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[目的]探讨槲皮苷对核因子κB受体激动剂配体(receptor activator of nuclear factor kappa B ligand,RANKL)诱导的破骨细胞形成及骨吸收功能的影响。[方法]通过CCK-8法观察不同浓度槲皮苷(0~800μmol/L)干预不同时间(48 h、96 h)对RAW 264.7细胞的生存影响,确定合适的体外用药浓度;利用体外RANKL诱导RAW 264.7细胞形成破骨细胞体系,通过抗酒石酸酸性磷酸酶(tartrate resistant acid phosphatase,TRAP)染色计数评价槲皮苷(200、400μmol/L)对破骨细胞形成和生存的影响;通过骨片吸收实验对骨凹陷和骨吸收面积统计分析评价槲皮苷(200、400μmol/L)3 d内对成熟破骨细胞骨吸收功能的影响;釆用实时定量(Real-Time)PCR技术,检测槲皮苷(200、400μmol/L)对RANKL诱导的破骨细胞特异性基因NFATc1、TRAP和c-fos表达水平的影响。[结果]细胞生存实验发现槲皮苷干预96 h后,槲皮苷(0~800μmol/L)对RAW 264.7细胞4 d内生存未发现显著影响;通过TRAP染色发现200、400μmol/L槲皮苷能显著抑制体外RANKL诱导的破骨细胞形成;通过骨片吸收实验发现200、400μmol/L槲皮苷3d内能显著降低骨吸收面积,提示其抑制成熟破骨细胞骨吸收功能;同时,槲皮苷能呈剂量依赖性抑制RANKL诱导活化T细胞核因子(nuclear factor of activated T cells,NFAT)c1、TRAP和c-fos基因表达。[结论]槲皮苷通过抑制NFATc1,TRAP和c-fos的表达,来抑制体外RANKL诱导的破骨细胞形成和骨吸收功能,是一种潜在治疗骨质疏松药物。
[Objective] To investigate the effect of quercetin on osteoclast formation and bone resorption induced by receptor activator of nuclear factor kappa B ligand (RANKL). [Method] The effects of different concentrations of quercitrin (0 ~ 800μmol / L) on the survival of RAW 264.7 cells at different time (48 h, 96 h) were observed by CCK-8 method to determine the appropriate drug concentration in vitro. The formation of osteoclast system by RAW 264.7 cells and the effect of quercetin (200,400 μmol / L) on the formation and survival of osteoclasts were evaluated by staining with tartrate resistant acid phosphatase (TRAP) The effect of quercetin (200,400μmol / L) on the bone resorption of mature osteoclasts within 3 days was evaluated by statistical analysis of bone dents and bone resorption area. Real-time PCR (200,400μmol / L) on RANKL-induced osteoclast-specific gene expression of NFATc1, TRAP and c-fos. [Results] The cell survival experiments showed that quercitrin (0 ~ 800μmol / L) had no significant effect on the survival of RAW 264.7 cells within 4 days after quercetin intervention. The results of TRAP staining showed that the concentration of 200,400μmol / L quercetin Can significantly inhibit the RANKL-induced osteoclastogenesis in vitro; bone resorption experiments found that 200,400μmol / L quercetin 3d can significantly reduce bone resorption area, suggesting that it inhibits bone maturation of mature osteoclasts; the same time, quercetin Glycosides inhibited the expression of c-reactive protein (NFAT) c1, TRAP and c-fos in a dose-dependent manner by RANKL. [Conclusion] Quercetin inhibits osteoclast formation and bone resorption induced by RANKL in vitro by inhibiting the expression of NFATc1, TRAP and c-fos, which is a potential therapeutic agent for osteoporosis.