Efficacy of the herbal pair, Radix Achyranthis Bidentatae and Eucommiae Cortex, in preventing glucocorticoid-induced osteoporosis in the zebrafish model.

OBJECTIVE: In traditional Chinese medicine, the herbal pair, Radix Achyranthis Bidentatae (RAB) and Eucommiae Cortex (EC), is widely used to treat osteoporosis. Herein, we determined whether this herbal pair can be used to ameliorate glucocorticoid (GC)-induced osteoporosis (GIOP) and find its optimal dosage in zebrafish. METHODS: The characteristics of the aqueous extract of RAB and EC were separately characterized using high-performance liquid chromatography. Osteoporosis was induced in 5-day post-fertilization zebrafish larvae by exposing them to 10 µmol/L dexamethasone (Dex) for 96 h. Seven combinations of different ratios of RAB and EC were co-administered. Treatment efficacy was determined by calculating zebrafish vertebral area and sum brightness, via alizarin red staining, and by detecting alkaline phosphatase (ALP) activity. Multiple regression analysis was conducted to test the optimal dosage ratio. RESULTS: According to the Chinese Pharmacopoeia (2015), ß-ecdysone (ß-Ecd) is a major bioactive marker in RAB extract, while pinoresinol diglucoside (PDG) is the major marker in EC extract. Both of ß-Ecd and PDG content values aligned with the Chinese Pharmacopoeia standards. Treatment with 10 µmol/L Dex reduced zebrafish vertebral area, sum brightness, and ALP activity, but RAB and EC attenuated these effects. Combining 50 µg/mL RAB and 50 µg/mL EC was optimal for preventing GIOP in zebrafish. Reverse transcription-quantitative polymerase chain reaction was used to evaluate the mRNA expression of osteogenesis-related genes. A treatment of 10 µmol/L Dex decreased runt-related transcription factor 2 (Runx2), osteogenic protein-1 (OP-1), bone γ-carboxyglutamic acid-containing protein (BGLAP), and ß-catenin levels. This effect was counteracted by RAB and EC co-treatment (P < 0.05). Additionally, the effect of using the two herbal extracts together was better than single-herb treatments separately. These results demonstrated that RAB and EC preserve osteoblast function in the presence of GC. The best mass ratio was 1:1. CONCLUSION: RAB and EC herbal pair could ameliorate GC-induced effects in zebrafish, with 1:1 as the optimal dosage ratio.

Beta-Ecdysone Protects Mouse Osteoblasts from Glucocorticoid-Induced Apoptosis In Vitro.

Glucocorticoid-induced osteoporosis is a common form of secondary osteoporosis. Glucocorticoids affect both bone formation and resorption, and prolonged glucocorticoid exposure can suppress osteoblast activities. beta-Ecdysone, found in many plants, is involved in protein synthesis, carbohydrate and lipid metabolism, and immunologic modulation. Here, we evaluated the effects of beta-ecdysone on osteoblast viability by assessing apoptosis following treatment with excess glucocorticoids. Mouse bone marrow stromal cells were induced to differentiate and grow into osteoblasts, and then treated with 10 µM glucocorticoid and 10, 1, or 0.1 µM beta-ecdysone. The expression levels of osteoblast growth and differentiation factors (runt-related transcription factor 2, osteogenic protein-1, and alkaline phosphatase), apoptosis-related genes (transformation-related protein 53, ataxia telangiectasia mutated protein, caspase-3, and caspase-8), and Akt1 and phospho-Akt (Thr308) were then assessed via alkaline phosphatase staining, acridine orange-propidium iodide staining, annexin V/PI apoptosis assay, real-time RT-PCR, and Western blot analyses. Notably, treatment with 10 µM glucocorticoid resulted in reduced osteoblast viability and the specific activity of alkaline phosphatase as well as reduced runt-related transcription factor 2, osteogenic protein-1, and alkaline phosphatase mRNA expression in vitro, indicating that glucocorticoid inhibited osteogenic differentiation. Moreover, glucocorticoid treatment yielded increased transformation-related protein 53, ataxia telangiectasia mutated protein, caspase-3, and caspase-8 expression and decreased Akt1 and phospho-Akt levels, indicating glucocorticoid-induced apoptosis. Meanwhile, beta-ecdysone inhibited glucocorticoid function, preserving the expression of Akt1 and phospho-Akt and reducing the expression of transformation-related protein 53, ataxia telangiectasia mutated protein, caspase-3, and caspase-8. Thus, beta-ecdysone prevented glucocorticoid-induced osteoblast apoptosis in vitro. These data highlight the potential for beta-ecdysone as a treatment for preventing the effects of glucocorticoid on bone growth.