Icariin prevents bone loss by inhibiting bone resorption and stabilizing bone biological apatite in a hindlimb suspension rodent model

Abstract

Bone loss induced by microgravity is a substantial barrier to humans in long-term spaceflight. Recent studies have revealed that icariin (ICA) can attenuate osteoporosis in postmenopausal women and ovariectomized rats. However, whether ICA can protect against microgravity-induced bone loss remains unknown. In this study, the effects of ICA on a hindlimb suspension rodent model were investigated. Two-month-old female Wistar rats were hindlimb suspended and treated with ICA (25 mg·kg^-1·d^-1, i.g.) or a vehicle for 4 weeks (n = 6). The bone mass density of the hindlimbs was analyzed using dual-energy X-ray absorptiometry and micro-CT. mRNA expression of osteogenic genes in the tibia and the content of bone metabolism markers in serum were measured using qRT-PCR and ELISA, respectively. The bone mineral phase was analyzed using X-ray diffraction and atomic spectrometry. The results showed that ICA treatment significantly rescued the hindlimb suspension-induced reduction in bone mineral density, trabecular number and thickness, as well as the increases in trabecular separation and the structure model index. In addition, ICA treatment recovered the decreased bone-related gene expression, including alkaline phosphatase (ALP), bone glaprotein (BGP), and osteoprotegerin/receptor activator of the NF-κB ligand ratio (OPG/RANKL), in the tibia and the decreased bone resorption marker TRACP-5b levels in serum caused by simulated microgravity. Notably, ICA treatment restored the instability of bone biological apatite and the metabolic disorder of bone mineral elicited by simulated microgravity. These results demonstrate that ICA treatment plays osteoprotective roles in bone loss induced by simulated microgravity by inhibiting bone resorption and stabilizing bone biological apatite.

Keywords: apatite; bone loss; bone resorption; hindlimb suspension; icariin; simulated microgravity.

Fig. 1

The chemical structure of icariin

Fig. 2

Body weight changes in the different groups. Ctrl control group, HLS hindlimb suspension group, HLS + ICA hindlimb suspension and treated with icariin group. Data are shown as the mean ± SD, n = 6 in each group

Fig. 3

The bone mineral density (BMD) and mechanical parameters of hindlimb bones. a Total femur BMD assessed using dual-energy X-ray absorptiometry; b, c Mechanical parameters of the bone samples from three-point bending tests; b the maximal force (N); c Elasticity modulus (N/mm²); Ctrl control group, HLS hindlimb suspension group, HLS + ICA hindlimb suspension and treated with icariin group. Data are shown as the mean ± SD, n = 6 in each group. *P < 0.05, **P < 0.01 vs the HLS group

Fig. 4

ICA treatment exhibits reduced damage of cancellous bone induced by hindlimb suspension. a Representative micro-CT reconstructive images of the distal femurs of the Ctrl, HLS, and HLS + ICA rats; b–g The bone micro-architectural parameters; BV/TV trabecular bone volume fraction, Tb.Th trabecular thickness, Tb.Sp trabecular separation, Tb.N trabecular number, SMI structure model index. Ctrl control group, HLS hindlimb suspension group, HLS + ICA hindlimb suspension and treated with icariin group. Data are shown as the mean ± SD, n = 6 in each group. *P < 0.05, **P < 0.01 vs the HLS group

Fig. 5

ICA treatment facilitates osteoblast activity and suppresses osteoclast activity in hindlimb suspension rats. The relative expression of osteoblast differentiation related genes ALP (a), BGP (b) and the ration of OPG/RANKL (c) was evaluated using qRT-PCR. Total RNA was extracted from the tibia. GAPDH was used to normalize the gene expressions and the results are presented as the relative expression compared with the Ctrl group. Ctrl control group, HLS hindlimb suspension group, HLS + ICA hindlimb suspension and treated with icariin group. Data are shown as the mean ± SD, n = 6 in each group; *P < 0.05 vs the HLS group

Fig. 6

ICA treatment suppresses bone resorption induced by simulated microgravity. The levels of serum ionic calcium and tartrate-resistant acid phosphatase 5b (TRACP-5b) were measured using ELISA. The concentration of ionic calcium (a) and TRACP-5b (b) were calculated with the standard curves, respectively. Ctrl control group, HLS hindlimb suspension group, HLS + ICA hindlimb suspension and treated with icariin group. Data are shown as the mean ± SD, n = 6 in each group. *P < 0.05, **P < 0.01 vs the HLS group

Fig. 7

ICA treatment attenuates the instability of the bone mineral phase induced by hindlimb suspension. a X-ray diffraction patterns of bone samples after the annealing at 950 °C for 1 h; * indicates the peak of β-TCMP; b–d concentrations of Na⁺ (b), Mg²⁺ (c), and K⁺ (d) in water medium after ultrasonic treatment of the annealed and powdered bone samples. The lines are drawn only to guide the eye. Ctrl control group, HLS hindlimb suspension group, HLS + ICA hindlimb suspension and treated with icariin group