| BackgroundDue to the lack of drugs that can effectively block the progression of pathological new bone formation in ankylosing spondylitis(AS),traditional Chinese medicine with multi-component and multi-target properties has been shown to be effective in improving joint inflammation and abnormal bone metabolism.The previous studies of our group showed that Bushen Qiangdu Zhilv Decoction,the representative prescription of Bushen Qiangdu,could not only improve the AS inflammation but also reduce sacroiliac joint osteosclerosis,but its specific mechanism is still unclear and needs further study.ObjectiveTo investigate the effect of Bushen Qiangdu Zhilv Decoction(BSQD)on osteogenic differentiation of AS’s pathological new bone formation cell model in vitro(MC3T3-E1),and to analyze and determine the active components and content of BSQD and to observe the effect of its high content compounds on osteogenic differentiation.Methods1 Experiments in Cellular Molecular BiologyAlizarin red S staining was used to detect osteogenic differentiationof osteoblastic precursor cells MC3T3-E1 induced by osteogenic inductionmedium in order to construct a cell model of new bone formation in vitro.Then,CCK-8(Cell count kit-8)cytotoxicity kit was used to detect the effect of BSQD on the activity of MC3T3-E1 cells.Alizarin red S staining wasused to detect the effect of BSQD on the cell mineralization capacity.ThemRNA expression levels of ALP,Runx2,β-catenin and BMP2 were detected by reverse transcription-polymerase chain reaction(RT-PCR).Western blottingwas used to detect Runx2 protein expression.Microplate method of ALP activity was used to detect the effect of BSQD on ALP activity.2 Component analysis of BSQD and activity detectionHigh performance liquid chromatography(HPLC)was used to separate the components of BSQD,and the chromatogram peak was obtained.Then,the chemical components in the decoction were identified by comparing the retention time of the chromatogram peak with that of the standard reference substance,and their contents were determined by standard curve.Moreover,the effect of the high content compound on the osteogenic differentiation of MC3T3-E1 cells derived from mouse was detected.Results1 Construction of new bone formational cell model in vitro.Under the optical microscope,alizarin red staining assay showed that mineralized nodules of MC3T3 cells were more widely distributed and the number of mineralized nodules increased significantly compared with the control group after 4 or 6 days of culture in osteogenic induction medium,indicating that the construction of new bone formational cell model in vitro was successful.2 Effect of BSQD on osteogenic differentiation and mineralization of MC3T3 cellsCompared with blank control group,the intervention of BSQD(0-20mg/ml)for 24h and 48h had no inhibitory effect on the activity of MC3T3-E1 cell-s,and the difference was not statistically significant(P>0.05).The inhibitory effect of celecoxib on MC3T3-E1 cells enhanced with the increase of drug concentration and treatment time,and from the demarcation line of 0.02mg/ml(P>0.05),the inhibitory effect of celecoxib on MC3T3-E1 cells in-creased rapidly with the increasing concentration.Compared with the osteoinductive group,BSQD(20mg/mL)and celecoxib(0.02mg/mL)could significantly inhibit the formation of cell’s mineralized nodules(P<0.01);The expression of ALP,Runx2,β-catenin and BMP2 mRNA were significantly decreasedand the activity of ALP on MC3T3-E1 was inhibited by BSQD(20mg/ml),and the difference was statistically significant(P<0.01).3 Active ingredients and their contents of BSQD,and effect of its higher content compounds on osteogenic differentiationNine compounds in BSQD identified were:chlorogenic acid,mangiferin,a-lbiflorin,psoralenoside,icariin,paeoniflorin,naringin,asperosaponin VI,o-sthole.Determination of co ntent(from the high to low):lmg/ml BSQD respectively contains 8.97ug/ml asperosap onin VI,5.08ug/ml paeoniflorin,2.77ug/ml chlorogenic acid,1.05ug/ml psoralenoside,0.68ug/ml mangiferin,0.66ug/ml albiflorin,0.59ug/ml naringin,0.40ug/ml icariin,and 60mg/ml BSQD contains 0.58ug/ml osthole,among which the content of asperosaponin VI was the highest.Therefore,the effect of asperosaponin VI on osteogenic differentiation wasthen observed.Cytotoxicity assay showed that asperosaponin VI(0-200ug/ml)had no inhibitory effect on the activity of MC3T3 cells(P>0.05).Asperosaponin VI could inhibit the activity of ALP and the formation of mineralized nodules(P<0.05 or P<0.01),and asperosaponin VI could decrease the expression of β-catenin,ALP and Runx2 mRNA in different degrees(P<0.05 or P<0.01)Conclusion1 BSQD can inhibit the osteogenic differentiation and mineralization function of MC3T3-E1 cells.2 BSQD can inhibit the expression of osteogenic genes ALP,Runx2 and β-catenin mRNA,and inhibit the activity of ALP and the expression of Runx2 protein,which may be the molecular mechanism of BSQD in inhibiting new bone formation and related to its inhibition of Wnt/β-catenin—Runx2 signaling pathway.3 Asperosaponin VI in BSQD has a high content,and can inhibit the osteogenic differentiation related factors,ALP activity and mineralization function of MC3T3 cells,which may be one of the effective and active components of BSQD in inhibiting new bone formation in AS... |
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