Female Sprague-Dawley (SD) rats (8-months old, 250–350 g, n=48) were obtained from the Experimental Animal Center of Dalian Medical University (Dalian, China). All rats were acclimatized for at least one week prior to the experiments. Animals were housed individually in standard laboratory cages with controlled temperature and humidity (22–25°C; 50–65%) under a 12-h light/dark cycle. Rats had access to food and water ad libitum. A total of 8 rats were randomly selected and used in the in vitro experiment, and BMSCs were obtained from them. Remaining rats (n=40) were used to conduct in vivo experiments. All experiments were approved by the Ethics Committee on Animals at Dalian University (Dalian, China) and performed in accordance with the standards set by the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (Bethesda, MD, USA).

BMSCs were isolated as previously described (13,14). SD rats were subjected to euthanasia by inhalation of 70% CO₂ and femurs and tibias were harvested. Bone marrow was removed, filtered through a 70-µm nylon mesh and centrifuged at 1,200 × g for 8 min at 4°C. Collected cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin (all, Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 37°C with 5% CO₂ for 3 days. Subsequently, adherent cells were washed three times with PBS and cultured in DMEM until 90% confluence was attained. Following 3 or 4 passages, BMSCs were treated with vehicle (soya bean oil) or various concentrations of CoQ10 (10, 20 or 100 µM, Sigma-Aldrich; Thermo Fisher Scientific, Inc.). CoQ10 was dissolved in soya bean oil at 40°C.

Viability of BMSCs was assessed by using 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). Cells (2×10⁴ cells/ml) were seeded in 96-well plates and treated with a vehicle or various concentrations of CoQ10 (10, 20 or 100 µM) for 24 h. On days 1, 3, 5 and 7 prior to the treatment, cells were incubated with an MTT solution (20 µl, 0.5 mg/ml) for 4 h at 37°C, followed by an addition of dimethylsulfoxide (Sigma-Aldrich; Merck KGaA). The absorbance was measured at a wavelength of 590 nm with a SpectraMax 340 microplate reader (Molecular Devices LLC, Sunnyvale, CA, USA).

The activity of ALP was determined as previously described (12). BMSCs (5×10³ cells/ml) were seeded in 96-well plates and incubated with vehicle or 10, 20 or 100 µM CoQ10 for 24 h at room temperature. Following pre-treatment with vehicle or CoQ10, 20 µl p-nitrophenyl phosphate (pNPP; 1 mg/ml; Sigma-Aldrich; Merck KGaA) was added to the RIPA buffer solution for 30 min at 37°C on days 1, 3, 5, and 7 of the experiment, according to the manufacturer's protocol. Enzymatic activity was quantified by ALP ELISA kit (cat. no. 687242; Antibody Research Corp., St. Peters, MO, USA) and absorbance measured at a wavelength of 405 nm with a SpectraMax Plus384 reader (Molecular Devices LLC).

Expression of osteogenic markers, runt-associated transcription factor 2 (RUNX-2) and osteocalcin (OCN), was assessed by RT-qPCR. Total RNA was isolated from BMSCs using the RNeasy Mini kit (Qiagen Inc., Valencia, CA, USA) according to the manufacturer's protocol. First-strand complementary DNA was synthesized using Superscript III Reverse Transcriptase (Invitrogen; Thermo Fisher Scientific, Inc.). Reverse transcription was performed at 55°C for 60 min. Expression levels were determined using SYBR green-based qPCR (SYBR Green PCR Master Mix; Applied Biosystems; Thermo Fisher Scientific, Inc.). PCR was undertaken according to the following parameters: 1 predenaturation cycle of 1 min at 94°C, 40 cycles of 95°C for 30 sec, 60°C for 30 sec, and 72°C for 2 min, and a final extension at 72°C for 5 min. All primers were supplied by Genepharma Co., Ltd. (Shanghai, China) and are listed in Table I. Expression was normalized to GAPDH using the comparative 2^−ΔΔCq method (15).

