Molecular Mechanisms And Preliminary Application Studies Of Natural Compounds On Osteoclast Related Osteolytic Diseases

Objective:To explore the biology effects of natural compounds Lycorine and Neogambogic acid(NGA)on osteoclastogenesis and bone resorption function,to elucidate the molecular mechanisms of natural compounds on osteoclastic bone resorption function,and to investigate the therapeutic effects of Lycorine on osteoclast related osteolytic diseases in vivo.Methods:(1)Primary osteoclast precursor cell BMMs were isolated from the whole long bone marrow of male,6-week-old,C57BL/6 mice.BMMs were induced by RANKL to differentiate into mature osteoclasts in vitro.The cytotoxic effects of Lycorine and Neogambogic acid on BMMs were determined using a CCK-8 assay,and the IC50 were calculated from the results;(2)To evaluate the impact of different concentrations of Lycorine and Neogambogic acid on osteoclastogenesis and bone resorption function;(3)Quantitative RT-PCR assay to evaluate the expression levels of osteoclast-specific genes;(4)Western blotting was used to examine the molecular mechanisms of Lycorine and Neogambogic acid in the inhibition of osteoclast differentiation;(5)To establishe an ovariectomy-induced murine.bone loss model and a Ti particle-induced murine calvarial osteolysis model,and investigate the therapeutic effects of Lycorine on local bone loss combining with micro-computed tomography(CT),histomorphologic analysis and RT-PCR.Results:(1)Lycorine at concentrations no more than 0.8 μM and Neogambogic acid at concentrations no more than 0.4μg/mL had no cytotoxic effects on the viability of BMMs.The differentiation of osteoclasts were both significantly inhibited in a dose-dependent manner.(2)When treated with different concentrations of Lycorine or Neogambogic acid,the bone resorption area on bovine bone slices both significantly reduced.When treated with Lycorine,quantitative analysis showed that the bone resorption area almost reduced to only 30%and 10%area of the control group at concentrations of 0.1 μM and 0.2μM,respectively,and the bone resorption almost completely disappeared at the concentration of 0.4μM.When treated with Neogambic acid,quantitative analysis showed that the bone resorption area almost reduced to 61%area of the control group at concentrations of 0.1μg/mL,and the bone resorption almost completely disappeared at the concentration of 0.4μg/mL.(3)Under the sitmulation of RANKL,the expression of osteoclast-specific genes including TRAP,CTR,CTSK,c-Fos,NFATc1,V-ATPase d2,V-ATPase a3 and DC-STAMP were significantly upregulated,but the expression of these genes was significantly suppressed in a time-dependent manner when treated with Lycorine and in a dose-dependent manner when treated with Neogambic acid.(4)Lycorine significantly attenuated the phosphorylation of ERK,P38 and JNK induced by RANKL,but the phosphorylation of IκB-α remained unchanged.Lycorine also significantly inhibited the phosphorylation of MEK1/2,MKK6 and MKK7,but the phosphorylation of the upstream kinase TAK1 did not change significantly.The phosphorylation of NFATc1 was also significantly attenuated when treated with Lycorine,suggesting that MKKs are likely the targets for Lycorine on osteoclastogenesis,rather than on the NF-κB pathway;(5)Western blotting revealed that Neogambic acid only significantly decreased phosphorylation of JNK,whereas phosphorylation of P38 was unaffected.The phosphorylation of p65 and NFATcl were also significantly attenuated.By fluorescent labeling of p65,the inhibition of nuclear import of p65 was confirmed and the expression of NFATc1 was attenuated.(6)An ovariectomy-induced murine bone loss model and a Ti particle-induced murine calvarial osteolysis model were successfully established.When treated with Lycorine:,the bone mass wers significantly elevated compared with the vehicle group and the bone microstructural indices wers reversed.Further histological analysis eonfirmed the number of osteoclasts was significantly decreased.Conclusion:Lycorine inhibited osteoclast differentiation and bone resorption via targeting MKKs in the MAPK signaling pathway,which can effectively prevent systemic bone loss induced by ovariectomy and local bone loss induced by titanium particles,suggesting that Lycorine has the potential value as a therapeutic drug for osteoclast related osteolytic diseases.Neogambic acid inhibited osteoclast differentiation and bone resorption via suppressing JNK and NF-κB activation,suggesting that NGA has the potential value as a therapeutic drug for osteoclast related osteolytic diseases.