Aucubin exerts anti-osteoporotic effects by promoting osteoblast differentiation

AU protected MG63 cells against Dex-induced damage via modulation of Nrf2 signaling

AU reduced the apoptotic rate of MG63 cells exposed to 4 μM of Dex for 24 h in a dose-dependent manner (Figure 1A). Mitochondrial function is one of the factors contributing to apoptosis and it plays a role in the feedback loop that responds to ROS accumulation [17]. The over-accumulation of intracellular ROS (Figure 2B) and the enhanced dissipation of MMP (Figure 2C) in MG63 cells caused by Dex were all strongly relieved by AU at doses of 1, 2.5 and 5 μM, as shown by the reduced green fluorescence intensity, and enhanced ratio of red/green fluorescence intensity, respectively. Bcl-2 family members contribute to cell apoptosis related to mitochondrial function [15]. Compared to MG63 cells exposed to Dex alone, AU significantly enhanced the expression levels of Bcl-2 and reduced the expression levels of Bax and cleaved caspase-3 (P <0.05) (Figure 1D).

Figure 1. AU protected MG63 cells against Dex damage. (A) AU reduced the apoptosis rate of MG63 cells caused by Dex after 24 h incubation. (B) AU suppressed the over-accumulation of ROS in MG63 cells caused by Dex after 24 h incubation. (C) AU inhibited the dissipation of MMP in MG63 cells caused by Dex. (D) AU enhanced the expression levels of Bcl-2, and reduced the expression levels of Bax and cleaved caspase-3 in MG63 cells exposed to Dex. The quantification data of the expression levels of Bcl2, casepase3 and Bax were normalized by corresponding GAPDH. Data are expressed as mean ± S.D. (n=6) and analyzed using a one-way ANOVA. # P<0.05 and ## P<0.01 vs. control cells, *P<0.05 and **P<0.01 vs. Dex-exposed cells.

According to our study and previous findings, proteins including collagen I, osterix, OPN, BMP-2, OCN, and Smads are biomarkers of osteoblast differentiation [16]. 24-h 4 μM DEX exposure strongly reduced the expression levels of all proteins related to osteoblast differentiation, including collagen I, osterix, OPN, BMP-2, OCN, and P-Smads (P <0.05) (Figure 2A), which were all up-regulated after AU incubation (P <0.05) (Figure 2A).

Figure 2. AU protected the Dex-caused MG63 cells apoptosis via regulation the Nrf2/HO-1 signaling. (A) AU up-regulated the expression levels of osteoblast differentiation related proteins including Osterix, OPN, BMP2, OCN and P-Smad in MG63 cells exposed to Dex. (B) AU increased the expression levels of proteins within the Nrf2/HO-1 signaling including P-DPR1, Nrf2, CAT, HO-1, HO-2, SOD-1 and SOD-2 in MG63 cells exposed to Dex. AU enhanced the expression levels of Nrf2 in both (C) nucleus and (D) cytoplasm of MG63 cells exposed to Dex. The quantification data of proteins were normalized by corresponding GAPDH and total proteins, respectively (n=4). (E) AU increased the mRNA levels of Nrf2 and NQO-1 in MG63 cells exposed to Dex. Marker size from top to bottom: 1000 bp, 700 bp, 500 bp, 400 bp, 300 bp, 200 bp and 100 bp. The data on quantified mRNA expression were normalized to the levels of β-actin (n=4). Data are expressed as mean ± S.D. and analyzed using a one-way ANOVA. # P<0.05, ## P<0.01 and ### P<0.001 vs. control cells, *P<0.05, **P<0.01 and ***P<0.001 vs. Dex-exposed cells.

