| ObjectiveBased on the previous work,this study was designed to investigate the effect of DTYMT on the inhibition of osteoclast differentiation by observing the effect of DTYMT on osteoclast differentiation,proliferation and function induced by RAW264.7 macrophage.The molecular mechanism of function provides an experimental basis for the clinical application of DTYMT in the treatment of rheumatoid arthritis,and helps to further promote the application of DTYMT to benefit more patients.MethodExperiment 1: The osteoclast-like cells induced by RAW264.7 macrophages were established and the blank group,model group,DTYMT400 mg · L-1,600 mg · L-1,800 mg · L-1,l000 mg · L-1 concentration group And methotrexate 2mg · L-1 group,the preparation of Radix Et Rhizoma Decoction freeze-dried powder,and the corresponding intervention in each group.Experiment 2: Identification of osteoclasts by TRAP staining and explore the effect of Radix et Rhizoma Decoction on osteoclast differentiation.Experiment 3: The cell survival rate of each group was detected by CCK-8 method,and the effect of Radix et Rhizoma Decoction on the proliferation of osteoclasts was explored.Experiment 4: The expression of key proteins of Wnt and RANKL signal pathway was detected by Western blotting,and the mechanism of action of Radix et Rhizoma Decoction on osteoclastogenesis was explored.Experiment 5: The level of MMP-9inflammatory cytokines was detected by ELISA,and the effect of Jiutong Yimu Decoction on inflammatory cytokines was exploredResultExperiment one and two:TRAP staining showed that compared with the model group,the number of fused cells in DTYMT400 mg · L-1,600 mg · L-1 group was decreased,but the difference was not statistically significant(P>0.05);DTYMT800mg · L-1,The number of fusion cells in the group of 1000 mg · L-1 significantly decreased(P <0.01),which suggested that the differentiation of OC could be effectively inhibited when the concentration of Radix et Rhizoma Rhei decoction reached a certain level.Experiment three:The results of CCK8 showed that the survival rates of DTYMT600 mg · L-1,800 mg · L-1,1000 mg · L-1 and methotrexate 2mg · L-1 significantly decreased compared with the model group(P<0.05,P <0.01),suggesting that a certain concentration of Fujito Yimu Decoction can significantly inhibit the proliferation of OC.Experiment four:Western blot results showed that compared with the model group,the expression of RANKL protein in the group of DTYMT 1000 mg · L-1 and methotrexate 2mg · L-1 decreased(P<0.05).The expression of OPG protein in DTYMT groups did not increase or decrease significantly(P>0.05).The expression of Wnt3 a and ?-catenin in DTYMT800ug/ml,1000ug/ml group and methotrexate 2ug/ml DKK1 protein expression decreased significantly,suggesting that a certain concentration of DTYMT can effectively reduce RANKL expression,increase ?-catenin protein expression and down-regulate DKK1 expression.Experiment five:The results of ELISA showed that compared with the model group,the expression levels of MMP-9 in DTYMD600 mg · L-1,800 mg · L-1,l000 mg · L-1 group and methotrexate group decreased,and the concentration of DTYMT The higher the decline,the more obvious,suggesting that DTYMT can significantly reduce the expression of MMP-9.Conclusion1.When DTYMT concentration reaches 800ug/ml and 1000ug/ml,OC differentiation can be effectively inhibited.2.When the concentration of DTYMT reached 600ug/ml,800ug/ml,1000ug/ml,it had an inhibitory effect on the proliferation of osteoclasts,and the higher the drug concentration,the more obvious the inhibitory effect.3.When DTYMT drug concentration reaches 800ug/ml,1000ug/ml,it can increase the expression of ?-catenin protein and down-regulate DKK1 expression.When the concentration of DTYMT drug reaches 1000mg·L-1,it can effectively reduce Result the expression level of RANKL.DTYMT concentrations did not significantly affect the expression of OPG protein.4.The DTYMT concentration of 600 mg · L-1,800 mg · L-1,1000 mg · L-1 can significantly reduce the expression of MMP-9. |
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