艾拉莫德对类风湿关节炎外周血破骨细胞分化及破骨细胞相关基因表达的影响

Effect of Iguratimod on osteoclast differentiation and osteoclast-associated genes in peripheral blood of the patients with rheumatoid arthritis

目的观察艾拉莫德对类风湿关节炎(rheumatoid arthritis,RA)外周血破骨细胞分化形成及破骨细胞相关基因表达的影响。方法使用人核因子kB受体活化因子配体(receptor activator of nuclear factor-κB ligand,RANKL)及人巨噬细胞集落刺激因子(macrophage colony-stimulating factor,M-CSF)诱导RA患者外周血淋巴细胞(Peripheral blood mononuclear cell,PBMCs)分化为成熟破骨细胞,抗酒石酸酸性磷酸酶(tartrate-resistant acid phosphatase,TRAP)染色进行鉴定。CCK-8法筛选艾拉莫德抑制破骨细胞形成的浓度。观察艾拉莫德25μg/mL、12.5μg/mL、6.25μg/mL对RA患者PBMCs生成破骨细胞的影响,RT-PCR法检测不同浓度艾拉莫德对破骨细胞分化过程中TRAP、组织蛋白酶K(Cathepsin K,CTSK)、细胞核因子κB受体活化因子(receptor activator of nuclear factor-κB,RANK)、激活蛋白-1(Activator protein-1,AP-1)mRNA表达的影响。结果 100 ng/mL RANKL和50 ng/mL M-CSF在第14天将RA患者PBMCs诱导为破骨细胞。CCK-8检测结果显示艾拉莫德25μg/mL、12.5μg/mL、6.25μg/mL三个浓度对PBMCs细胞活力无明显影响,且均能抑制破骨细胞的生成,以25μg/mL组最为显著。艾拉莫德能抑制TRAP、CTSK、RANK、AP-1 mRNA的表达水平,且随艾拉莫德浓度的升高抑制作用越明显。结论艾拉莫德通过抑制破骨细胞特异性基因表达来抑制破骨细胞分化、增殖。

Objective To observe the effect of Iguratimod on osteoclast differentiation and the expression of osteoclast-associated genes in peripheral blood of rheumatoid arthritis(RA)patients.Methods Peripheral blood lymphocytes(PBMCs)in RA patients were induced to mature osteoclasts by human nuclear factor-κB receptor activator ligand(RANKL)and human macrophage colonystimulating factor(M-CSF),and identified by using tartrate-resistant acid phosphatase(TRAP)staining.The concentration of Iguratimod to inhibit the formation of osteoclast was screened with CCK-8 method.The effect of different concentrations of Iguratimod on the expression of TRAP,cathepsin K(CTSK),receptor activator of nuclear factor-κB(RANK),and activator protein-1(AP-1)mRNA in the process of osteoclast differentiation were detected.Results PBMCs of RA patients were induced to osteoclast in 14 days with 100 ng/mL of RANKL and 50 ng/mL of M-CSF.CCK-8 detection showed that the 3 concentrations of Iguratimod(25,12.5,and 6.25μg/mL)had no significant effect on the activity of PBMCs cells,and all of them could inhibit the generation of osteoclasts.The 25 μg/mL group was the most significant.Iguratimod could inhibit the expression of TRAP,CTSK,RANK,and AP-1 mRNA,and the inhibitory effect was increased with the increase of the concentration of Iguratimod.Conclusion Iguratimod inhibits the differentiation and proliferation of osteoclast by inhibiting the expression of osteoclast-associated genes.