Geniposide (purity >98%) was purchased from Pure-one Bio Technology, Co., Ltd (Shanghai, China). Geniposide was dissolved in water, pH 7.4. Cadmium chloride (CdCl₂) was purchased from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany).

Rat MC-3T3-E1 cells (Riken Cell Bank, Tsukuba, Ibaraki, Japan) were cultured in Dulbecco's modified Eagle's medium (DMEM; Thermo Fisher Scientific, Inc., Waltham, MA, USA) with 10% (v/v) fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and 100 U/ml penicillin (or 100 µg/ml streptomycin) in a 37°C incubator with 5% CO₂ humidified atmosphere. The morphology of primary cultured MC-3T3-E1 cells was observed using an inverted microscope (×40).

The CCK-8 assay kit (Beyotime Institute of Biotechnology, Haimen, China) was used to measure cell viability. MC-3T3-E1 cells (5×10³ cells/well) were cultured in 96-well plates and were treated with CdCl₂ (0–20 µM). Geniposide (100, 200 and 400 µg/ml) was used as previously described (37) to treat the cells in order to detect its effect on CdCl₂-induced injury.

For the cell viability assay, 10 µl CCK-8 solution was added into each well, and the cells were incubated for another 3 h at 37°C. Cell viability was determined using a microplate reader as previously described (38) by reading the optical density at a wavelength of 450 nm, and at a reference wavelength of 630 nm.

Cell apoptosis was detected in MC-3T3-E1 cell cultures using a flow cytometer. The cells were harvested and re-suspended in Annexin binding buffer at 1×10⁵ cells/ml. Then, the suspension was incubated with Annexin V-FITC and propidium iodide (PI) [cat. no. 70-AP101-60; MultiSciences (Lianke) Biotech Co., Ltd., Hangzhou, China] in the dark for 15 min at 4°C. The apoptosis of the cell samples was analyzed by flow cytometry with BD CellQuest Pro Software version 1.2 (BD Biosciences, San Jose, CA, USA).

The ROS levels were measured using 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) as previously described (39). DCFH-DA (Sigma-Aldrich; Merck KGaA), without fluorescence, can enter the cell membrane and form DCFH in the cell. DCFH is then oxidized to form a fluorescent substance DCF in the presence of ROS. MC-3T3-E1 cells were stained with DCFDA and held for 30 min at room temperature. Finally, DCF fluorescence levels were measured by flow cytometry and the data were analyzed using Summit Software (version 4.3; Dako; Agilent Technologies, Inc., Santa Clara, CA, USA).

Oxidative stress-related factors malondialdehyde (MDA; cat. no. ml077384; Enzyme-linked Biotechnology Co., Ltd., Shanghai, China), lactate dehydrogenase (LDH; cat. no. ml076593; Enzyme-linked Biotechnology Co., Ltd.) and superoxidase dismutase (SOD; cat. no. ml077379; Enzyme-linked Biotechnology Co., Ltd.) were measured using ELISA. MC-3T3-E1 cells were seeded on a 24-well plate, and cell-free supernatants were harvested after 3 h. The concentrations of MDA, LDH and SOD in the supernatants of MC-3T3-E1 cells were determined using ELISA kits following the manufacturer's instructions.

RT-qPCR was performed for the purpose of examining gene expression profiles of Bax, Bcl-2, survivin, NF-κB ligand (RANKL), osteoprotegerin (OPG), osterix, nuclear factor erythroid 2-related factor (Nrf2), heme oxygenase-1 (HO-1) and NAD(P)H quinone dehydrogenase 1 (NQO1). Total RNA was extracted using TRIzol^® regent (Invitrogen; Thermo Fisher Scientific, Inc.) following the manufacturer's instructions. RNA was reverse transcribed into cDNA using GoScript™ RT kit (Promega Corporation, Madison, WI, USA). The RT temperature protocol consisted of 37°C for 15 min and at 85°C for 5 sec. RT-qPCR was conducted using SYBR Fast qPCR Mix (Invitrogen; Thermo Fisher Scientific, Inc.) The thermocycling conditions were: 94°C for 3 min for an initial denaturation, followed by 30 denaturation cycles at 94°C for 5 sec, annealing and elongation at 60°C for 30 sec; and final extension at 72°C for 10 min. The primer sequences are summarized in Table I. The quantity of RNA was calculated using the 2^−ΔΔCq method (40), and the level of expression of an RNA was normalized to GAPDH (denoted ‘relative expression’).

