墨旱莲提取物调控RANKL/RANK/NF-κB途径抑制破骨细胞骨吸收活性

Inhibition of osteoclast's bone resorption activity by extract of Ecliptaeherba through controlling RANKL/RANK/NF-κB expression

目的:探讨墨旱莲提取物(EAE)对前破骨细胞RAW264.7骨吸收活性的影响及机制。方法:苏木精染色法观察骨片骨吸收陷窝数量及侵蚀面积;Phalloidin-Atto 565荧光染色法检测肌动蛋白环的形成;q RT-PCR法检测cathepsin K、c-Src表达;Western blot检测NF-κB p65及磷酸化p65的表达。结果:2.5、5、10μg/m L EAE分别抑制了RANKL诱导的RAW264.7骨吸收陷窝的数量从28±2减少到13±2、8±2、3±2;侵蚀面积从(0.024±0.004)cm2减少到(0.014±0.001)、(0.003±0.000)、(0.001±0.001)cm2。5μg/m L EAE作用6d后,明显抑制了RANKL诱导的RAW264.7细胞肌动蛋白环的形成。q RT-PCR检测显示骨吸收活性相关基因cathepsin K、c-Src表达下降(P<0.01);经EAE刺激后,RANKL诱导的p-NFκB p65表达下调(P<0.05)。结论:EAE抑制了破骨细胞骨吸收活性和功能,其机制是可能与调控RANKL/RANK/NF-κB途径有关。

Objective:To investigate the effects of ethyl acetate extract of Ecliptaeherba(EAE) on osteoclast RAW264.7's bone resorption and the biological mechanism.Methods:The number and area of resorption lacunae on RAW264.7 bone slice were counted after being stained with Hematoxylin Solution.Actin ring was assayed by method of Phalloidin-Atto 565 staining.Expressions of cathepsin K and c-Src mRNA were detected by quantitative reverse transcription polymerase chain reaction(q RT-PCR), and expressions of NF-κB p65 and phosphorylation p65 were determined by Western blot.Results:EAE of 2.5, 5, 10 μg/m L respectively inhabited the number of resorption lacunae on the bone surfaceof RANKL induced RAW264.7 decreased from 28 to 13±2, 8±2, 3±2, and the area reduced from(0.024±0.004) to(0.014±0.001),(0.003±0.000),(0.001±0.001)cm2.After 6 hours' reaction, EAE at 5μg/m L remarkably inhibited the formation of actin-ring in RAW264.7 cells.The result of the q RT-PCR indicated that expressions of bone resorption activity related genes of cathepsin K and c-Src were reduced.After EAE stimulation, the expression of RANKL induced p-NFκB p65 was also down-regulated(P<0.05).Conclusion:EAE inhibited osteoclast's activity of bone resorption and function, whose mechanism could be related by controlling RANKL/RANK/NF-κB expressions.