| Objective: To observe the effect of compound Duzheng decoction extract on the differentiation and proliferation of mouse mononuclear macrophage(RAW264.7),a precursor cell of osteoclast(Osteoclast,OC),and to explore its possible mechanism,so as to provide experimental basis for clinical treatment of rheumatoid arthritis.Methods: RAW264.7,from mouse mononuclear macrophages in logarithmic growth phase.Cell proliferation and toxicity test kit(CCK-8)was used to detect the effect of compound Duzheng decoction extract(50,100,200?g/ml)on RAW264.7proliferation at 24 h,48 h,72 h,96 h and 120 h.RAW264.7.From mouse mononuclear macrophages in logarithmic phase were induced and cultured with different concentrations(6.25,12.5,25,50,100 ng / ml)of nuclear factor kappa B receptor activating factor ligands(Receptor Activator of Nuclear Factor-?B Ligand,RANKL),After 5 days,the morphological changes were observed and OC was identified by tartrate resistant acid phosphatase(tartrate resistant acid phosphatase,TRAP)staining.OC were divided into control group(RAW264.7 cells),model group(RAW264.7 cells + 50ng/ml RANKL)and drug high,medium and low treatment groups(RAW264.7 cells + 50ng/ml RANKL + drugs),the effect of extract of compound Duzheng decoction(50,100,200?g/ml)on OC differentiation was detected by TRAP staining.Western blot was used to detect the expression of RANK,RANKL,TRAF-6 and NF-kappa B protein in each group after 5 days of intervention,Western blot was used to detect the expression of PI3 K and AKT protein in each group after intervention for 1 day.Results: 1.CCK-8 results showed that the extract of compound Duzheng decoction had no obvious toxicity to RAW264.7 cells at the concentration of 50?g/ml-200?g/ml.2.TRAP staining showed that the number of positive OC(nucleus ? 3)induced by OC inducer RANKL in 50ng/ml was similar to that induced by 100ng/ml and there was no statistical significance between the two groups(P > 0.05),and the number of OC induced by 6.25ng/ml-25ng/ml RANKL was much more than that induced by OC inducer RANKL,so that 50ng/ml RANKL was selected to induce OC in RAW264.7 cells.3 The extract of compound Duzhengdecoction(50,100,200?g/ml)inhibited the differentiation of RAW264.7 cells into OC.The extract of compound Duzheng decoction was used to interfere with osteoclasts,after TRAP staining,it was found that: There was no OC production in the control group,but obvious large multinucleated OC production could be seen in the model group and each drug group.The number of OC in each drug group was significantly lower than that in the model group,and there was a statistical significance among the drug groups(P < 0.05),which was negatively correlated with the drug concentration.The number of OC in each drug group decreased with the increase of drug concentration.4 WB showed that compared with the model group,200?g/ml drug group could inhibit the expression of RANK,TRAF-6,NF-?B,PI3 K and AKT protein(P < 0.05),while 50?g/ml and 100?g/ml drug group had little effect on RANK,NF-? B,PI3 K and AKT protein(P > 0.05),while the extact of compound Duzheng decoction had no significant change on the erxpression of RANKL and and the difference was not statistically significant(P > 0.05).Conclusion: 1.Compound Duzheng decoction has no obvious toxicity to RAW264.7 cells at the concentration of50?g/ml-200?g/m.2.Compound Duzheng decoction can inhibit the production of OC when the drug concentration is 50?g/ml-200?g/m.The higher the drug concentration is,the less the number of OC is,the better the effect is.3.When the concentration of compound Duzheng decoction reached 200?g/ml,it could down-regulate the expression of RANK,NF-?B,TRAF6,PI3 K and AKT protein,while each concentration of compound Duzheng decoction had no significant effect on the expression of RANKL protein,and compound Duzheng decoction might inhibit the differentiation of OC by down-regulating the expression of RANK,NF-?B,TRAF6,PI3 K and AKT protein,so as to prevent and delay the occurrence and development of bone destruction in RA. |
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