不同浓度西红花苷对RANKL诱导的小鼠破骨细胞分化的影响

摘要

Objective To investigate the effects of different concentrations of crocin on receptor activator of nuclear factor kappa B ligand (RANKL)-induced osteoclastogenesis using the monocyte-macrophage cell line RAW264.7. Methods The monocyte-macrophage cell line RAW264.7 was cultured routinely.After treatment with 0,6.25,12.5,25, 50,100,200 and 400 μmol/L crocin,cell counting kit-8(CCK-8)assay was used to analyze the viability of RAW264.7 cells to screen out the appropriate experimental concentration. RAW264.7 cells were induced by RANKL (100 ng/L) to form osteoclasts. After treated with 0, 12.5, 50 and 100 μmol/L crocin respectively, the number of osteolasts was counted by tatrate resistant acid phosphatasec (TRAP) staining. Real-time PCR was used to detect the mRNA expression levels of calcitonin receptor(CTR),nuclear factor of active T cells 1(NFATC1),C-fos and TRAP.Results No significant effects of crocin (within 0-100 μmol/L) were found on the viability of RAW264.7 cells (P>0.05). However, When crocin concentration was over 100 μmol/L,the cell proliferation was decreased,and which showed a significant inhibitory effect on proliferation (P<0.05). Thus, 0-100 μmol/L crocin was selected as the experiment concentration. The amount of differentiated osteolasts and the expression levels of CTR,NFATC1,C-fos and TRAP mRNA were decreased significantly with the increased concentrations of crocin(P<0.05).Conclusion At a certain concentration(0-100 μmol/L),the higher levels of crocin could inhibit RANKL-induced osteoclastogenesis.%目的 研究不同浓度的西红花苷对核因子κB受体活化因子配体(RANKL)诱导的破骨细胞分化的影响.方法 常规培养小鼠巨噬细胞RAW264.7,经0、6.25、12.5、25、50、100、200、400 μmol/L浓度的西红花苷处理后,采用CCK-8法检测细胞生长活力的变化,筛选出合适的实验浓度.RAW264.7细胞经RANKL(100 ng/L)诱导形成破骨细胞系后,分别经0、12.5、50、100 μmol/L的西红花苷处理,抗酒石酸酸性磷酸酶(TRAP)染色观察各浓度组破骨细胞的生成数量;实时定量PCR(real-time PCR)检测破骨细胞的特异性基因降钙素受体(CTR)、活化T细胞核因子1 (NFATC1)、C-fos、TRAP mRNA的表达水平变化.结果 在0~100 μmol/L范围内,西红花苷对RAW264.7细胞的生长无明显影响(P>0.05).当西红花苷浓度≥100 μmol/L时,细胞增殖活力下降,显示出明显的抑制增殖作用(P<0.05),选取0~100 μmol/L西红花苷进行实验.随着西红花苷的处理浓度的升高,RAW264.7分化成破骨细胞的数量明显减少,同时破骨细胞的特异性基因CTR、NFATC1、C-fos和TRAP mRNA的表达水平也明显下降(P<0.05).结论 在一定浓度范围内(0~100 μmol/L),高水平的西红花苷可明显抑制破骨细胞的分化.