姜黄素对类风湿关节炎破骨细胞分化中NF-κB p65和NFATc1表达的影响

The effect of curcumin on the expression of NF-κB p65 and NFATc1 in osteoclast differentiation of rheumatoid arthritis patients

目的研究姜黄素(curcumin,Cur)对类风湿关节炎(rheumatoid arthritis,RA)破骨细胞(osteoclast,OC)分化中核因子-κB(nuclear factor kappa B,NF-κB)p65、活化T细胞核因子c1(nuclear factor of activated T cells cytoplasmic 1,NFATc1)表达的影响。方法密度梯度离心法分离RA患者外周血单个核细胞(peripheral blood mononuclear cell,PBMC),经核因子κB受体活化因子配体(receptor activator of nuclear factor-κB ligand,RANKL)和巨噬细胞集落刺激因子(macrophage colony-stimulating factor,M-CSF)诱导培养为OC,通过抗酒石酸酸性磷酸酶(tartrate-resistant acid phosphatase,TRAP)染色及骨吸收功能检测鉴定OC,分别用不同浓度(0、2.5、5及10μmol/L)的Cur进行干预。采用CCK-8法进行Cur细胞毒性检测;计数TRAP染色阳性细胞数;Western-blotting检测NF-κB p65、NFATc1蛋白的表达;免疫荧光染色检测NF-κB p65核易位。结果 CCK-8结果示,Cur2.5、5和10μmol/L的浓度对PBMC未产生细胞毒性;TRAP染色结果示,Cur抑制RANKL诱导的RA-OC分化;Westernblotting结果示,经不同浓度Cur干预后,NF-κB p65在细胞浆中的表达明显升高,在细胞核中的表达明显降低,NFATc1的蛋白表达明显降低;免疫荧光染色结果示Cur抑制NF-κB p65核易位。结论 Cur能明显抑制NF-κB的入核表达,降低NFATc1的表达,从而抑制RA-OC的分化。

Objective To observe the effect of curcumin（ Cur） on the expression of NF-κB p65 and NFATc1 in osteoclast（ OC）differentiation of rheumatoid arthritis patients. Methods Peripheral blood mononuclear cells（ PBMCs）,separated from RA patients by density gradient centrifugation in vitro,were cultured into OCs with the presence of receptor activator of nuclear factor-κB ligand（ RANKL） and macrophage colony-stimulating factor（ M-CSF）. OCs were identified by tartrate-resistant acid phosphatase（ TRAP） staining and detecting the function of bone resorption. Each group was given different concentrations of Cur（ 0,2. 5,5 and 10 μmol/L） during the culture. The cytotoxicity of Cur was assessed using CCK-8. TRAP staining was used to mark mature OCs. Western blotting was used to detect the expression of NF-κB p65 and NFATc1 in different groups.Immunofluorescence staining was used to detect the nuclear translocation of NF-κB p65. Results The result of CCK-8 indicated that Cur had no toxicity on PBMCs at the concentrations of 2. 5,5 and 10 μmol/L. The result of TRAP staining indicated that Cur inhibited osteoclast differentiation induced by RANKL. The result of Western-blotting indicated that the expression of NF-κB p65 in cytoplasm was significantly increased while the expression of NF-κB p65 in nucleoprotein was significantly decreased after the intervention of different concentrations of Cur. The expression of NFATc1 was significantly decreased. The result of immunofluorescence staining indicated that Cur could inhibit the nuclear translocation of NF-κB p65. Conclusion Curcumin can inhibit osteoclast differentiation in RA patients by suppressing nuclear translocation of NF-κB p65 and decreasing the expression of NFATc1.