MC-3T3-E1 cells were washed three times with PBS, and detached from the dishes by scraping. Cells were centrifuged at 12,000 × g for 5 min at 4°C and re-suspended in RIPA lysis buffer (Thermo Fisher Scientific, Inc.) with phenylmethanesulfonyl fluoride (PMSF) at 1:200 dilution. The homogenate was centrifuged at 12,000 × g for 10 min at 4°C. Protein concentrations were quantified using a bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology). Equivalent amounts of total protein (20 µg/lane) were loaded on a 10% Tris-glycine, 10% SDS-PAGE (Beyotime Institute of Biotechnology) for separation. Proteins were then transferred onto a polyvinylidene difluoride membrane, which were blocked with 5% milk in TBS containing 0.2% Tween-20 (TBST) at room temperature for 2 h, and incubated with primary antibodies as follows: Rabbit anti-Bcl-2 (cat. no. ab32124, 1:1,000) anti-Bax (cat. no. ab32503, 1:1,000), anti-survivin (cat. no. ab76424, 1:1,000); rabbit anti-RANKL (cat. no. ab9957, 1:1,000), anti-OPG (cat. no. ab73400, 1:1,000), anti-osterix (cat. no. ab94744, 1:1,000); mouse anti- HO-1 antibody (ab13248, 1:1,000), NQO1 antibody (ab34173, 1:1,000), Nrf2 antibody (ab137550, 1:1,000) and anti-GAPDH (ab9485, 1:1,000) overnight at 4°C, all purchased from Abcam (Cambridge, UK). The membranes were washed with TBST, and then incubated with horseradish peroxidase-conjugated secondary antibodies goat anti-rabbit (cat. no. ab205718; 1:2,000; Abcam) and goat anti-mouse (cat. no. ab205719; 1:5,000; Abcam) at 4°C for 1 h. The blots were visualized using enhanced chemiluminescence (ECL; Thermo Fisher Scientific, Inc.). An ECL system (Amersham; GE Healthcare, Chicago, IL, USA) was used to detect the bands. Quantity one software version 4.6.2 (Bio-Rad Laboratories, Inc., Hercules, CA, USA) was used for densitometry analysis.

GraphPad Prism version 6.0 software (GraphPad Software, Inc., La Jolla, CA, USA) was used for conducting statistical analysis. Data are presented as the mean ± standard deviation. Statistical significance was analyzed using one-way analysis of variance, followed by Turkey's multiple comparison test. P<0.05 was considered to indicate a statistically significant difference.

After MC-3T3-E1 cells were inoculated and cultured for 24 h, the growth of adherent cells was observed using an inverted microscope (Fig. 1A). The cells showed a fibroblast-like appearance. To be more specific, the cells appeared to be irregular fusi-formed, triangular, or polygonal, and no fusion between cells was identified. As the duration of the culture time prolonged, cell bodies grew larger and the cell morphology was stretched. It appeared fiber bundles, triangles and polygons with more protuberances. Some elongated protuberances were often found to link cells with distant protuberant cells and to increase cell-to-cell contact. Meanwhile, the number of cells was found to increase, and cells were mostly spindle-shaped or cubic.

The cytotoxic effects of different CdCl₂ concentrations (0–20 µM) at different time points (3, 6, 12 and 24 h) in MC-3T3-E1 cells were determined using CCK-8 assay. The results showed that CdCl₂ decreased MC-3T3-E1 cell viability in time- and dose-dependent manners (20 µM, 3 h, P<0.05; 20 µM, 24 h, P<0.01; Fig. 1B). Based on this findings, 20 µM of CdCl₂ was employed in all subsequent experiments. Moreover, the cytotoxic effects of geniposide were also detected, and that geniposide had no cytotoxic effects at a concentration of 100–400 µg/ml (Fig. 1C). As showed in Fig. 1D, geniposide was able to ameliorate the CdCl₂ injury in cells and increase viability in a dose-dependent manner, and treatment using 400 µg/ml geniposide significantly increased cell viability (P<0.05).

