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目的:探讨骨碎补单体成分柚皮苷对小鼠单核细胞RAW264.7诱导分化为破骨细胞的影响。方法:通过100μg·L-1核因子κB受体活化因子配基(RANKL)诱导RAW264.7细胞株分化为成熟破骨细胞,经TRAP特异性染色和骨吸收陷窝对破骨细胞进行鉴定。采用MTT法筛选抑制破骨细胞最强的浓度。在诱导过程中,采用筛选后含柚皮苷的培养基,诱导5 d后,通过TRAP阳性细胞计数和骨吸收面积分析来观察柚皮苷对破骨细胞的形成和骨吸收功能情况;流式细胞术检测柚皮苷对破骨细胞增殖的影响,RT-PCR检测柚皮苷对破骨细胞分化过程中核因子κB受体活化因子(RANK)、抗酒石酸酸性磷酸酶(TRAP)、基质金属蛋白酶9(MMP-9)、活化T细胞核因子1(NFATc1)与C-fos mRNA表达的影响。结果:采用100μg·L-1的RANKL可成功诱导成熟的、有功能的破骨细胞。柚皮苷可以抑制破骨细胞的分化和骨吸收功能;抑制破骨细胞的增殖活性;柚皮苷可明显下调破骨细胞分化过程中的RANK,TRAP,MMP-9,NFATc1 mRNA的表达,上调C-fos mRNA表达。结论 :柚皮苷可抑制破骨细胞分化、增殖和骨吸收功能,其机制可能是通过抑制破骨细胞分化过程中特异性基因表达实现的。
OBJECTIVE: To investigate the effect of naringin, a component of Osteopractica, on the differentiation of mouse monocyte RAW264.7 into osteoclasts. METHODS: RAW264.7 cells were induced to differentiate into mature osteoclasts by 100μg · L-1 nuclear factor-κB receptor activator ligand (RANKL). The osteoclasts were identified by TRAP-specific staining and bone resorption. Using MTT method to inhibit the strongest concentration of osteoclasts. During the induction process, the culture medium containing naringin was screened for 5 days, and then the osteoclast formation and bone resorption function of naringin were observed by TRAP positive cell count and bone resorption area analysis. Flow cytometry Cytometry was used to detect the effect of naringin on the proliferation of osteoclasts. The effects of naringin on the expression of RANK, TRAP, MMP-9 and TIMP-1 in osteoclast differentiation were detected by RT- 9 (MMP-9), nuclear factor-kappa B (NFATc1) and C-fos mRNA expression. Results: Mature and functional osteoclasts were successfully induced with 100μg · L -1 RANKL. Naringin inhibited osteoclast differentiation and bone resorption, inhibited the proliferation activity of osteoclasts. Naringin significantly down-regulated the expression of RANK, TRAP, MMP-9 and NFATc1 mRNA during osteoclast differentiation C-fos mRNA expression. CONCLUSION: Naringin can inhibit the differentiation, proliferation and bone resorption of osteoclasts, and its mechanism may be through inhibiting the expression of specific genes in osteoclast differentiation process.