In Dex-alone exposed MG63 cells, the levels of anti-oxidative proteins related to Nrf2 signaling were strongly reduced (P <0.05) (Figure 2B). Compared with Dex-damaged cells, AU treatment resulted in 89.8%, 40.2%, 22.5% 237.2%, 159.1%, 27.6%, and 98.5% increases in the expression levels of PDRP1, Nrf2, CAT, HO-1, HO-2, SOD-1, and SOD-2 in MG63 cells, respectively (P <0.05) (Figure 2B). Furthermore, in Dex-damaged MG63 cells, AU increased the expression levels of Nrf2 in both nucleus (P <0.001) (Figure 2C) and cytoplasm (P <0.001) (Figure 2D). Compared with Dex-damaged cells, AU enhanced the mRNA levels of Nrf2 (P <0.001) and NAD(P)H dehydrogenase [quinone] 1 (NQO1) (P <0.001) in MG63 cells (Figure 2E).

AU protected MG63 cells against H₂O₂-induced damage related to Nrf2 signaling

In H₂O₂-induced apoptotic MG63 cells, AU was protective against H₂O₂ damage via reducing the apoptosis rate (Figure 3A), suppressing the accumulation of ROS (Figure 3B), and inhibiting the dissipation of MMP (Figure 3C). Compared with H₂O₂-damaged MG63 cells, AU incubation resulted in a 13.6% increase in the expression of Bcl-2 (P <0.01; Figure 3D), and 25.7% and 29.2% reductions in the expression of Bax (P <0.001) (Figure 3D) and cleaved caspase-3 (P <0.01) (Figure 3D).

Figure 3. AU restored the damage of H₂O₂ on MG63 cells. (A) AU reduced the apoptosis rate of MG63 cells caused by H₂O₂. (B) AU suppressed the over-accumulation of ROS in MG63 cells caused by H₂O₂. (C) AU inhibited the dissipation of MMP in MG63 cells caused by H₂O₂. (D) AU enhanced the expression levels of Bcl-2, and reduced the expression levels of Bax and cleaved caspase-3 in MG63 cells exposed to H₂O₂. The quantification data of the expression levels of Bcl2, casepase3 and Bax were normalized by corresponding GAPDH. Data are expressed as mean ± S.D. (n=6) and analyzed using a one-way ANOVA. # P<0.05 and ### P<0.001 vs. control cells, *P<0.05, *P<0.01 and ***P<0.001 H₂O₂-exposed cells.

Compared with H₂O₂-damaged MG63 cells, AU up-regulated the expression of biomarkers of osteoblast differentiation, including collagen I, osterix, OPN, BMP-2, OCN, and P-Smads by 42.3%, 34.7%, 62.3%, 30.6%, 38.5%, and 62.3%, respectively (P <0.05) (Figure 4A). AU resulted in 654.5%, 35.9%, 33.4%, 69.5%, 99.1%, 213.7%, and 85.0% increases in the expression of PDRP1, Nrf2, CAT, HO-1, HO-2, SOD-1, and SOD-2 in H₂O₂-damaged MG63 cells (P <0.05) (Figure 4B). Furthermore, in H₂O₂-damaged MG63 cells, AU increased the expression levels of Nrf2 in both nucleus (P <0.01) (Figure 4C) and cytoplasm (P <0.01) (Figure 4D). Compared with H₂O₂-damaged cells, AU increased the mRNA levels of Nrf2 (P <0.001) and NQO1 (P <0.01) in MG63 cells (Figure 4E).

Figure 4. AU protected the H₂O₂-caused MG63 cells apoptosis via regulation the Nrf2/HO-1 signaling. (A) AU up-regulated the expression levels of osteoblast differentiation related proteins including Osterix, OPN, BMP2, OCN and P-Smad in MG63 cells exposed to H₂O₂. (B) AU increased the expression levels of proteins within the Nrf2/HO-1 signaling including P-DPR1, Nrf2, CAT, HO-1, HO-2, SOD-1 and SOD-2 in MG63 cells exposed to H₂O₂. AU enhanced the expression levels of Nrf2 in both (C) nucleus and (D) cytoplasm of MG63 cells exposed to H₂O₂. The quantification data of proteins were normalized by corresponding GAPDH and total proteins, respectively (n=4). (E) AU increased the mRNA levels of Nrf2 and NQO-1 in MG63 cells exposed to H₂O₂. Marker size from top to bottom: 1000 bp, 700 bp, 500 bp, 400 bp, 300 bp, 200 bp and 100 bp. The data on quantified mRNA expression were normalized to the levels of β-actin (n=4). Data are expressed as mean ± S.D. and analyzed using a one-way ANOVA. # P<0.05, ## P<0.01 and ### P<0.001 vs. control cells, *P<0.05, **P<0.01 and ***P<0.001 vs. Dex-exposed cells.

