Mechanism Of Ligustilied On RANKL-induced Osteoclastogenesis And Titanium Particle-induced Osteolysis Of Mice Calvaria

Bone is a dynamic organic tissue.Bone homeostasis depends on the balance of the function of osteoclast and osteoblast.Osteoclasts are the only cells involved in bone resorption and hold the post of essential role in bone density regulation.Dysfunction of osteoclasts may cause a great diversity of diseases such as osteoarthritis,osteoporosis,rheumatoid arthritis,speriodontitis,Paget,s disease,bone metastasis and so on.Therefore,the knowledge involved in the regulation of abnormal osteoclasts may be important for treating pathological osteolysis.Artificial joint replacement is one of the most important innovations in the field of surgery and an effective method for the treatment of various end-stage joint diseases.Aseptic loosening of prosthesis caused by wear particles induced osteolysis is a long-term complication of artificial joint replacement.Wear particles activate osteoclasts directly and indirectly,causing bone resorption and ultimately leading to aseptic loosening of prostheses.Although recent researches have been devoted to improving materials to reduce wear particles and prolong the life of prostheses,the generation of wear particles can not be completely avoided.Therefore,the research on development of new drugs is also the current hot spot.Currently,clinical drugs used to treat aseptic loosening of prostheses,such as bisphosnates,Denosumab and so on,have lots of side effect.Therefore,the discovery of effective drugs from natural products for the treatment of aseptic loosening of prostheses has become a hot area of research in recent years.Ligustilide?LIG?,the main component of the volatile oil isolated and purified from the root of A.sinensis,has been proved to exhibit several pharmacological properties,such as anti-inflammatory,antibacterial,analgesis,antioxidant,antitumor,and neuroprotection effects.However,its effect on osteoclasts and the mechanism of treating wear particle-induced osteolysis remain unclear.Our previous studies showed that LIG inhibited osteoclastogenesis in a dose-dependent manner.Therefore,this research will study the prevention and treatment effects of LIG on osteoclast related osteolysis diseases,providing a molecular biological and zoological research basis for future clinical application of ligustilide.Part I:Effects of ligustilide on differentiation and function of osteoclasts Objective:To study the effect of ligustilide on osteoclast formation and function in vitro.Methods:Bone marrow macrophages?BMMs?of C57BL/6 mice were extracted and cultured in vitro,then treated with different concentrations of LIG in the presence of RANKL for 5 days.TRAP staining was performed and the number of osteoclasts in each group was counted.BMMs were inoculated into 96-well plate,and treated with different concentrations of LIG for 48 hours.The cytotoxicity of LIG to osteoclast precursors was measured by MTS method,and IC₅₀0 was calculated.BMMs were inoculated into 96-well plate,and treated with different concentrations of ligustilide in the presence of RANKL for 5 days.Phalloidine/DAPI staining was performed and formation of F-actin ring was observed under confocal microscopy.BMMs were inoculated into 6-well plate to generate small osteoclasts,and transfered onto hydroxyapatite bone plate.The cells were treated with different concentrations of LIG.Three days later,bone resorption area was observed under microscope and calculated by image J software.Results:Osteoclastogenesis experiment showed that LIG?2.5,5,10,20?M?inhibited osteoclastogenesis in a dose-dependent manner.MTS test showed that LIG had no toxic effect on osteoclast precursor cells at concentrations below or equal to 40?M for 48 hours,IC₅₀ was 602.95?M.F-actin staining showed that LIG inhibited the formation of F-actin ring in a dose-dependent manner,which was supported by statistical analysis.The results of bone resorption experiment showed that LIG?2.5,5,10?M?inhibited the bone resorption area of osteoclasts in a dose-dependent manner.Part II The mechanism of ligustilide