rate was significantly lower in the HG + E2 group compared with the HG-only group, whereas cells in the HG + E2 + siNrf2 group exhibited a higher apoptotic rate compared with the HG + E2 + siNC group (Fig. 5B and C).