Icariin attenuates hypoxia-induced oxidative stress and apoptosis in osteoblasts and preserves their osteogenic differentiation potential in vitro

Abstract

Objectives: Icariin, a prenylated flavonol glycoside isolated from traditional Chinese medicinal herb of the genus Epimedium, has been demonstrated to be a potential alternative therapy for osteoporosis, and its action mechanism so far has been mainly attributed to its phytoestrogenic property. As blood supply to bone is considerably reduced with ageing and by the menopause, we hypothesized that icariin treatment would reduce bone loss by preventing ischaemia-induced hypoxic damages to bone.

Materials and methods: To investigate effects of icariin treatment on cultured rat calvarial osteoblasts exposed to hypoxic conditions (2% oxygen).

Results: Compared to normoxic control, cell viability decreased with time to 50% by 48 h in the hypoxic group, and icariin attenuated the reduction, dose dependently, with 10(-6) and 10(-5) m concentrations showing significant protective effects. Icariin also inhibited increase of lactate dehydrogenase activity in culture media. Measurements on oxidative stress, cell cycling and cell survival indicated that icariin protected osteoblasts by reducing production of reactive oxygen species and malondialdehyde, increasing superoxide dismutase activity, arresting the cell cycle and inhibiting apoptosis. Icariin also preserved osteogenic differentiation potential of the hypoxic cells in a dose-dependent manner, compared to the hypoxia alone group, as revealed by increased levels of RUNX-2, OSX and BMP-2 gene expression, alkaline phosphatase activity, and formation of mineralized nodules.

Conclusions: Our results demonstrated that icariin attenuated oxidative stress and apoptosis and preserved viability and osteogenic potential of osteoblasts exposed to hypoxia in vitro, and suggested that its anti-osteoporotic effect may be attributed to its anti-hypoxic activity and phytoestrogenic properties.

Figure 1

Cell viability and oxidative stress of osteoblasts after hypoxic treatment with or without icariin presence, over different times. The subcultured rat calvarial osteoblasts grown to 70% confluence were supplemented with 10⁻⁷, 10⁻⁶, 10⁻⁵ and 10⁻⁴ m icariin and switched to hypoxia (2% oxygen) for 12, 24, 36 and 48 h respectively. The group supplemented with vehicle (1 μl/l DMSO) under normoxia, was used as normal normoxia control (NC). The group with the vehicle under hypoxia was hypoxia control (HC). Cell viability was assayed by the MTT method (a). lactate dehydrogenase activity in culture media (lactate dehydrogenase release) was used as an indicator for degree of cell injury (b). Intracellular MDA content (c) and superoxide dismutase activity (d) represent oxidative and anti‐oxidative levels, respectively. Values for cell viability were means ± SD from six replicate cultures, and the others are means ± SD from triplicate cultures. *P < 0.05, **P < 0.01 versus NC group; #P < 0.05, ##P < 0.01 versus HC group.

Figure 2

Intracellular reactive oxygen species ( ROS ) stained by fluorescence probe DCFH ‐ DA after hypoxic treatment, with or without icariin for 36 h (a), scale bar = 100 μm. Images of fluorescent signals and signal intensities were analysed by Image‐Pro Plus 6.0 software (b). Results are means ± SD from triplicate cultures. **P < 0.01 versus normoxia control (NC); #P < 0.05, ##P < 0.01 versus hypoxia control (HC).

Figure 3

Effects of hypoxic treatment with or without icariin, on proliferating cell nuclear antigen ( PCNA ) expression of osteoblasts. PCNA expression was examined by immunohistochemical staining (a), scale bar = 50 μm. Positive signal intensity was analysed by Image‐Pro Plus 6.0 software (b). Results are means ± SD from triplicate cultures. **P < 0.01 versus normoxia control (NC); #P < 0.05, ##P < 0.01 versus hypoxia control (HC).

Figure 4

Effects of hypoxic treatment with or without icariin on osteoblast apoptosis and associated gene expression. Flow cytometric analysis of apoptosis (a), showing that hypoxic treatment sharply increased percentage of apoptotic cells, while icariin supplements inhibited increase in a dose‐dependent manner (b). Expression levels of pro‐apoptotic gene caspase‐3 (c) and anti‐apoptotic gene Bcl‐2 (d). Data are means ± SD from triplicate cultures. **P < 0.01 versus normoxia control (NC); #P < 0.05, ##P < 0.01 versus hypoxia control (HC).

Figure 5

Effects of hypoxic treatment with or without icariin, on osteoblast differentiation potential as analysed by expression levels of genes associated with osteogenesis and alkaline phosphatase ( ALP ) activity. Expression levels of growth factor BMP‐2 (a), transcription factors Runx‐2 (b) and OSX (c). Intracellular ALP activities were measured after 6 days (d). Results are means ± SD from triplicate cultures. *P < 0.05, **P < 0.01 versus normoxia control (NC); #P < 0.05, ##P < 0.01 versus hypoxia control (HC).

Figure 6

Effects of hypoxic treatment with or without icariin, on osteoblast mineralization potential. Mineralized nodules were stained with alizarin red after 12 days (a). Numbers (b) and areas (c) of mineralized nodules were analysed by Image‐Pro Plus 6.0 software. Results are means ± SD from triplicate cultures. **P < 0.01 versus normoxia control (NC); #P < 0.05, ##P < 0.01 versus hypoxia control (HC).