Effect Of Isorhamnetin Extracted From Ginkgo Biloba On The Differentiation Of RAW264.7 Cells Into Osteoclasts And Its Molecular Mechanism

Objective:Osteoporosis is a common chronic disease that is commonly accompanied by osteoclast-mediated bone resorption that is greater than osteoblast-induced bone formation during disease development,resulting in imbalanced bone metabolism.Osteoclasts are also the main cells in alveolar bone metabolism,what's more,using natural drugs to inhibit osteoclast activity is one of the ways to prevent and improve osteoporosis.The objective of this experiment is to investigate the effect of isorhamnetin extracted from Ginkgo biloba on the differentiation of osteoclasts and its possible molecular mechanisms.Method:1?CCK8 assay was used to evaluate the effect of the toxicity of isorhamnetin(ISO)at different concentrations(0-10?M)to RAW264.7cells for 1,3 and 5 days.2?The RAW264.7 cell line was induced by different concentrations of RANKL,and then was stained with tartrate-resistant acid phosphatase(TRAP)to bserve cell morphology on day 5,and the mature osteoclasts were quantified.3 ? In the process of RANKL induced RAW264.7 cell lineVII differentiation into mature osteoclasts,we use different concentrations of ISO to stimulate the cells,then TRAP staining on the 7th day was used to observe mature osteoclast cell morphology of the different experimental groups.4 ? In the process of RANKL induced RAW264.7 cell line differentiation into mature osteoclasts,we use different concentrations of ISO to stimulate the cells,then counting the the number of bone resorption lacuna scanned by SEM on the 7th day was used to observe mature osteoclast cell morphology of the different experimental groups.5 ? In the process of RANKL induced RAW264.7 cell line differentiation into mature osteoclasts,we use the RT-PCR method to detect the m RNA expression of Ctsk ?MMP-9?NFATC1?c-Fos and p65 by osteoclast that stimulated by ISO(0-10?M)on 7day.7 ? The experimental results statistics were used by SPSS20.0software statistics,CCK8 results using two-factor analysis of variance,real-time quantitative PCR results using independent samples t test analysis,P <0.05 was considered statistically significant.Results:1 ? The CCK8 assay result showed that the ISO with the concentration ranged from 0 to 10 ? M is non-cytotoxic on the RAW264.7 cell line at 1,3,5 days.2 ? RANKL at a concentration of 50 ng / ml induced the differentiation of RAW264.7 cells into mature osteoclasts after 5 days of treatment with different concentrations of RANKL in inducingRAW264.7 cells to differentiate into osteoclasts.3?In the process of 50 ng / ml RANKL induced RAW264.7 cell line differentiation into mature osteoclasts,The Trap staining results showed that the number of matured osteoclasts of experimental group ISO(1-10?M)on the 7th day was significantly smaller than the ISO(0?M)group.4 ? During the differentiation of RAW264.7 cells into osteoclasts induced by 50 ng / ml RANKL with the stimulation of different concentrations of ISO(1-10?M),the experimental group ISO(1-10?M)mature osteoclasts was counted,and its number was significantly smaller than the ISO(0 ?M)group on the 7th day.5?Real-time PCR test showed that the expression of Ctsk,MMP-9,NFATC1,c-Fos and the downstream of osteoclast differentiation NF-KBp65 signaling pathway phosphorylation level of the experimental group ISO(1-10 ? M)was lower than ISO(0 ? M)group m RNA expression.Conclusion:ISO extracted from Ginkgo biloba extract showed an inhibitory effect on osteoclast diferentiation,and its mechanism may be related to inhibition of classical NF-k B pathway...