As showed in Fig. 2A, the effect of geniposide on CdCl₂-induced apoptosis was investigated using flow cytometric analysis. We found that the apoptosis induced by CdCl₂ was significantly decreased in a concentration-dependent manner after being pretreated with geniposide (100 and 200 µg/ml: P<0.05, 400 µg/ml: P<0.01).

As showed in Fig. 2B, CdCl₂ exposure increased the ROS generation in MC-3T3-E1 cells (P<0.01). However, pretreatment of MC-3T3-E1 cells with geniposide significantly decreased the generation of ROS in a dose-dependent manner (P<0.01).

As shown in Fig. 3, the effects of geniposide on CdCl₂-induced oxidative stress-related factors were assessed using ELISA assay. We found that the level of MDA was not significantly increased by exposure of the cells to CdCl_2, and that pretreatments at different concentrations of geniposide also showed no significant effect on the level of MDA (P>0.05, Fig. 3A). However, LDH was significantly increased following exposure of the cells with CdCl₂. By contrast, a medium concentration of geniposide pretreatment significantly decreased the level of LDH, compared to the cells incubated with CdCl₂ (P<0.05, Fig. 3B). Our results also showed that antioxidant key enzyme SOD was decreased following CdCl₂ exposure, but SOD was significantly higher following pretreatment with a high concentration of geniposide (P<0.05, Fig. 3C).

We determined the expression levels of Bax, Bcl-2 and survivin in MC-3T3-E1 cells using both western blotting and qPCR analyses. As shown in Fig. 3D-G, compared with levels in cells exposed to CdCl₂, pretreatment with geniposide increased the expression of Bcl-2 and survivin both at mRNA and protein levels in a concentration-dependent manner (survivin, 200 µg/ml P<0.05; 400 µg/ml P<0.01) and reduced the expression of Bax at the mRNA and protein levels (400 µg/ml, P<0.01). Both western blot and qPCR analysis showed that medium and high concentrations of geniposide could inhibit Bax, and increase Bcl-2 and survivin. These results showed that geniposide strongly antagonized the apoptotic process of CdCl₂-induced MC-3T3-E1 cells.

In order to investigate whether geniposide could reverse the inhibition of CdCl₂ on osteoblast formation, we assessed the expression of osteoblast-related factors, RANKL, OPG and osterix, by carrying out western blot and qPCR analyses in MC-3T3-E1 cells. The results showed that exposure to CdCl₂ significantly inhibited osteoblast formation by increasing the expression of OPG and by decreasing the expression of RANKL and osterix both at the mRNA and protein levels. Medium concentration of geniposide significantly reversed of the inhibition mediated by CdCl₂ on osteoblast formation through upregulating the expression of RANKL and osterix (P<0.01) and downregulating the expression of OPG (P<0.05). The protein levels (Fig. 4A-C) were consistent with the expression of mRNA (Fig. 4D).

In order to understand the mechanism of geniposide in Cd-induced osteoblast injury, the Nrf2 signaling pathway was evaluated in MC-3T3-E1 cells. As shown in Fig. 5, both qPCR and western blot analysis identified an increase in Nrf2, HO-1 and NQO1 expression in a dose-dependent manner following pretreatment with geniposide in the CdCl₂-injured MC-3T3-E1 cells. We found that a low concentration of geniposide significantly increased the mRNA expression of Nrf2 in comparison to that in cells exposed to CdCl₂ (P<0.05, Fig. 5A). However, both the increase of HO-1 and NQO1 mRNA expression required treatment with a medium concentration of geniposide (P<0.05, Fig. 5B and C). Western blot analyses also showed that a low concentration of geniposide increased the protein expression of Nrf2, HO-1 and NQO1 compared to that in cells exposed only to CdCl₂ (P<0.01, Fig. 5D).