| Osteoporosis is a systemic metabolic bone disease with the characteristic of bone micro-structural damage and bone loss, leading bone fragility and fracture risk increase. Excessive ROS induced by aging can destroy the balance in bone metabolism by promoting osteoclastic bone absorption and inhibiting osteoblastic bone formation, resulting in development of Osteoporosis eventually.Originating from the rhizome of Curculigo orchioides Gaertn, Traditional Chinese medicine Curculiginis Rhizoma has long been used in China and is considered to have attributes beneficial to the kidney, liver channels, bones and muscles. As the main active ingredient in Curculiginis Rhizoma, curculigoside is reported to possess immune enhancement, anti-osteoporosis, anti-depression and neural protection effects and so on.Previous studies of research group found that curculigoside doesn’t have estrogenic effects on estrogen-lacking osteoblasts, which was cultured with charcoal stripped FBS and phenol red-free medium. Through the replication of Iron-overload mouse model, however, curculigoside was proved to effectively improve the bone mineral density of iron-overload mice and regulate the expression of proteins related to bone metabolism, indicating that curculigoside can protect bone tissue from oxidative stress, but its mechanism is still not clear. On the other hand, curculigoside belongs to phenolic glycosides with a very low oral absolute bioavailability, yet the material basis for the efficacy remains unclear. Therefore, this study explored the mechanism of protective effects of curculigoside on osteoblasts with H2O2 stimulation, and elucidated the metabolites of curculigoside in rats:The effects of curculigoside on cell functions and Antioxidant system of osteoblasts with oxidative injury was investigated in this paper. In order to explore the mechanism of protective effects of curculigoside, the influence of curculigoside on expression of FoxO1 pathway proteins was studied. Meanwhile, LC-Q-TOF/MS was applied to identify the metabolites of curculigoside in rats and explain the metabolic mechanism of curculigoside with a very low oral absolute bioavailability, and antioxidant activities of the main metabolite M2 on osteoblasts with H2O2 stimulation was tested to explore the material basis for the efficacy of curculigoside.1. The antioxidant effects of curculigoside on osteoblasts with H2O2 stimulationThe antioxidant effects of curculigoside were investigated using osteoblast model with H2O2 induced oxidative injury. The cell proliferation rate, alkaline phosphatase (ALP) activity, bone mineralization rate, superoxide dismutase (SOD) activity and catalase (CAT) activity were measured, and ROS level, cell cycle and mitochondrial membrane potential were tested by flow cytometry. The results showed that curculigoside (10-8,10-7 and 10 mol/L) can significantly improve cell vialibity, ALP activity, the rate of bone mineralized nodule, SOD and CAT activity.The level of ROS was reduced and the mitochondrial membrane potential was improved, and optimization of curculigoside on cell cycle was observed.2. Antioxidant mechanism of curculigoside on osteoblasts with H2O2 stimulationThe probable antioxidant mechanism of curculigoside was investigated in this study. The results of western blot experiments showed that curculigoside could up-regulate expressions of FoxOla, SOD, OPG and down-regulate expression of Rankl in total protein, and improve the ratio of FoxOl protein in the nucleus/cytoplasm protein with an increase SOD expression. Fluorescent immunohisto -chemistry experiment results show that curculigoside could significantly improve the amount of nuclear FoxOla, and promote the interactions between FoxOla and ATF4 factors.3. Characterization of metabolites of curculigoside in rats using HPLC/QTOF-MSIn order to thoroughly characterize the metabolism of curculigoside in rats, an effective and reliable HPLC/QTOF-MS analytical method was developed for detection of curculigoside and its metabolites in bile, plasma, urine and selected tissues samples of dosed rats. Three phase I metabolites and four phase Ⅱ metabolites were observed and their chemical structures were carefully elucidated based on MS/MS spectrometry.4. Analysis of relative content of metabolites of curculigoside in ratsEfforts in comparing concentration of curculigoside with that of significant metabolites M2 indicated that low absorption of curculigoside provides explanation for its very low absolute bioavailability. Furthermore, the abundant distribution of M2 in bone marrow, spleen, thymus, ovary and uterus indicates that M2 may be a bioactive metabolite.5. Effects of metabolite M2 on osteoblasts with H2O2 stimulationMTT colorimetric assay and ALP activities test were carried out to observe effects of metabolite M2 on osteoblasts with H2O2 stimulation. The results showed that M2 (10-8,10-7 and 10-6mol/L) can improve cell vialibity and ALP activity, suggesting a role for M2 as a metabolite of curculigoside can protect osteoblast from oxidative damage. |
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