Observation on the Effect of Erzhiwan on Zebrafish Osteoporosis Model and Study on Autophagy Mechanism of Osteoclast

OBJECTIVE To observe the treatment efficacy of Erzhiwan on prednisolone-induced osteoporosis of zebrafish model and to clarify its regulation of autophagy mechanism on osteoclast differentiation. METHODS The young zebrafishes of good energy after 3 days， normal development were selected. The zebrafish osteoporosis model was established with 25 μmol/L prednisolone. The zebrafishes were divided into control group， model group， etidronate disodium group (300 mg/L)， Mohanlian group (50 μg/mL)， Nvzhenzi group (50 μg/mL) and Erzhiwan group (100 μg/mL). After 4 days， the calcein staining was adopted to measure the vertebral bone fluorescence area of zebrafish; qPCR was adopted to detect the osteogenic marker genes ALP， BMP-2b， Runx2 and osteoclast marker genes CTSK， TRAP， NFATC-1 expression. RAW264.7 cells were divided into control， LPS induction (10 μg/L)， EZW(100 μg /mL)， MHL(50 μg/mL)， and NZZ group (50 μg/mL) after they were cultured to 80%~90% density. The mRNA expression of TRAP， CTSK and autophagy-related genes 5 (ATG5)， ubiquitin-binding protein 62 (p62)， mammalian target of rapamycin(mTOR)，and Beclin were detected by qPCR. RESULTS Compared with the control group， the model group had significant declines in both vertebral bone fluorescence area and osteogenic marker gene expression and had significant rise in osteoclast marker genes（P<0.05，P<0.01）. Compared with the model group， the ED group， the Nvzhenzi group and the Erzhiwan group all remarkably improved zebrafish osteoporosis and acted better than both Mohanlian group and Nvzhenzi group（P<0.01）. qPCR showed that compared with the control group， the model group's osteogenic marker gene expression significantly declined and osteoclast marker genes rose conspicuously（P<0.05，P<0.01）. Compared with model group， the ED group， Mohanlian group and the Nvzhenzi group as well as Erzhiwan group had osteogenic marker gene expression rose significantly（P<0.05，P<0.01）， and Erzhiwan group has osteoclast marker genes reduced significantly（P<0.01）. The in-vitro study showed that the expression of osteoclast marker genes CTSK， TRAP， ATG5 and NFATC-1 induced by LPS as well as autophagy-related genes mTOR， ATG5， Beclin， p62 all rose（P<0.01）. The Erzhiwan group， Mohanlian group and Nvzhenzi group significantly decreased the expressions of osteoclast marker genes and autophagy-related genes（P<0.05， P<0.01）. CONCLUSION Erzhiwan can significantly improve the clinical symptoms of osteoporosis in zebrafish model， its mechanism may be related to the inhibition of the osteoclast-differentiation by reducing autophagy-related gene expressions.