Tao Hong Si Wu decoction reversed glucocorticoid-induced osteoporosis in zebrafish

To assess the therapeutic effect of Tao Hong Si Wu decoction (THSWD) on glucocorticoid-induced osteoporosis and its mechanism,the standard products of nine active components in THSWD were accurately weighed and the reserve liquid of standard product was prepared. Then a series of concentration standard liquid was obtained by dilution, and the standard curve was established. The formula of THSWD was proportionally extracted and the nine active components were determined by high performance liquid chromatography (HPLC). Naturally mated to obtain zebrafish embryos after incubation, the zebrafish larvae were cultured in blank E₃ medium until the 3 days post fertilization (DPF), then divided into control group (blank E₃ medium), prednisolone (PNSL) group (25 μmol·L^-1 PNSL), disodium ethydronate (DE) group (15 μmol·L^-1 DE and 25 μmol·L^-1 PNSL), THSWD group (0.1, 1.0 or 10.0 μg·mL^-1 THSWD and 25 μmol·L^-1 PNSL), 15 in each group. The culture was continued until the 10 DPF. The zebrafish was anesthetized and sacrificed, stained with alizarin red S, then the bone staining was observed under a microscope. Quantitative analysis of bone mineralization area and cumulative optical density. Expression of alkaline phosphatase (AKP), type I collagen (type I), runt-related transcription factor 2 (RUNX-2), osteoprotegerin (OPG), tartrate-resistant acid phosphatase 5b (TRACP) and osteocalcin (OCN) were assessed by quantitative real-time PCR. The mineral contents in zebrafish were measured using inductively coupled plasma-mass spectrometry. In the THSWD water extract, the content of 9 compounds in descending order was as follows:gallic acid > hydroxysafflor yellow A > chlorogenic acid > senkyunolide H > ferulic acid > caffeic acid, senkyunolide I > coumaric acid > benzoic acid. Compared with the control group, treatment of zebrafish with 25 μmol·L^-1 prednisolone significantly reduced the area of mineralized bone and cumulative optical density, and the expression of AKP, type I, RUNX-2, OPG, OCN gene was also significantly reduced. The levels of Ca, P, K, Mg, Zn and Fe in zebrafish were reduced by 2.8, 2.4, 13.8, 6.6, 8.0 and 8.8 times, respectively. Compared with the prednisolone group, after treating with THSWD, the area of mineralized bones and cumulative optical density value of zebrafish increased significantly and was dose-dependent, and the expression of related genes also increased. The Ca, P, K, Mg, Zn, Fe levels were significantly higher than the prednisolone group. Result show that THSWD has the potential to reverse the effect of glucocorticoid osteoporosis, the mechanism of which is to enhance the activity of osteoblast, promote the expression of bone gelatin and bone mineralization, and increase bone mass.