on osteoclast differentiation and functionObjective:To study the mechanism of ligustilide on osteoclast formation and function in vitro.Methods:qRT-PCR was carred out to detect the expression of osteoclast-related genes,including Vacuolar ATPase d2?V-ATPase d2?,Tartrate-resistant Acid Phosphatase?TRAP?,Dendritic Cells-Specific Transmembrane Protein?DC-STAMP?,Cathepsin K?CTSK?,Receptor Activator of NF-?B?RANK?,and Nuclear Factor of Activated T cells cl?NFATc1?.The effect of LIG on the RANKL-induced signaling pathway?including the signal pathways of NF-?B,MAPK and ITAM?was detected using Western Blot technique in order to find the target of LIG.Results:qRT-PCR showed that LIG inhibited the expression of osteoclast-relative genes,such as V-ATPase d2,DC-STAMP,TRACP,CTSK,RANK and NFATc1 in a dose-dependent manner compared with the control group.Western Blot results showed that LIG could inhibit the degradation of I?B and phosphorylation of p65 in NF-?B singnaling pathway,the phosphorylation of ERK and p38 in MAPK signaling pathway,the phosphorylation of Gab2 and PLC?2 in ITAM signaling pathway.Furthermore,LIG down-regulate the expression of TRAF6 protein.Part III Effects of ligustilide on differentiation and function of osteoblasts Objective:To study the effects of ligustilide on osteoblast formation and function in vitro.Methods:Osteoblast precursor cells were extracted and cultured from the skull of neonatal SD mice in vitro.The cytotoxicity of LIG to osteoblast precursor cells was measured by MTS and IC₅₀ was calculated.Osteoblast precursor cells were inoculated into 48-well plates and added with osteogenic medium?including?-glycerol,dexamethasone and vitamin C?.Alkaline phosphatase staining was performed on 14 days,and alizarin red staining was performed on21 days.Results:MTS test showed that LIG had no toxic effect on osteoblast precursor cells at concentrations below or equal to 40?M for 48 hours.IC₅₀ was 265.3?M.Alkaline phosphatase staining and alizarin red staining showed that ligustilide?2.5,5,10 and 20?M?had no significant effect on the generation and mineralization of osteoblasts.Part IV Protective effects of ligustilide on skull osteolysis induced by titanium particlesObjective:To study the protective effect of LIG on titanium particles-induced skull osteolysis model.Methods:Mouse models of skull osteolysis induced by titanium particles were established and divided into four groups,including sham-operated group,vehicle group,low dose group?2.5mg/kg?and high dose group?5mg/kg?.The calvaria of each group was collected and analyzed by microCT scanning after 14days of intervention with different concentrations of LIG.The numbers of osteoclasts were analyzed by Image J in histopathological section staining.Metabolic toxicity of LIG was analyed by pathlogical sections of liver and kidney.The levels of serum bone resorption related marker and inflammatory cytokines were analyzed by ELISA.Results:In the model of titanium-induced skull osteolysis,micro-CT showed that LIG could reduce the osteolysis induced by titanium particles,which showed that decreased of the percent of porosity and the number of porosity,and increased of Bone volume fraction?BV/TV?.Histological sections showed that ligustilide reduced the number of osteoclasts in the region of interest.Pathological sections of liver and kidney showed that LIG at therapeutic dose had no hepatorenal toxicity on mice.ELISA results showed that LIG decreased the levels of serum TNF-?,IL-1?,IL-1?,RANKL,OSCAR,and CTX-1,and increased the levels of serum OPG.Conclusion:?1?Ligustilide inhibited osteoclastogenesis,F-actin ring formation,bone resorption function and the expression of osteoclast-related genes,such as V-ATPase d2,DC-STAMP,TRACP,CTSK,RANK and NFATc1 in a dose-dependent manner.?2?Ligustilide may inhibit osteoclast formation and activation by inhibiting NF-?B/ERK/p38/ITAM signaling pathway.The drug target may be RANK.?3?Ligustilide had no effect on osteoblast formation and mineralization.?4?Ligustilide has protective effect on skull osteolysis induced by titanium particles in mice without hepatorenal toxicity.