The Ameliorate Effect Of 7,8-Dihydroxyflavone On Osteoporosis And Its Mechanism

Bone tissue achieves self-renewal through bone remodeling,which integrates osteoblast-mediated bone formation and osteoclast-mediated bone resorption.Disrupting the balance between bone formation and resorption leads to osteoporosis.7,8-Dihydroxyflavone(7,8-DHF),a structurally rare flavonoid derived from plants,has been identified as a small-molecule mimetic of brain-derived neurotrophic factor(BDNF).7,8-DHF can selectively activate the tropomyosin receptor kinase B(Trk B)and exert beneficial effects in various BDNF/Trk B-related disease models.Previous studies have shown that BDNF could promote osteoblast differentiation,migration,and fracture healing.However,it remains unclear whether 7,8-DHF can mimic the function of BDNF and exhibit positive regulatory effects on osteoblasts or/and osteoclasts,thus affecting the bone remodeling processes.To investigate the role of 7,8-DHF in regulating osteoporosis caused by bone remodeling imbalance,a systematic and deep study was conducted to explore the relevant efficacy and mechanism using cell models in vitro and whole animal models in vivo.The main research contents and results are as follows:(1)MC3T3-E1 cells were used as the osteoblast cell model to investigate the effects of 7,8-DHF on osteoblast differentiation and mineralization.Treatment of MC3T3-E1 cells with 0.5 μM,1 μM and 5 μM 7,8-DHF could promote cell proliferation by up-regulating the m RNA level of cyclin D1,thus stimulating more cells at S phase but less at G0 phase to active DNA synthesis.During the osteogenic differentiation of MC3T3-E1 cells,7,8-DHF could effectively increase the activity of alkaline phosphatase(ALP),an indicator of early-stage osteoblastic differentiation,and enhance mineralized nodule formation in a dose-dependent pattern.(2)Focusing on the Wnt/β-catenin signaling pathway,the molecular mechanism underlying the osteogenic effects of 7,8-DHF was investigated.7,8-DHF could inactivate GSK3β by increasing its phosphorylation of the Ser 9 residue and increase the levels of β-catenin to a certain extent,thereby inducing the m RNA and protein expression of osteogenic target genes,including Runx2 and Osterix.In addition,a β-catenin knockdown experiment was performed to demonstrate the importance of the Wnt/β-catenin signaling pathway in the 7,8-DHF-induced upregulation of osteogenic target genes.When β-catenin was silenced,7,8-DHF was unable to induce the upregulation of Runx2 and Osterix expression.(3)The Trk B inhibitor K252 a was used to further investigate the role of Trk B activation in 7,8-DHF-mediated osteogenesis.MC3T3-E1 cells were pretreated with K252 a to effectively inhibit Trk B and then added 0.5 μM or 1 μM of 7,8-DHF.Results showed that K252 a significantly suppressed 7,8-DHF-induced cell proliferation and the ALP activity.As for the Wnt/β-catenin signaling pathway,the p-GSK3β/GSK3β ratio was reduced due to Trk B inhibition,indicating that the inactivated state of GSK3β was reversed.The expression of β-catenin,Runx2,and Osterix,were all reduced conspicuously when K252 a was added prior to 7,8-DHF.(4)RAW264.7 cells were used as the osteoclast cell model to investigate the effects of 7,8-DHF on osteoclast formation,differentiation,and functional expression.The above three concentrations of 7,8-DHF did not exhibit cytotoxicity on RAW264.7cells.The formation of TRAP-positive osteoclasts induced by RANKL was sharply reduced by 0.5 μM and 1 μM of 7,8-DHF,though 5 μM 7,8-DHF did not show any obvious effects.Besides,0.5 μM and 1 μM,not 5 μM of 7,8-DHF could notably decrease the protein levels of bone resorption markers,CTSK and MMP-9.This may be associated with the downregulation of the expression of c-Fos and c-Jun,which are two important transcription factors in the osteoclast-related signaling pathways.(5)The OVX-induced osteoporotic rat model was constructed to further evaluate the effects of continuous oral gavage of 7,8-DHF at doses of 5 mg/kg·BW/day and 10mg/kg·BW/day for 12 weeks on the improvement of osteoporosis.Firstly,7,8-DHF was devoid of any uterotrophic activity.The 10 mg/kg·BW/day of 7,8-DHF could significantly increase the BMD and improve the microstructure of trabecular bone of the femur in ovariectomized rats,leading to a more complete and compact trabecular bone.While 5 mg/kg·BW/day of 7,8-DHF showed a better effect on enhancing bone biomechanical parameters such as the maximum load,the fracture deflection and the failure strain of the tibia.What’s more,intervention with 7,8-DHF could reduce the number of TRAP-positive osteoclasts in bone tissue,suppress the excretion of urinary calcium and lower the levels of serum bone turnover biochemical markers such as ALP and BGP,improving the high bone turnover state caused by ovariectomy.In summary,7,8-DHF has a fascinating dual regulatory effect of promoting osteoblast differentiation and maturation while inhibiting osteoclast formation and functional expression.The osteogenic effect of 7,8-DHF is mainly mediated by Trk B,which acts as a‘bridge’to activate the Wnt/β-catenin signaling pathway,thereby inducing the transcription and translation of downstream osteogenic target genes,and ultimately promoting the differentiation and mineralization of osteoblasts.In addition,7,8-DHF also demonstrates a good improvement effect on BMD,microstructure of bone tissue,bone biomechanical strength,and bone metabolism in osteoporosis animal models.Therefore,7,8-DHF has the potential to be used as a functional ingredient for the prevention,improvement,and treatment of osteoporosis in health food and medicine,providing a new idea and strategy for the treatment of osteoporosis.