咖啡酸苯乙酯对RAW264.7细胞向破骨细胞分化的影响

Effect of caffeic acid phenethyl esteron osteoclast differentiation in RAW264.7 macrophages

背景:近年来,骨质疏松症、磨损颗粒介导的骨溶解等骨吸收类疾病的发生率越来越高。目前临床上治疗骨溶解性疾病的药物以西药为主,而中草药成分的研制还处于起步阶段。目的:观察天然蜂胶提取物咖啡酸苯乙酯(CAPE)对核因子(NF)-κB受体活化因子配体(RANKL)诱导的小鼠单核/巨噬细胞株RAW264.7向破骨细胞分化的影响。方法:利用不同浓度的CAPE与RANKL单独或共同处理RAW264.7细胞。CCK-8法测定细胞增殖活性,抗酒石酸酸性磷酸酶(TRAP)染色法观察TRAP阳性多核细胞。逆转录-聚合酶链(RT-PCR)测定破骨细胞TRAP、MMP-9基因m RNA的含量。Western blot检测NF-κB p65及核转录因子κB抑制蛋白(IκB)-α表达水平。结果:CCK-8结果表明75、150、300 nmol/LCAPE对RAW.264.7细胞增殖能力无影响。CAPE可抑制RANKL诱导的TRAP阳性多核细胞形成,下调RANKL诱导的TRAP和MMP-9基因m RNA表达。Western blot检测显示,CAPE能下调RANKL诱导的NF-κB表达水平。结论:CAPE能有效地抑制RANKL诱导的RAW264.7向破骨细胞分化。

Background：In recent years, the incidence of bone resorptive diseases such as osteoporosis, wear particle medi-ated osteolysis is getting higher and higher. Drugs currently used in clinical treatment of bone resorptive diseases are west-ern medicine, and the development of Chinese herbal medicine components is still in the emerging stage. Objective：To ob-serve the effect of caffeic acid phenethyl ester （CAPE, a propolis extract） on receptor activator of NF-κB ligand （RANKL） induced osteoclast differentiation using the monocyte-macrophage cell line RAW264.7. Methods：Mouse mononuclear cell line RAW264.7 were cultured. CAPE and/or RANKL were added. Cell Counting Kit-8 （CCK-8） assay was used to analyze the viability of RAW264.7 cells which were exposed to different concentrations of CAPE （75, 150 and 300 nmol/L）. Tar-trate-resistant acid phosphatase （TRAP） staining was used to observe the formation of osteoclasts. RT-PCR was used to mea-sure the mRNA expression of TRAP and matrix metalloproteinase 9 （MMP-9）. Western blot was used to determine the nu-clear factorκB （NF-κB） pathway and IκB-α. Results：Different concentrations of CAPE did not affect RAW264.7 cell viabil-ity. Mature osteoclasts were obtained from RAW264.7 cells stimulated with RANKL for 6 days and detected by TRAP stain-ing. CAPE could inhibit the formation of TRAP-positive cells and result in an obvious reduction of TRAP and MMP-9 mRNA expression. Western blot showed that CAPE suppressed RANKL-induced IκB-α degradation and NF-κB nuclear translocation in RAW264.7 cells. Conclusions：CAPE can inhibit the ososteoclast differentiation of mature osteoclasts from RAW264.7 induced with RANKL.