Total protein was extracted from BMSCs or rat femurs using radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China). Protein concentration was assessed using a Bicinchoninic Acid protein assay kit (Beyotime Institute of Biotechnology) according to the manufacturer's protocol. Proteins were separated by 10–12% SDS-PAGE and transferred to nitrocellulose membranes (EMD Millipore, Billerica, MA, USA) and blocked with 5% dried skimmed milk in Tris buffered saline with Tween-20. Membranes were then probed with: Anti phosphatidylinositol 4,5-bisphosphate 3-kinase (PI3K, 1:1,000, cat. no. mAb4249), phosphorylated (p)-PI3K (1:1,000, cat. no. 4228), RAC-alpha serine/threonine-protein kinase (AKT, 1:1,000, cat. no 4691), p-AKT (1:1,000, cat. no 4060, all Cell Signaling Technology, Inc., Danvers, MA, USA) or phosphatidylinositol 3,4,5-trisphosphate 3-phosphatase and dual-specificity protein phosphatase PTEN (PTEN; cat. no. ab32199; Abcam, Cambridge, USA) primary antibodies overnight at 4°C. Anti-GAPDH (1:2,000, cat. no. sc-25778) Santa Cruz Biotechnology, Inc., Dallas, TX, USA) was used as a loading control and was incubated overnight at 4°C. Following washing with PBS, membranes were incubated with a horseradish peroxidase-conjugated secondary antibody (1:5,000, cat. no. sc-2372, Santa Cruz Biotechnology, Inc.). Immunoreactive protein bands were visualized by enhanced chemiluminescence Western blotting substrate (Roche Applied Science, Penzberg, Germany).

The rats were randomly divided into 5 groups (n=8 per group): i) the sham group, where rats were subjected to a sham surgery that involved exposing but not removal of ovaries, and received normal feedstuff for 3 months; ii) the OVX group, where rats were subjected to OVX operation and received normal feedstuff for 3 months; iii) the OVX+1 mg/kg CoQ10 group, where rats were subjected to OVX and treated with 1 mg/kg CoQ10 by intraperitoneal injection every 5 days for 3 months; iv) the OVX+10 mg/kg CoQ10 group where, rats were subjected to OVX and treated with 10 mg/kg CoQ10 by intraperitoneal injection every 5 days for 3 months; and v) the OVX+20 mg/kg CoQ10 group, where rats were subjected to OVX and treated with 20 mg/kg CoQ10 by intraperitoneal injection every 5 days for 3 months.

Rats were subjected to OVX as previously described (16). Animals were anesthetized by an intraperitoneal injection of 8% chloral hydrate solution (Sigma-Aldrich; Merck KGaA). Following anesthesia, rats were subjected to bilateral OVX by the dorsal approach. Six hours after the last dose of CoQ10, all animals were sacrificed. Blood samples (4 ml) were collected from hearts and stored at −20°C. Blood samples were then centrifuged at 1,200 × g for 10 min at room temperature. Whole femurs were dissected and fixed in 4% paraformaldehyde for a subsequent micro-computed tomography (CT) analysis.

Levels of serum estrogen (E2) (cat. no. IB79329, IBL-America Inc., Spring Lake, MN, USA) and serum bone metabolism markers, including calcium, parathyroid hormone (PTH) (cat. no. 60–2500, Immutopics Inc., San Clemente, CA, USA) and osteoprotegerin (OPG) (cat. no. BE69058; IBL-America Inc.), were measured by ELISA. Serum calcium levels were determined using an Accucare Calcium Arsenazo III kit (Serotec Co., Ltd., Hokkaido, Japan).

High-resolution micro CT scans (SCANCO viva; version 4.0; Scanco Medical AG, Brüttisellen, Switzerland) were performed on the sham- and CoQ10-treated joints. Changes in BMD and bone volume/total volume (BV/TV) ratio were evaluated using the Image-Pro software (version 7.0; Media Cybernetics Inc., Rockville, MD, USA) and SCANCO microCT software packages (version 6.0). The average trabecular number (Tb.N), average trabecular thickness (Tb.Th) and average trabecular separation (Tb.Sp) were assessed by the plate model 16 set-up: Tb.N=0.5 bone surface (BS)/TV; Tb.Th=2 BV/BS; Tb.Sp=2 (TV-BV)/BS (17).

All experiments were repeated at least three times. The data are expressed as the mean ± standard error. Student's t-test was used to determine differences between the two groups, and one-way analysis of variance was performed to compare differences among three groups, followed by the Student-Newman-Keuls post-hoc test. P<0.05 was considered to indicate statistically significant differences. Results were analyzed using SPSS software (version 18.0; SPSS, Chicago, IL, USA).

