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Objectives Excessive reactive oxygen species (ROS) play an important role in the development of osteoporosis.Ophiopogonin D (OP-D), isolated from the traditional Chinese herbal agent Radix Ophiopogon japonicus, is a potent anti-oxidative agent.Previous studies show that OP-D is involved in some pharmacological activities, such as inhibition of venous thrombosis, anti-inflammation, anti-tussive and anti-oxidative activities.We hypothesized that OP-D demonstrates anti-osteoporosis effects via decreasing ROS generation in mouse pre-osteoblast cell line MC3T3-E1 subclone 4 cells and a macrophage cell line RAW264.7 cells.Methods We investigated OP-D on osteogenic and osteoclastic differentiation under oxidative status.Hydrogen peroxide (H2O2) was used to establish an oxidative damage model.We examined OP-D effects on MC3T3-E1 osteogenic differentiation by calcium mineralization, ALP activity and the mRNA expression of osteogenic genes including BGLAP, COL1A1 and SPP1.We also tested OP-D effects on RAW264.7 osteoclastic differentiation by TRAP staining and the mRNA expression of osteoclastic genes including NFATc1 and TRAP.In vivo, we established a murine ovariectomized (OVX) osteoporosis model.The bone micro-architecture of the 4th lumbar vertebrae (L4) and distal femur were measured by micro-CT and HE staining.Then, we searched the molecular mechanism of OP-D against osteoporosis.