AU protected the Dex-damaged mice against osteoporosis

Compared with Dex-damaged mice with osteoporosis, 8-week AU administration made the cortical bone more continuous and reduced the number of osteoclasts, as detected by HȦE staining (Figure 5A). Giemsa staining revealed that AU treatment enhanced the number of trabecular osteoblasts in the mice (Figure 5B).

Figure 5. The effects of AU on the femoral histological changes of osteoporotic mice were detected by (A) H&E staining and (B) Giemsa staining (n=6).

The structural parameters of femur trabecular and cortical regions, and of the tibia cortical region, were detected via micro-CT (Figures 6 and Supplementary Figure 1). Compared with the control mice, thinner cortical bones and sparser trabecular bone were observed in the model mice with osteoporosis (Figure 6A). Comparatively, E2 and AU enhanced the thickness of the bone cortex and the density of the trabecular bone, as shown by the increased brightness (Figure 6A). Using standard 3D microstructural analysis, the bone mineral density (BMD), bone volume fraction (BV/TV), trabecular thickness (Tb.Th), trabecular spacing (Tb.Sp) and trabecular number (Tb.N) were calculated for each group. Compared with the control mice, reduced levels of BMD (P <0.01) (Figure 6B), BV/TV (P <0.05) (Figure 6C), Tb.Th (P <0.05; Figure 6D) and Tb.N (P <0.05) (Figure 6F), and the increased levels of Tb.Sp (P <0.05) (Figure 6F) and BS/BV (P <0.05) (Figure 6G) were noted in Dex-damaged mice with osteoporosis. Similar to the effects of E2, 8-week AU administration significantly enhanced the levels of BMD (P <0.01) (Figure 6B), BV/TV (P <0.05) (Figure 6C), Tb.Th (P <0.01) (Figure 6D), and Tb.N (P <0.05) (Figure 6F), and reduced the levels of Tb.Sp (P <0.05) (Figure 6F) and BS/BV (P <0.05; Figure 6G) in the osteoporotic mice.

Figure 6. The effects of AU on the femoral bone morphological changes and the levels of osteoporotic indexes of osteoporotic mice. (A) The photographs of femur in osteoporotic mice detecting via micro-CT. The levels of (B) BMD, (C) BV/TV, (D) Tb.Th, (F) Tb.N and (G) BS/BV among all groups were analyzed. Data are expressed as mean ± S.D. (n=6) and analyzed using a one-way ANOVA. # P<0.05 and ## P<0.01 vs. CTRL mice, *P<0.05 and **P<0.01 Dex-treated mice.

Administration of AU for 8 weeks at 15 mg/kg had no significant effects on the bone structures of healthy mice compared with the control mice (Figures. 5 and 6).

AU promoted osteoblast differentiation in osteoporotic mice

Dex injection caused a significant reduction in the levels of factors related to osteoblast differentiation, including ALP, collagen I, OCN, OPN, BMP2 and BMPR2, and in the levels of Ca, Pi and E2 in the peripheral blood of Dex-injected mice with osteoporosis (P <0.05) (Table 1), which were strongly enhanced after 8 weeks’ AU and E2 administration (P <0.05) (Table 1). Administration of AU for 8 weeks at 15 mg/kg had no significant effects on the serum levels of these factors (Table 1).

