Effects Of Icariin On MAPK Signal Pathway In Osteoclast Differentiation Of RAW264.7 Cells

ObjectiveThis essay will detect how dose effect and time effect are related when the inhibition effect of ICA on osteoclasts differentiation occurs.Through studying how ICA affects MAPK signaling pathways,the Molecular biology mechanism in this process will be understood,which can potentially provide the fundamental basis for ICA's further clinical application.Materials and MethodsThis study consists of two experiments.Experiment ? focuses on the dose-effect relationship of the inhibition effect of ICA.RAW264.7 cells ware divided into four groups in the light of drug concentration.Westernblot is adopted simultaneously to test the expression of key protein and the phosphorylation levels.Experiment ? concerned the time-effect relationship of the inhibition effect of ICA.Both the control group and ICA group are inducted,and are observed by TRAP staining in every twelve hours for six times to know the shape and count of osteoclasts.Also,westernblot works here to test the expression of key protein and the phosphorylation levels.ResultsMultinucleated cells(number of cell nucleus?3)are seen after forty-eight hours of in vitro induction and culture of RAW264.7.TRAP staining shows that,while the concentration of ICA elevates,the cells and their nucleus dwindles in numbers and cell bodied become smaller.In model control group high level of phosphorylation is observed in P38,JNK and ERK,while no statistic difference are shown on expressive level among the three.In comparison with control groups without ICA model,the phosphorylation level of P38,JNK and ERK of the ICA testaments negatively related to the concentration,while no tatistic difference are shown on expression level.In the control group,TRAP positive osteoclasts could be seen after 12 hours of induction,and the number of osteoclasts increased with the increase of induction time.However,compared with the control group with the same induction time,the number of osteoclasts in ICA group was very small,and the cell morphology was small.There was no significant difference in the levels of P38,JNK and ERK between the control group and ICA treatment group,and the expression levels of P38,JNK and ERK were not statistically different.The phosphorylation level of P38,JNK and ERK in the control group increased gradually with the induction time,but there was no significant difference in the expression of P38,JNK and ERK.There was no significant difference in the levels of P38,JNK and ERK phosphorylation and protein expression between ICA groups in the induction time.ConclusionsIcariin can inhibit the osteoclast differentiation of mononuclear cells,and the inhibitory effect was enhanced with the increase of icariin concentration.Icariin had no effect on the expression of p38,ERK and JNK in the MAPK signaling pathway in the differentiation of osteoclasts.However,icariin could significantly inhibit the phosphorylation level,and the inhibition was positively correlated with the concentration.The optimal concentration of icariin in inhibiting osteoclast differentiation in vitro was 10-5mol/L.The inhibitory effect of Icariin on osteoclast differentiation has reached a stable level after 12 hours of induction.