Coenzyme Q10 inhibits RANKL‐induced osteoclastogenesis by regulation of mitochondrial apoptosis and oxidative stress in RAW264.7 cells

Abstract

Coenzyme Q10 (CoQ10) has been reported to improve bone density and the number of trabeculae in postmenopausal osteoporosis, but the mechanism remains to be elucidated. We aimed to investigate the effects of CoQ10 on receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis and the underlying molecular mechanisms. RAW264.7 cells were treated with different concentrations of RANKL to differentiate into osteoclasts, and then these cells were treated with different concentrations of CoQ10 with or without H 2 O 2 . Tartrate-resistant acid phosphatase staining was performed to detect osteoclasts. Cell viability was tested by 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, cell apoptosis was examined by flow cytometry, and the effects of CoQ10 on protein and messenger RNA expression of mitochondrial apoptosis-associated proteins and osteoclast marker proteins were measured by quantitative reverse transcription polymerase chain reaction and western blot, respectively. Furthermore, enzyme-linked immunosorbent assay was conducted to analyze the activities of malondialdehyde (MDA), superoxide dismutase (SOD), and catalase (CAT). RANKL significantly induced osteoclastogenesis in RAW264.7 cells, with the greatest efficiency at 50 ng/ml. CoQ10 had no significant effects on cell viability but it significantly increased the percentages of cell apoptosis.Mechanically, CoQ10 statistically decreased the levels of Bcl-2 and cytochrome C in mitochondria and upregulated the levels of Bax, cleaved caspase 3, and cytochrome C in the cytoplasm. Moreover, CoQ10 significantly decreased RANKL-induced osteoclastogenesis regardless of exposure to H 2 O 2 . In addition, CoQ10 statistically reduced MDA activity and elevated the activities of SOD and CAT, as well as the expression of oxidative stress-related proteins. CoQ10 may inhibit RANKL-induced osteoclastogenesis by regulation of mitochondrial apoptosis and oxidative stress in RAW264.7 cells.

Oxidative Stress and Inflammation in Osteoporosis: Molecular Mechanisms Involved and the Relationship with microRNAs

“…Nrf2 downregulates the expression of cytokines involved in osteoclastogenesis and induces the transcriptional activation of antioxidant genes; for this, the reduced expression of Nrf2, present in oxidative stress conditions and detected in osteoporotic rats, causes an increase in osteoclastic formation [ 50 , 51 , 52 , 53 ]. H 2 O 2 stimulates osteoclastogenesis by increasing the expression of osteoclastic differentiation factors by activating the transcriptional factor nuclear factor kappa B (NF-kB) and reducing the expression of Nrf2 in mouse mononuclear macrophage (RAW264.7) cells [ 54 , 55 ]. ROS-activated NF-κB can stimulate the expression of transcriptional factors c-Fos and nuclear factor of activated T-cell cytoplasmic 1 (NFATc1), which regulates the genes involved in osteoclastogenesis and bone resorption, such as tartrate-resistant acid phosphatase and cathepsin K. Moreover, ROS induce the expression of an activator of NF-kB, TNF receptor associated factor 6 (TRAF-6), which RANKL recruits through RANK with a consequent increase in osteoclastic formation [ 55 , 56 ].…”

Coenzyme Q10 prevents RANKL-induced osteoclastogenesis by promoting autophagy via inactivation of the PI3K/AKT/mTOR and MAPK pathways

“…RANKL is an important regulator of osteoclastogenesis. On the basis of our previous study, we confirmed that the optimal concentration of RANKL for promoting osteoclastogenesis was 50 ng/mL (11). To further determine the effects of CoQ10 on osteoclastogenesis, 10 -3 M CoQ10 was administered, after which TRAP staining and MTT assays were performed.…”

“…An increasing body of data suggests that CoQ10 contributes to the inhibition of osteoclast differentiation by decreasing the expression of genes encoding osteoclast markers, but the exact mechanism is not known. A number of mechanisms have been reported, including a reduction in bone malondialdehyde levels along with an increase in superoxide dismutase levels, regulation of mitochondrial apoptosis, and suppression of ROS production (11,27,28). On the basis of our previous study, CoQ10 may inhibit RANKL-induced osteoclastogenesis by regulating mitochondrial apoptosis and oxidative stress in RAW264.7 cells (11).…”

“…To promote differentiation into osteoclasts, RAW264.7 cells were treated with RANKL (Sigma-Aldrich, USA) for six days. After treatment with 50 ng/mL RANKL, CoQ10 (10 -3 M, Aladdin Reagent Co., Ltd., China) with or without 10 -4 M H 2 O 2 was added to the cell line (11). These cells were preconditioned with the inhibitors PI3K (LY294002, 30 mM; #L832989; Macklin, China) for 1 h (19), ERK (PD98059, 10 mM; MedChem Express, USA) for 1 h (20), p38MAPK (SB203580, 10 mM; Yuanye Bio-Technology Co., Ltd., China) for 1 h (21), or mitophagy agonist Torin1 (4 nM; #T861013; Macklin) for half an hour.…”

“…Recently, a growing body of evidence has suggested that CoQ10 can concurrently escalate osteoblastogenesis and reduce osteoclastogenesis (10). Our previous studies revealed that the antioxidant CoQ10 could repress osteoclastogenesis induced by receptor activator of NF-kB ligand (RANKL) by modulating mitochondrial apoptosis and oxidative stress (11). However, the underlying mechanism(s) still need to be further clarified.…”