Different from E2, AU showed no significant effects on the levels of TRACP-5b (Supplementary Figure 2A). AU at 15 mg/kg and E2 at 15 μg/kg strongly enhanced the levels of TNF-α compared with model mice (P<0.01) (Supplementary Figure 2B) Similarly, remarkably low expression levels of collagen I, osterix, OCN, OPN, BMP2, P-Smads and P-Akt were noted in the lysed tibias and fibulas of Dex-induced osteoporotic mice (P<0.05) (Figure 7A and 7B). Both E2 and AU treatment increased all of the detected proteins (P <0.05) (Figure 7A and 7B). AU administration resulted in 40.5%, 21.6%, 31.5%, 112.9%, 238.7%, 83.1%, and 56.2% increases in the expression levels of collagen I, osterix, OCN, OPN, BMP2, P-Smads and P-Akt, respectively, at 8 weeks (P <0.05) (Figure 7A and 7B). Among all of the detected proteins, administration of AU alone strongly enhanced the expression of osterix (P <0.05), but had no significant effects on other factors in the bone tissues of healthy mice compared with the control group (Figure 7A).

Figure 7. AU enhanced the osteoblast differentiation of femoral bone in Dex-ineduced osteoporotic mice. (A) AU enhanced the expression levels of osteoblast differentiation related proteins including collagen I, Osterix, OCN, OPN, BMP2, and P4HB in femoral bone tissues of Dex-ineduced osteoporotic mice. (B) AU enhanced the expression levels of P-Smad and P-Akt in femoral bone tissues of Dex-ineduced osteoporotic mice. (C) The effects of AU on the expression levels of Nrf2/HO-1 signaling related proteins in femoral bone tissues of Dex-ineduced osteoporotic mice, including P-DRP1, Nrf2, CAT, HO-1, HO-2, SOD-1, SOD-2, P-Smad and P-Akt. The quantification data of proteins were normalized by corresponding GAPDH and total proteins, respectively, expressed as mean ± S.D. (n=6) and analyzed using a one-way ANOVA. # P<0.05, ## P<0.01 and ### P<0.001 vs. CTRL mice, *P<0.05, **P<0.01 and ***P<0.001 Dex-treated mice.

Nrf2 signaling is involved in AU-mediated anti-osteoporotic activity

It is well known that oxidative stress inhibits bone cell differentiation and impairs bone integrity [18]. In Dex-induced osteoporotic mice, high levels of ROS and low levels of SOD and CAT were noted in peripheral blood (P <0.05) (Table 2). Compared with the osteoporotic mice, AU resulted in an 18.0% reduction in ROS, and 15.6% and 35.1% increases in SOD and CAT levels in peripheral blood (P <0.05) (Table 2). In the lysed tibias and fibulas of the Dex-induced osteoporotic mice, the expression levels of Nrf2 signaling proteins were all strongly reduced (P <0.05) (Figure 8). Comparatively, E2 and AU relieved these reductions (P <0.05) (Figure 8). AU, especially at doses of 15 mg/kg, caused 16.4%, 94.5%, 101.2%, 12.5%, 182.3%, 173.6%, and 30.2% increases in the expression levels of P-DPR1, Nrf2, CAT, HO-1, HO-2, SOD-1, and SOD-2 in bone tissues compared with those of the control mice (P <0.05) (Figure 7C).

Figure 8. The Nrf2-siRNA transfection strongly abolished the effects of AU on the protein expressions in Dex and H₂O₂ damaged MG63 cells. The up-regulation of AU on the expressions of Nrf2, SOD1, HO-1, OCN and BMP were strongly abolished in Nrf2-siRNA transfected MG63 cells after (A) Dex and (B) H₂O₂ exposure. The quantification data of proteins were normalized by corresponding GAPDH, respectively, expressed as mean ± S.D. (n=4) and analyzed using a one-way ANOVA. # P<0.05, ## P<0.01 and ### P<0.001 vs. control cells, *P<0.05, **P<0.01 and ***P<0.001 vs. Dex or H₂O₂-exposed cells.

Further experiments were performed in Nrf2-siRNA transfected MG63 cells. The enhancing effects of AU on the expressions of Nrf2, SOD1, HO-1, Collagen 1, OCN, and BMP2 were all suppressed by Nrf2-siRNA transfection in both Dex and H₂O₂ damaged transfected MG63 cells (Figure 8A and 8B). In contrast, the negative siRNA transfection failed to influence the modulatory effects of AU on the expressions of Nrf2, SOD1, HO-1, Collagen 1, OCN, and BMP2 in both Dex and H₂O₂ damaged MG63 cells (Supplementary Figure 3A and 3B).

