CoQ10 suppression of oxidative stress and cell senescence increases bone mass in orchiectomized mice

Abstract

Numerous studies support the detrimental effects of oxidative stress and cell senescence on skeletal homeostasis. Coenzyme Q10 (CoQ10) acts as a scavenger for oxidative stress and protects mitochondrial activity from oxidative damage. However, it is unclear whether CoQ10 has a protective effect on osteoporosis caused by orchiectomy. To investigate suppression effect of antioxidant CoQ10 on osteoporosis in orchiectomized (ORX) mice, ORX mice were supplemented with/without CoQ10, and were compared with each other and with sham-operated mice. Our results showed that CoQ10 could prevent ORX-induced bone loss by inhibiting oxidative stress and cell senescence, subsequently promoting osteoblastic bone formation and inhibiting osteoclastic bone resorption. The results of this study not only reveal the mechanism of CoQ10 supplementation in anti-osteoporosis, but also provide experimental and theoretical basis for the clinical application of CoQ10 in the prevention of osteoporosis.

Keywords: ORX-induced bone loss; cell senescence; coenzyme Q10; oxidative stress.

Figure 1

The effect of CoQ10 on ORX-induced bone loss. Eight-week-old male C57/BL6J mice were randomly performed ORX or Sham surgery and euthanize after 40 weeks. (A) Representative radiographs of tibias. (B) Representative Micro-CT-scanned and 3D reconstructed sections along the longitudinal direction of tibias. Analysis of the distal femoral trabecular bone parameters by micro-CT, (C) Bone mineral density, (D) Trabecular bone volume relative to tissue volume (BV/TV, %), (E) Thickness of trabecular bone (Tb.Th), (F) Trabecular number (Tb.N), (G) Trabecular separation (Tb.Sp). Data are presented as the mean ± SEM of determinations, each data-point was the mean of five specimens. **P < 0.01, ***P < 0.001, versus sham mice; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 versus ORX mice.

Figure 2

The effect of CoQ10 on the osteoblastic bone formation in ORX mice. (A) Representative photomicrographs of paraffin sections of tibias from 48-week-old mice of each group stained for HE. (B) The number of osteoblasts (N.Ob) per mm² trabecular area (T. Area) was measured and presented. (C) Representative photomicrographs of paraffin sections of tibias from 48-week-old mice of each group stained immunohistochemically for type I collagen. (D) The percentage of the Col I-positive area was measured and presented by image analysis (%). Real-time RT-PCR was performed on bone tissue extracts from 48-week-old mice of each group. Gene expression of (E) OPG, (F) RUNX2 and (G) OCN are shown. Messenger RNA expression, assessed by real-time RT-PCR analysis, was calculated as a ratio to the GAPDH mRNA level and expressed relative to levels in sham mice. Data are presented as the mean ± SEM of determinations, each data point is the mean of five specimens. *P < 0.05, ***P < 0.001, versus sham mice; ^#P < 0.05, ^##P < 0.01 versus ORX mice.

Figure 3

The effect of CoQ10 on the osteoclastic bone resorption in ORX mice. A. Representative photomicrographs of paraffin sections of tibias from 48-week-old mice of each group stained histochemically for TRAP. B. The number of TRAP-positive osteoclasts (N.Oc) per mm bone perimeter (B.Pm) was measured and presented. C. Osteoclast surface relative to bone surface (Oc.S/BS, %). D. Real-time RT-PCR was performed on bone tissue extracts from 48-week-old mice of each group. Gene expression of RANKL/OPG ratio are shown. Messenger RNA expression, assessed by real-time RT-PCR analysis, was calculated as a ratio to the GAPDH mRNA level and expressed relative to levels in sham mice. Data are presented as the mean ± SEM of determinations, each data point is the mean of five specimens. *P < 0.05, **P < 0.01, versus sham mice; ^#P < 0.05, ^##P < 0.01 versus ORX mice.

Figure 4

The effect of CoQ10 on BM-MSCs proliferation and differentiation into osteoblasts. A. Ex vivo primary cultures of BM-MSCs were stained cytochemically for ALP to detect CFU-ALP+ colonies. B. Ex vivo primary cultures of BM-MSCs were stained with methylene blue to show total CFU-F colonies. C. The number of CFU-ALP+ colonies per well (#/well) were counted. D. The number of CFU-F colonies per well (#/well) were counted. Data are presented as the mean ± SEM of determinations, each data point is the mean of five specimens. *P < 0.05, ***P < 0.001, versus sham mice; ^#P < 0.05 versus ORX mice.

Figure 5

The effect of CoQ10 on redox balance in ORX mice. (A) Representative flow cytometric analysis of ROS levels of bone marrow cells from 48-week-old mice of each group. (B) Relative fluorescence intensity (RFI) of ROS was calculated and expressed relative to the sham mice. (C) Biochemistry analysis of bone tissue extracts from 48-week-old mice of each group for the total antioxidant capacity (T-AOC). Real-time RT-PCR was performed on long bone extracts from 48-week-old mice of each group. Gene expression of (D) CAT, (E) SOD1, (F) SOD2, (G) GSR and (H) Prdx1 were shown. Messenger RNA expression, assessed by real-time RT-PCR analysis, was calculated as a ratio to the GAPDH mRNA level and expressed relative to levels in sham-operated mice. Data are presented as the mean ± SEM of determinations, each data-point was the mean of five specimens. *P < 0.05, versus sham mice; ^#P < 0.05, ^##P < 0.01 versus ORX mice.

Figure 6

The effect of CoQ10 on cell senescence in ORX mice. A. Representative micrographs of paraffin sections of tibias from 48-week-old mice of each group stained immunohistochemically for p16^INK4a. B. Representative micrographs of paraffin sections of tibias from 48-week-old mice of each group stained immunohistochemically for β-gal. C. The percentages of p16^INK4a-positive cells were determined by image analysis. D. The percentages of β-gal-positive cells were determined by image analysis. E. Real-time RT-PCR was performed on long bone extracts from 48-week-old mice of each group. Gene expression of p16^INK4a were shown. Messenger RNA expression, assessed by real-time RT-PCR analysis, was calculated as a ratio to the GAPDH mRNA level and expressed relative to levels in sham-operated mice. Data are presented as the mean ± SEM of determinations, each data-point was the mean of five specimens. *P < 0.05, **P < 0.01, ***P < 0.001, versus sham mice; ^#P < 0.05, ^##P < 0.01 versus ORX mice.