The Anti-osteoporosis Effect Of Oroxin A In Zebrafish And Its Mechanism Of Inhibiting Osteoclast Differentiation And Bone Resorption

Objective: Oroxylum indicum(L.)Vent,a traditional Chinese medicine,has abundant chemical compounds and a variety of pharmacological effects,including anti-inflammatory,anti-microbial,anticancer,anti-oxidant,hepatoprotective and antidiabetic activity,etc.But the anti-osteoporosis effect of Oroxylum indicum has not been extensively investigated yet.As one of the main bioactive components of Oroxylum indicum,Oroxin A(OA)has been proved to have anti-cancer and antidiabetic effects,etc.However,the anti-osteoporosis activity of OA remains unknown.Therefore,this study aims to explore the antiosteoporosis activity and mechanism of OA in vitro and in vivo,which will provide experimental basis for its further drug development and clinical application.Methods:(1)To investigate the anti-osteoporosis effect of OA in zebrafish.The larvae of zebrafish at 3 days after fertilization were incubated with OA to explore the safe dose of OA in vivo.Then,prednisolone was used to induce zebrafish osteoporosis,and alizarin red staining was performed to evaluate the effect of OA in prednisoloneinduced osteoporosis zebrafish.Finally,q RT-PCR was used to detect the expression of osteoclast-specific marker genes trap,ctsk,mmp-9 and the expression of anti-osteoclastic genes,such as runx2 b,sp7,bmp4,alp,col1α1 in zebrafish larvae.(2)To investigate the anti-osteoclastic activity of OA.Bone marrowderived mononuclear macrophages(BMMs)were isolated and extracted from the femur and tibia of C57 mice at 4-6 weeks.The effect of OA on the proliferation of BMMs and RAW264.7 cells was investigated by CCK-8.Within the safe dose,the effect of OA on osteoclast formation and bone resorption was investigated by TRAP staining and pit formation assay.The osteoclast-related markers were detected by q RT-PCR and Western Blot(WB),such as TRAP,CTSK,MMP-9.(3)To investigate the effect of OA on osteoclast ER/RANK/NFATc1 signaling pathway.The regulatory effect of OA on the key signaling genes of osteoclast differentiation Erα,Rank,c-fos and Nfatc1 was assessed by q RT-PCR.After treatment with OA,the expressions of ERα,RANK,TRAF6,c-Fos,NFATc1 and the phosphorylation levels of ERK-MAPK,JNK-MAPK,p38-MAPK,p65,Akt pathways in RAW264.7 cells were detected by WB.Results:(1)The zebrafish toxicity test showed that after OA intervention for 5 days,there was no obvious drug toxicity within the range of 32 μM.Compared with the control group and the solvent group,the area of alizarin red staining and integrated optical density(IOD)of the zebrafish skull in the prednisolone group were significantly reduced,which indicated the prednisolone-induced osteoporosis model using zebrafish was established successfully.After OA treatment,the staining area and IOD of the zebrafish skull significantly increased,reflecting the anti-osteoporosis activity of OA.The results of q RT-PCR showed that OA inhibited the expression of mmp-9 and up-regulated the expression levels of osteoblastrelated genes runx2 b,sp7,alp,bmp4 and col1α1,which suggested OA improves osteoporosis in zebrafish by promoting bone formation.(2)OA had no obvious cytotoxicity in the range of 8 μM as detected by CCK-8 experiment.TRAP staining showed that OA significantly inhibited the formation of osteoclasts.The pit formation assay showed that OA significantly inhibited the bone resorption function of osteoclasts.In addition,OA significantly down-regulated the expressions of osteoclastrelated markers TRAP,CTSK,and MMP-9 at m RNA and protein levels in a dose-dependent manner.(3)As detected by q RT-PCR and WB,OA up-regulated the expressions of ERα,a key signaling molecule for osteoclast differentiation,and down-regulated RANK with no statistical significance.Simultaneously,OA significantly down-regulated the m RNA and protein expression levels of TRAF6,c-Fos and NFATc1.After inducing by RANKL,OA significantly inhibited the phosphorylation levels of ERK,JNK,p38 and NF-κB pathways,but had no significant effect on Akt pathway.Conclusion: OA effectively improves prednisolone-induced osteoporosis in zebrafish,and significantly inhibits BMMs,RAW264.7cells differentiation into osteoclasts and bone resorption induced by RANKL,indicating that OA has application potential in the treatment of osteoporosis.14 figures,9 tables,140 references...