地拉罗司通过NF-κB信号抑制小鼠单核细胞RAW264.7向破骨细胞分化

Deferasirox inhibit differentiation of mouse RAW264.7 monocytes into osteoclasts through NF-κB signal

目的探讨地拉罗司(deferasirox,DFS)体外对小鼠单核细胞RAW264.7向破骨细胞分化的影响及相关机制。方法体外培养小鼠单核细胞RAW264.7,在核因子κB受体活化因子配体(receptor activator of NF-κB ligand,RANKL)的作用下诱导分化为破骨细胞,并使用不同浓度DFS(0、5、10、20μmol/L)进行干预,用CCK-8法检测细胞的增生活性,抗酒石酸酸性磷酸酶(tartrate-resistant acid phosphatase,TRAP)染色法观察TRAP阳性破骨细胞的形态并计数,实时定量PCR法检测细胞转录因子c-Fos、转录因子活化T细胞核因子(nuclear factor of activated T cell c1,NFATc-1)和组织蛋白酶K(cathepsin K,CTK)mRNA的表达,双荧光素酶报告基因系统检测核转录因子κB(nuclear transcription factor kappa B,NF-κB)报告基因的表达,蛋白免疫印迹(Western blotting)法分别检测细胞质蛋白、核蛋白NF-κB P65的表达。结果 DFS 0、5、10、20μmol/L组TRAP染色阳性的数目分别为63.67±3.78、55.33±3.21、34.00±5.00、16.00±4.58,各浓度组组间比较,差异有统计学意义(P<0.05),提示DFS在实验浓度围内可减少RANKL诱导的RAW264.7细胞TRAP染色阳性的数目;DFS 0、5、10、20μmol/L组c-Fos mRNA表达量分别为1.83±0.11、1.46±0.13、0.88±0.13、0.30±0.09,各浓度组组间比较,差异有统计学意义(P<0.05),NFATc-1 mRNA表达量分别为4.09±0.20、3.21±0.22、2.28±0.23、1.47±0.22,各浓度组组间比较,差异有统计学意义(P<0.05),CTK mRNA表达量分别为3.51±0.08、3.21±0.19、2.55±0.22、1.50±0.19,各浓度组组间比较,差异有统计学意义(P<0.05),以上结果提示DFS在实验浓度围内可下调c-Fos、NFATc-1、CTK mRNA的表达;在RANKL的基础上加入DFS后NF-κB报告基因的表达量为0.119±0.019,与RANKL组0.202±0.017比较明显下降,差异有统计学意义(P<0.05),提示DFS可抑制RAW264.7细胞NF-κB报告基因的表达;在RANKL的基础上加入DFS后细胞质内NF-κB P65蛋白表达量为0.56±0.08,与RANKL组0.42±0.09比较明显增加,差异有统计学意义(P<0.05),细胞核内NF-κB P65蛋白表达量为1.49±0.12,与RANKL组1.71±0.08比较明显减少,差异有统计学意义(P<0.05),提示DFS可抑制RAW264.7细胞NF-κB P65向细胞核转位。结论DFS可能通过抑制NF-κB信号活性来抑制小鼠单核RAW264.7细胞向破骨细胞分化。

Objective To investigate the effects of deferasirox （DFS） on differentiation of mouse RAW264.7 monocytes into osteoclasts and its related mechanism. Methods RAW264.7 cells were treated with DFS in the presence of receptor activator of NF-κB ligand （RANKL）. Cell viability was assessed by CCK-8. The number of tartrate-resistant acid phosphatase （TRAP） -positive cells were counted under light microscopy. The levels of transcription factor c-Fos, nuclear factor of activated T cell el （NFATc-1 ） and cathepsin K （CTK） mRNA were analyzed by real-time PCR. The levels of reporter gene of nuclear transcription factor kappa B （NF-κB） was examined by luciferase reporter assay. NF-κB P65 were detected by Western blotting. Results The number of TRAP-positive MNCs in RAW264. 7 cells of 0, 5, 10, 20 μmol/L groups were 63.67 ±3.78, 55.33 ±3.21, 34. 00 ±5.00, 16. 00 ±4. 58 respectively. The significant differ- ence （ P 〈 O. 05） indicated that DFS could significantly decrease the number of TRAP-positive MNCs. The mRNA expres- sion of c-Fos in the 4 groups were 1.83 ±0. 11, 1.46 ±0.13, 0. 88 ±0. 13, 0.30 ±0.09, and the mRNA expression of NFATc-1 were 4. 09 ±0.20, 3.21 ±0.22, 2.28 ±0.23, 1.47 ±0.22. The mRNA expression of CTK were 3.51 ±0. 08, 3.21 ±0. 19, 2. 55 ±0. 22, 1.50 ±0.19. The significant difference among the 4 groups in c-Fos, NFATc-1, and CTK genes （P〈0.05） indicated that DFS could down-regulate their mRNA expression. The level of receptor gene of NF-κB P65 in RANKL ± DFS group （0. 119 ±0.019 ） was obviously less than that in RANKL group （0.202 ±0. 017, P〈0.05）. The results indicated that DFS could suppress expression of NF-κB reporter gene. The protein expression of NF- KB P65 in cytoplasm of RAW264. 7 cells in RANKL ± DFS group （0.56 ± 0.08 ） was obviously more than that in RANKL group （0. 42±0.09） with significant difference （P〈0.05）, and the protein expression of NF-κB P65 in nucleus of RAW264. 7 cells in RANKL ± DFS group （1.49 ±0. 12） was obviously less than that in RANKL group （1.71 ±0. 08, P 〈 0. 05）. The results indicated that DFS could hinder translocation of NF-κB P65 to cell nucleus in RAW264. 7 ceils. Conclusion DFS can significantly inhibit differentiation of RAW264.7 cells into osteoclasts, and the mechanism behind inhibition may involve suppressed NF-κB activity.