Effect Of Osthole On The Differentiation And Maturation Of Osteoclast In Vitro And Mechanism

Objective:To observe the effect of osthole(OST)on osteoclast(OC)differentiation and maturation,and study its molecular mechanism,so as to clarify the mechanism of osthole against osteoporosis.Methods:The rat bone marrow monocytes to establish induced osteoclast culture system:using a special device for separating bone marrow monocytes extracted bone marrow monocytes;morphologic observation of bone marrow mononuclear cell growth;CCK-8 was adopted to observe the growth characteristics of bone marrow monocytes;mononuclear cell purity by flow cytometry method validation get through this method;staining by tartrate resistant acid phosphatase(TRAP)and calcitonin receptor(CTR)immunofluorescence staining was used to identify osteoclasts;construct induced osteoclast culture system by mouse RAW264.7 cell lines:To observe the morphological characteristics of the growth of RAW264.7 cells;identification of osteoclasts by tartrate resistant acid phosphatase staining;The use of CCK-8 was screened had no cytotoxicity on bone marrow monocytes and RAW264.7 cell lines 10-6mol/L,10-5mol/L concentration in group,solvent control group(DMSO)as negative group,receptor activator of nuclear factor kappa B ligand(RANKL)group;OST staining was used to observe the two kinds of cells into osteoclasts when the cell differentiation changes of TRAP positive cells and the fusion index using TRAP;use the lacunae of toluidine blue staining and scanning electron microscopy observation of two kinds of cells to break the process of bone cell differentiation of bone lacuna in the number,total area and average area changes;use of cytoskeletal actin ring(FITC-Phalloidin)staining method to observe the effect of OST the bone marrow monocytes induced to osteoclast morphology;osteoclast apoptosis staining method to observe the effect of OST on bone marrow monocytes induced by using acridine orange;Effect of using the luciferase assay to observe the expression of OST in RAW264.7 cells and NF-NFAT transcription factor kappa B;to study the effect of OST application of q-PCR technology on osteoclast cell line RAW264.7 gene NFATc1,CTSK,MMP-9,TRAP,INTE-BETA3,C-SRC expression;effect of RAW264.7 cell line NF-kappa B signal pathway related protein expression of OST was detected by Western blotting technology.Results:we successfully established the osteoclast culture system to induce rat bone marrow monocytes;rat bone marrow monocytes proliferation in vitro stability in time for a week or so;flow cytometry showed that the expression of CD11b in primary cultured rat bone marrow monocytes in the rate of 98.6%,higher purity;and by TRAP staining and CTR immunofluorescence staining identified successfully;successful establishment of mouse RAW264.7 cell line induced by osteoclast culture system;and through TRAP staining method successfully identified.OST can significantly inhibit the bone marrow monocytes and RAW264.7 cells generated under RANKL stimulation of TRAP positive cells and the fusion index,the differences were statistically significant(P<0.05),and the two concentration difference between the two groups was statistically significant(P<0.05);OST can differentiate bone lacuna number and bone lacuna the total area of induced inhibition of bone marrow monocytes and RAW264.7 cells,the difference was statistically significant(P<0.05),bone marrow monocytes of bone lacuna.The average area of OST group than in the control group decreased,the difference was statistically significant(P<0.05),but there was no significant difference between the two concentration(P>0.05).Lacunaes average area of RAW264.7 cells between several groups were not statistically significant(P>0.05);appearance of osteoclast function structure of OST can also inhibit the differentiation of bone marrow monocytes;in addition,OST can promote the apoptosis of bone marrow monocytes differentiate into osteoclasts,and high concentration group of apoptosis the effect is better,the differences were statistically significant(P<0.05).OST can also inhibit the transcription factor NFAT and NF-kappa B promoter,accompanied by decreased expression of downstream genes and fluorescence values for the OST group compared with the control group,the difference was statistically significant(P<0.05),but the effect is not as good as the positive control group;OST can also inhibit the expression of NFATc1,CTSK,MMP-9,TRAP C-SRC,INTE-BETA3,these osteoclast specific genes,except Integrin-BETA3 and C-SRC,the concentration of two groups,the differences were statistically significant(P<0.05);in addition,OST can NF-kappa B signaling pathway on NF-kappa B nuclear translocation and I kappa p65 to B phosphorylation,inhibit the expression of related signal the pathway of protein.Conclusion:This study confirms that osthole can inhibit the differentiation and maturation of osteoclasts by inhibiting the expression of NF-kappa B and NFATcl related transcription factors.Moreover,the best concentration interval of OST inhibiting osteoclast differentiation is 10-6 mol/L-10-5 mol/L,which lays a foundation for the new drug research with osthole as active ingredient.