左归丸抗乳腺癌破骨性骨转移机制探讨

Effects of Zuogui Wan on Osteolytic Metastasis Induced by Breast Cancer Cells

目的:研究左归丸的抗乳腺癌破骨性骨转移作用,并探讨其机制。方法:采用左心室注射人乳腺癌MDA-MB^(-2)31细胞法建立裸鼠乳腺癌实验性骨转移模型,实验分为空白组、左归丸(21,42 g·kg^(^(-1)))组,采用巢式PCR特异性扩增裸鼠骨髓中人细胞角蛋白(CK19),确定肿瘤细胞在骨髓组织中的转移情况。体外分离小鼠破骨细胞前体细胞小鼠骨髓巨噬细胞(BMMs),外源性加入核转录因子-κB(NF-κB)受体活化因子配体(sRANKL),模拟肿瘤条件下破骨细胞活化,观察左归丸对破骨细胞活化、组织蛋白酶K(Cathepsin K)分泌以及骨片吸收的影响。采用免疫印迹法(Western blot)检测破骨细胞前体细胞NF-κB受体活化因子(RANK)表达的影响。结果:与空白组比较,左归丸(21,42 g·kg^(^(-1)))组均可显著抑制乳腺癌骨转移动物骨髓组织中人CK19表达的影响(P<0.01)。体外血清药理学研究表明,左归丸含药血清可显著抑制BMMs分化为具有多细胞核的破骨细胞(P<0.05),同时可明显抑制破骨细胞分泌Cathepsin K(P<0.05)。10%左归丸含药血清组的骨片吸收窝的数量和面积明显低于正常大鼠血清组(P<0.05)。Western blot实验结果表明,左归丸含药血清可明显抑制RANK蛋白表达(P<0.05)。结论:左归丸具有明显的抗乳腺癌破骨性骨转移作用,RANKL/RANK系统活性可能是其作用机制之一。

Objective： To investigate the anti-osteolytic metastasis effects of Zuogui Wan for breast cancer. Method： Balb / C Nu mice were used to establish in vivo experimental bone metastasis models by left ventricle injection of human breast cancer MDA-MB-231 cells. The mice in the experiment was divided into three groups： blank group,Zuogui Wan groups（ 21,42 g·kg-1）. The cytokeratin 19（ CK19） in bone marrow were detected by nest PCR assay. The bone marrow-derived macrophages（ BMMs） were in vitro isolated as osteoclast precurosors,and soluble receptor activator of nuclear factor-κB ligand（ sRANKL） was exogenously added to simulate the activation of osteoclasts under tumor conditions,and then the effects of Zuogui Wan on activation of osteoclasts,Cathepsin K secretion levels and bone slices absorption were observed. Western blot assay was used to detect its effect on RANK expression levels in BMMs. Result： As compared with the blank group,Zuogui Wan（ 21,42 g·kg-1） exhibited significant inhibition on human CK19 expression levels in bone marrow tissues of animals with breast cancer bone metastasis（ P〈0. 01）. In vitro serum pharmacological studies showed that Zuogui Wan significantly inhibited RANKL-induced osteoclastogenesis in murine BMMs（ P〈0. 05） and significantly inhibited the secretion of Cathepsin K in RANKL-induced osteoclasts（ P〈0. 05）. Bone slices experiment indicated that,the number and area of bone slices resorption pits in 10% Zuogui Wan group were significantly lower than those in normal group（ P〈0. 05）. Western blot assay indicated that medicated serum of Zuogui Wan could significantly inhibit the expression of RANK in RANKL-induced osteoclasts. Conclusion： Zuogui Wan have significant anti-osteolytic metastasis effects for breast cancer cells,and the mechanism may be associated with inhibiting RANKL / RANK activation.