The Mechanisms For Erigeron Breviscapus To Accelerate Orthodontic Tooth Movement

Objective:To study the expression and distribution of Osteoprotegerin(OPG),receptor activator of nuclear factor-κB ligand (RANKL) in orthodontic rebuilding of the periodontal tissues,and the effection of erigeron breviscapus(EB) injection on it.Methods:64 Wistar mice were randomly divided into 4 groups(groups A,B,C and D).Group A was given orthodontic appliance and injected with EB.Group B was only given orthodontic appliance. Group C was only injected EB injection.Group D was the control group. Groups A,B and C was observed 1,3,7,10,14d respectively,with 4 mice in each group.We selected the maxillary first molar as experimental tooth and observe the periodontal tissues of the mesi-glossia root.After killed on schedule,each mice of all groups was made into the sections of the periodontal tissues,the sections were studied histologically by HE stain and examined the expression and distribution of OPG and RANKL by hybridization in situ.Results:.â‘ The histological results were in line with normal orthodontic remodeling of the alveolar bone.Osteoblastic new bone formation on the tension side of A and B groups,and osteoclastic bone resorption on the pressure side of A and B groups.From A groups,the another effection of EB on orthodontic remodeling of the periodontal tissues was neovascularization.â‘¡From 1 to 7 days,the expression of OPG was gradually enhanced in the periodontal tissues on the tension side and on the pressure side.OPG was seen in the cytoplasm of osteoblasts,bone stave cells,cementoblasts and fibrablasts on the tension side,also seen in the cytoplasm of cementoblasts and fibrablasts on the pressure side,but was not seen in the cytoplasm of osteoclasts.EB did not change the distribution of OPG,but inhibited the expression of OPG.â‘¢From 1 to 7 days,the expression of RANKL was gradually enhanced in the periodontal tissues on the tension side and on the pressure side. RANKL was seen in the cytoplasm of osteoblasts,osteoclasts,bone stave cells,cementoblasts and fibrablasts on the tension side and on the pressure side.EB did not change the distribution of RANKL,but increased apparently the expression of RANKL.Conclusions:OPG and RANKL can play an important role in orthodontic rebuilding of the periodontal tissues.EB can inhibit the expression of OPG and increase the expression of RANKL,thus accelerate tooth movement. Objective:To study the effection of erigeron breviscapus(EB) with different concentration on proliferation or differentiation of MG-63 osteoblast-like cells and RAW264.7 pre-osteoclast cells in vitro by methyl thiazolyl tetrazolium(MTT) approach,[³H]TdR incorporation assay and Trypan blue excluding test.Methods:MG-63 osteoblast-like cells and RAW264.7 pre-osteoclast cells were cultured in vitro.Induction and differentiation of osteoblast:RAW264.7 cells were planted in a 6-well culture plate and every well contained 1×10⁵ cells. They were cultured with RANKL(50ng/ml),M-CSF(25ng/ml) and EB(0 mg/ml,0.001mg/ml,0.01mg/ml,0.1mg/ml,1mg/ml),TRAP staning and counting multinucleated osteoclast.MTT approach:MG-63 osteoblast-like cells or RAW264.7 pre-osteoclast cells were planted in a 96-well culture plate and every well contained 1×10⁴ cells.After 24h,they were cultured with EB(0 mg/ml,0.001 mg/ml,0.01 mg/ml,0.1 mg/ml,1mg/ml).After 36h,they were dyed by MTT method and OD(optical density) value of every well was detected by enzyme linked immunodetection meter.[³H]TdR incorporation assay:MG-63 osteoblast-like cells or RAW264.7 pre-osteoclast cells were planted in a 24-well culture plate and every well contained 2×10⁴ cells.After 24h,they were cultured with EB(0 mg/ml,0.001mg/ml,0.01mg/ml,0.1mg/ml,1mg/ml).After 32h,they were incorporated by[³H]TdR,and were detected[³H]TdR.Trypan blue excluding test:MG-63 osteoblast-like cells or RAW264.7 pre-osteoclast cells were planted in a 24-well culture plate. Experimental group were cultured with EB,they were dyed by Trypan blue,and counted cells from 1to 6 day.Then the curve of growth was drawed.Results:EB could not induce directly RAW264.7 pre-osteoclast cells into multinucleated osteoclast of TRAP positive staining,but EB(0.01-1mg/ml) could promote RANKL and M-CSF to induce RAW264.7 into multinucleated osteoclast.The result of MTT and[³H]TdR incorporation assay indicated that EB could promote dose-dependently the proliferation of MG-63 osteoblast-like cells and RAW264.7 pre-osteoclast cells in vitro.Trypan blue excluding test:The curve of growth of MG63 osteoblast-like cells and RAW264.7 pre-osteoclast cells rised gradually in the control groups and the experiment groups,and the experiment groups were apparent than the control groups.Conclusions:With some range of its concertration,EB could promote the proliferation of osteoblast