Cell culture

MG63 human osteoblast-like cells (CRL-1427, passage < 10) were obtained from the American Type Culture Collection. The cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin at 37°C in a humidified incubator with 5% CO₂. All cell culture reagents were obtained from Gibco BRL (USA).

Measurement of cell apoptosis, MMP, and intracellular ROS

MG63 cells were seeded into 6-well plates at a density of 2 × 10⁵ cells / well. The cells were then treated with 1, 2.5, or 5 μM AU for 2 h followed by 4 μM Dex or 200 μM of H₂O₂ for 24 h. Following the incubation, the cells were harvested, resuspended in solution at a concentration of 1×10⁶ cells / mL, and stained with propidium iodide and/or Annexin V for 20 min at room temperature. The rate of apoptosis was measured using a Muse Cell Analyzer (Millipore, USA).

The MMP was evaluated by staining the cells with 2 μmoL / L 5,5’,6,6’-Tetrachloro-1,1’,3,3’-tetraethylbenzimidazolylcarbocyanine iodide (JC-1; Sigma-Aldrich, USA) at 37°C for 15 min in the dark. The cells were then washed three times with phosphate buffered saline (PBS) and red and green fluorescence recorded using a fluorescence microscope (20×; CCD camera, TE2000, Nikon, Japan).

Intracellular ROS was quantified by staining the cells with 10 μM 2’-7’-dichlorodihydrofluorescein diacetate (DCFH-DA, Sigma-Aldrich) for 15 min at 37°C in the dark. The cells were then washed three times with PBS and green fluorescence, which reflects the intracellular ROS level, recorded using a Nikon Eclipse TE 2000-S fluorescence microscope (Nikon, Japan).

Transfection of siRNA

MG63 cells were seeded into 6-well plates (2 × 10⁵ cells/well). The cells were then transiently transfected with 20 nM Nrf2-siRNA (5’-GGATGAAGAGACCGGAGAA-3’) (R10043.8, RiboBio, China) for 30 min using the riboFECT^TMCP Reagent (RiboBio) according to the manufacturer’s protocol. Following transfection, the cells were treated with 2.5 μM AU for 2 h and then incubated with either 4 μM Dex or 200 μM H₂O₂ for 24 h. The cells were harvested and the expression of Nrf2, SOD1, HO-1, Collagen I, OCN, BMP2, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) analyzed by western blotting.

Animal experiments

All animal protocols were approved by the Animal Ethics Committee of Jilin University (20160809). C57BL/6 mice (6–8 weeks old; 18–22 g) were obtained from Yis Laboratory Animal Technology Co., Ltd. (China). Mice were maintained at 23 ± 1°C with a 12-h photoperiod. Food and tap water were provided ad libitum. A total of 140 male mice were randomly divided into seven groups (n = 20). Control mice were injected intraperitoneally and intragastrically with 10 mL / kg of 0.9% normal saline every other day for 8 weeks. AU-treated mice were intraperitoneally injected with 10 mL/kg of 0.9% normal saline and intragastrically injected with 15 mg / kg AU every other day for 8 weeks. Osteoporotic mice were generated by intraperitoneal injection of 30 mg / kg of Dex sodium phosphate every other day for 8 weeks. The osteoporotic mice were intragastrically injected with either 10 mL / kg of 0.9% normal saline (n = 20) (model mice), or 5 mg / kg (n = 20), 15 mg / kg (n = 20), or 45 mg / kg (n = 20) AU, and intraperitoneally injected with 15 μg / kg estradiol (E2) (n = 20) (positive control mice) every other day for 8 weeks. The bodyweights of the mice were recorded on days 1, 4, 11, 18, 25, 32, 39, 48, and 55. Mice were euthanized after the last treatment, and tibia and femur tissue collected and weighed immediately.

