ABSTRACT
Objective::To evaluate the intervention effect of Bushen Tongluo formula on zebrafish osteoporosis model and to clarify its regulation of autophagy mechanism on osteoclast differentiation. Method::The 260 young zebrafishes (3 dpf) were selected, the zebrafish osteoporosis model was established with 25 mmol·L-1 prednisolone (Pred) for 3 days, confirming the successful model by calcein staining. The zebrafishes were divided into control group, Pred group (25 mmol·L-1), etidronate disodium (ED) group (300 mg·L-1), Bushen (BS) group (180 mg·L-1), Tongluo (TL) group (30 mg·L-1), and Bushen Tongluo (BSTL) group (210 mg·L-1), 40 tails per group. After intervened with medicine for 4 days, the calcein staining was adopted to count the vertebral bone fluorescence area of zebrafish, Real-time PCR was adopted to detect the mRNA expression of akaline phosphatase (ALP), bone morphogenetic protein 2b (BMP-2b), runt-related protein 2 (Runx2) and cathepsin K (CTSK), phosphorus and tartrate-resistant acid phosphatase (TRAP), nuclear factor of activated T-cells, cytoplasmic-1 (NFATC-1). Divided RAW264.7 cells into control, Rankl induction (10 μg·L-1), BS (180 mg·L-1), TL (30 mg·L-1), and BSTL group (210 mg·L-1) after they were cultured to 80%-90% density. The expression of actin ring was detected by phalloidin cytoskeleton staining. The mRNA expression of TRAP, CTSK and autophagy-related genes 5 (ATG5), autophagy-related genes 7 (ATG7), and ubiquitin-binding protein p62 (p62) were detected by Real-time PCR. Result::Compared with the control group in vivo, the vertebral area and ALP, BMP-2b, and Runx2 expression of zebrafish in the Pred group were significantly decreased (P<0.01), and CTSK, TRAP, and NFATC-1 expression of zebrafish were significantly increased (P<0.01). Compared with the Pred group, the vertebral area of the TL (P<0.05) and BSTL group (P<0.01) increased significantly. The expressions of ALP, BMP-2b, and Runx2 in the BS, TL, and BSTL group were significant increased (P<0.01). The expression of CTSK, TRAP, and NFATC-1 in BSTL group were significant decreased (P<0.05, P<0.01). Compared with the control group in vitro, the number of actin rings (P<0.01) and CTSK, TRAP, ATG5 (P<0.01) expression and ATG7, p62 (P<0.01) expression were significantly increased in the Rankl-induced group. Compared with the Rankl induction group, the number of actin rings and CTSK and TRAP expression were significantly decreased in the BS, TL, and BSTL group (P<0.05, P<0.01), while the expression of ATG5, ATG7, and p62 were significant decreased in the BSTL group (P<0.05). Conclusion::BSTL can significantly improve prednisolone-induced zebrafish osteoporosis and inhibit osteoclast differentiation by reducing autophagy-related gene expression.