in vitro in dose and time dependent.EB could promote RANKL and M-CSF to induce pre-osteoclast cells into multinucleated osteoclast. Objective:To study the effection of erigeron breviscapus(EB) with different concentrations and different intervention time on the expression of OPG/RANKL/RANK in MG-63 osteoblast-like cells and RAW264.7 pre-osteoclast cells in vitro,thus to propose the role that EB can play in bone rebuilding.Methods:MG-63 osteoblast-like cells and RAW264.7 pre-osteoclast cells were cultured in vitro.The 3^th passage of cells were divided into the control group and the different experiment group.The different EB,including 0,0.001,0.01,0.1,1.0 mg/mL concentration,were join respectively into the different groups.Total RNA and protein were respectively isolated from cells after treatment with different concentration of EB for 12,24,and 48 hours.Expressions of OPGmRNA and RANKLmRNA in MG-63 osteoblast-like cells and RANKmRNA in RAW264.7 pre-osteoclast cells were detected by semiquantitative RT-PCR..Expressions of OPG protein and RANKL protein in MG-63 osteoblast-like cells and RANK protein in RAW264.7 pre-osteoclast cells were detected by Western blot.Results:RT-PCR and Western blot indicated that EB could inhibit the expression of OPGmRNA and protein in MG-63 osteoblast-like cells in dose dependent,and EB could promote the expression of RANKLmRNA and protein in MG-63 osteoblast-like cells in dose dependent.Western blot also indicated that EB could promote the expression of RANKL protein in MG-63 osteoblast-like cells in time dependent,and RANKL protein was increased remarkbly by EB intervention after 12,24,48h.RT-PCR and Western blot indicated that EB could promote the expression of RANKmRNA and protein in RAW264.7 pre-osteoclast cells in dose dependent from 0,0.001,0.01,0.1 to 1.0 mg/mL concentration.Western blot also indicated that EB could promote the expression of RANK protein in RAW264.7 pre-osteoclast cells in time dependent,and RANK protein was increased remarkbly by EB intervention after 12,24,48h.Conclusions:EB could inhibit the secretion of OPG in osteoblasts,and could promote the secretion of RANKL in osteoblasts and the secretion of RANK in pre-osteoclast,thus indicated EB could promote bone resorption. Objective:To study the effection of erigeron breviscapus(EB) and(or) mechanical force on the expression of OPG/RANKL/RANK mRNA and protein in MG-63 osteoblast-like cells and RAW264.7 pre-osteoclast cells in vitro,thus to propose the role that EB can play in bone rebuilding.Methods:MG-63 osteoblast-like cells and RAW264.7 pre-osteoclast cells were cultured in vitro,and were stimulated by a mechanical instrument called SXG4201 model.The 3th passage of cells were divided into the control group and the different experiment group.The different groups were intervented respectively by EB(1mg/ml),mechanical force(2000μstrain,0.2Hz), EB(1mg/ml) combined with mechanical force(2000μstrain, 0.2Hz).Total RNA and protein were respectively isolated from cells after 3,6,12 and 24 hours.Expressions of OPGmRNA and RANKLmRNA in MG-63 osteoblast-like cells and RANKmRNA in RAW264.7 pre-osteoclast cells were detected by semiquantitative RT-PCR.Expressions of OPG protein and RANKL protein in MG-63 osteoblast-like cells and RANK protein in RAW264.7 pre-osteoclast cells were detected by Western blot.Results:From 3,6,12 to 24 hours after intervented by mechanical force,the expression of OPG protein in MG-63 osteoblast-like cells was increased in time dependent,but the expression of RANKL protein in MG-63 osteoblast-like cells and the expression of RANK protein in RAW264.7 pre-osteoclast cells did not be changed apparently.Mechanical force(2000μstrain,0.2Hz) increased the expression of OPG protein in MG-63 osteoblast-like cells,but could not change the expression of RANKL protein in MG-63 osteoblast-like cells.EB(1 mg/ml) decreased the expression of OPG protein in MG-63 osteoblast-like cells,and increased the expression of RANKL protein in MG-63 osteoblast-like cells.EB(1mg/ml) combined with mechanical force (2000μstrain,0.2Hz) decreased the expression of OPG protein in MG-63 osteoblast-like cells,and increased apparently the expression of RANKL protein in MG-63 osteoblast-like cells.Mechanical force(2000μstrain,0.2Hz) could not change the expression of RANK protein in RAW264.7 pre-osteoclast cells.EB(1mg/ml) increased the expression of RANK protein in RAW264.7 pre-osteoclast cells.EB(1mg/ml) combined with mechanical force (2000μstrain,0.2Hz) increased apparently the expression of RANK protein in RAW264.7 pre-osteoclast cells.Conclusions:EB could inhibit the secretion of OPG in osteoblasts by mechanical force,and could promote the secretion of RANKL in osteoblasts and could also promote the secretion of RANK in pre-osteoclast by EB combined with mechanical force.