Cytokine detection

Peripheral blood was collected from the caudal veins of the mice. The levels of plasma cytokines including ALP (YY02947B), BMP2 (YY02939B), BMP receptor type II (BMPR-2; YY02936B), OCN (YY03287B), OPN (YY03172B), collagen I (YY02771B), ROS (CK-E91516M), tumor necrosis factor α (TNF-α; YY02868B), E2 (YY03302B), platelet activating factor (PAF; S0000-96T), tartaric acid phosphatase 5b (TRACP-5b; YY03144B), SOD (YY03125B), malonaldehyde (MDA; YY03124B), catalase (CAT) (CK-E92636M), Ca²⁺ (C004-2), and Pi (C006) detected using enzyme-linked immunosorbent assays according to the manufacturer’s protocols (Shanghai Yuanye Biological Technology Co., Ltd, China).

Histological analysis of femur tissue

Femur tissue was collected immediately after the mice were euthanized and fixed in 4% paraformaldehyde. After incubation with a decalcification solution for 7 days, the tissue was embedded in paraffin, sectioned (5 μm thickness), and stained with hematoxylin and eosin and Giemsa. Sections were examined under a light-microscope equipped with a digital camera (Nikon, Japan).

Micro-computed tomography

Femurs and tibias were collected from mice immediately after euthanasia. The structure of the trabecular and cortical regions of the femur, and the cortical regions of the tibia were evaluated by micro-computed tomography (micro-CT) using a micro-CT μCT50 (Scanco, Switzerland). Standard 3D microstructural analysis was performed to analyze parameters including the trabecular bone mineral density (BMD), bone volume fraction (BV/TV), trabecular thickness (Tb.Th), trabecular spacing (Tb.Sp), and trabecular number (Tb.N).

Western blotting

MG63 cells were seeded into 6-well plates at a density of 2 × 10⁵ cells / well and then treated with 1, 2.5, or 5 μM AU for 3 h followed by 4 μM Dex or 200 μM H₂O₂ for 24 h. The cells were harvested and homogenized in RIPA lysis buffer (Sigma-Aldrich) containing 1% protease inhibitor cocktail and 2% phenylmethanesulfonyl fluoride (Sigma-Aldrich). Bone tissue lysates from AU-treated mice were generated by grinding tibias and fibulas from the mice into a powder under liquid nitrogen and homogenizing the powder in RIPA buffer as described. All lysates were then centrifuged at 10,000 rpm for 10 min. Protein concentrations were estimated using the BCA protein kit (Merck Millipore, USA). A total of 30 μg of protein was separated by 10%–12% SDS-PAGE and electrophoretically transferred onto nitrocellulose membranes (0.45 μm) (Bio Basic, Inc., Canada). The membranes were incubated with the following primary antibodies (1:2000 dilution) overnight at 4°C: collagen I (ab34710), OPN (ab91655), OCN (ab93876), osterix (ab22552), BMP2 (ab6285), integrin β1 (ab3167), Akt (ab131443), GAPDH (ab181602) (Abcam, USA), P-Akt (CST 4060S), and Smad1/5 (bs-4253R) (Bioss Inc., China). The following day, the membranes were washed and then incubated with horseradish peroxidase-conjugated secondary antibodies (1:2000) (Santa Cruz Biotechnology, USA). Protein bands was visualized using a BioSpectrum 600 imaging system (Bioss Inc., China). The pixel density was quantified using the Image J software (National Institutes of Health, USA).

Reverse transcription-polymerase chain reaction (RT-PCR)

The RNA was isolated from MG63 cells using Trizol (Invitrogen, USA), and then synthesized by QuantScript RT Kit (Tiangen Biotech Co. Ltd., Beijing China). β-actin primers were used as an internal control. The conditions of PCR amplification were shown as follows: denaturation at 95 °C for 5 min, followed by 36 cycles at 95 °C for 45 s, 57 °C for 45 s and 72 °C for 45 s. The primer sequences are listed in Supplementary Table 1.

Statistical analysis

Data are expressed as the mean ± standard deviation. One-way analysis of variance (ANOVA) followed by post-hoc Dunn’s multiple comparison tests was performed using SPSS 16.0 software (IBM Corporation, USA). P < 0.05 was considered significant.