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

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.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.According to the Chinese Pharmacopoeia (2015), β-ecdysone (β-Ecd) is a major bioactive marker in RAB extract, while pinoresinol diglucoside (PDG) is the major marker in EC extract. Both of β-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 β-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.RAB and EC herbal pair could ameliorate GC-induced effects in zebrafish, with 1:1 as the optimal dosage ratio.