The viability of BMSCs was analyzed by MTT following 1, 3, 5 and 7 days of treatment with various concentrations of CoQ10 (10, 20 or 100 µM). As presented in Fig. 1, 10 µM CoQ10 increased cell viability but the difference was not statistically significant. Cell viability was not significantly altered on days 1 and 3 at any dose. CoQ10 administrated at a dose of 20 or 100 µM exhibited significant stimulatory effects on cell viability on days 5 and 7 (P<0.05 or P<0.01) in a dose-dependent pattern. The data suggests that CoQ10 promotes viability of BMSCs.

RUNX-2, OCN and ALP are osteogenic markers. Following treatment with different concentrations of CoQ10 (10, 20 or 100 µM), the expression levels of RUNX-2, OCN and ALP in BMSCs on days 1, 3, 5, and 7 were measured by RT-qPCR and ELISA. As indicated in Fig. 2, the expression of RUNX-2, OCN and ALP was significantly increased by the treatment with 20 or 100 µM CoQ10 on days 3, 5, and 7 (P<0.05, P<0.01, or P<0.001), in a dose-dependent manner. No significant differences were observed on day 1 and following treatment with 10 µM CoQ10 on any day. The results suggest that 20 and 100 µM CoQ10 can promote osteogenic differentiation of BMSCs.

To investigate the effects of CoQ10 on osteoporosis, a rat model of osteoporosis was established by OVX followed by the administration of CoQ10. Serum levels of E2 and bone metabolism markers (calcium, OPG and PTH) were determined following administrating different concentrations of CoQ10. As presented in Fig. 3, the levels of E2, calcium and OPG was significantly decreased in the OVX group compared with the sham group (all P<0.05), while PTH was significantly increased in the OVX group, compared with the sham group (P<0.05). Administration of 20 mg/kg CoQ10 significantly increased the levels of calcium and OPG and decreased the levels of PTH (all P<0.05) but exhibited no significant effect on the levels of E2 compared with the OVX group. Furthermore, no significant differences were observed in the abundance of E2, calcium, OPG and PTH between the OVX group and OXY+1 or 10 mg/kg treatment groups. The results demonstrated that 20 mg/kg CoQ10 can increase serum calcium and OPG and decrease PTH levels, which may be beneficial for patients with osteoporosis.

Micro-CT analysis was performed to evaluate bone morphology parameters. The results are presented in Table II. BMD, BV/TV, Tb.N. and Tb.Th. were significantly decreased in the OVX group compared with the sham group, while Tb.Sp. significantly increased (all P<0.001). Administration of CoQ10 (1, 10, or 20 mg/kg) significantly increased BMD and Tb.N. and reduced Tb.Sp, compared with the OVX group (all P<0.001). Only administration of 20 mg/kg CoQ10 exhibited significant stimulatory effects on BV/TV and Tb.Th, compared with the OVX group (all P<0.001). Fig. 4A presents micrographs of the vertical section of distal femur in different groups. The analysis revealed poor trabecular structure in the OVX group compared with the sham group. CoQ10 treatment improved the trabecular microstructure (Fig. 4A) and trabecular thickness (Fig. 4B) in a dose-dependent manner compared with OVX group. The results demonstrated that CoQ10 may protect against osteoporosis by improving bone formation.

It has been previously demonstrated that activation of the PI3K/AKT pathway protects against osteoporosis and controls multiple cell processes, including cell proliferation, differentiation and cell apoptosis. PTEN is a known negative regulator of PI3K/AKT. Therefore, mRNA and protein expression levels of PTEN/PI3K/AKT key factors, were analyzed. The results revealed that OVX significantly decreased the levels of phosphorylated to total ratio (p/t)-PI3K and p/t-AKT but significantly increased the levels of PTEN compared with the sham group (P<0.05 or P<0.01). There were no significant differences in the levels of p/t-PI3K, p/t-AKT and PTEN following administration of 1 or 10 mg/kg CoQ10 compared with the OVX group. However, significant differences were observed in the relative protein expression of p/t-PI3K, p/t-AKT and PTEN following administration of 20 mg/kg CoQ10, compared with the OVX group (P<0.05 or P<0.01; Fig. 5A and B). The above results indicate that CoQ10 activates the PTEN/PI3K/AKT signaling pathway.