吴茱萸次碱抗骨质疏松作用研究

Rutaecarpine exerted anti-osteroporosis

骨保护素(osteoprotegerin,OPG)是骨转换的标志物,由成骨细胞等分泌。OPG与核因子-κB受体活化因子配体(receptor activator of nuclear factor-κB ligand,RANKL)结合发挥抑制破骨细胞作用。本研究发现吴茱萸次碱(rutaecarpine,RUT)具有上调OPG表达的活性,并能显著增加小鼠胚胎成骨前体细胞MC3T3-E1及人骨肉瘤细胞U-2OS中OPG的蛋白水平。钙化结节染色实验结果表明,RUT显著促进MC3T3-E1细胞向成骨细胞分化;抗酒石酸磷酸酶(tartrate resistant acid phosphatase,TRAP)染色结果表明,RUT显著抑制RANKL诱导的小鼠巨噬细胞RAW264.7向破骨分化。体内研究发现,与卵巢去势(ovariectomized,OVX)大鼠模型组相比,RUT低剂量组(5 mg·kg^(-1)·day^(-1))和高剂量组(45 mg·kg^(-1)·day^(-1))灌胃给药3个月,显著增加OVX大鼠骨密度;钙黄绿素双标实验及甲苯胺蓝染色实验结果表明,RUT低剂量组在体内促进骨形成并减少骨量丢失;免疫组化结果显示RUT低剂量组能够增加大鼠股骨中OPG的表达。动物福利和实验过程均遵循中国医学科学院医药生物技术研究所动物伦理委员会的规定。综上,本研究表明RUT能上调OPG表达并在体内外促进成骨分化和抑制破骨分化。

Osteoprotegerin(OPG),secreted by osteoblasts,is a marker of bone turnover.OPG can inhibit osteoclastic differentiation by binding receptor activator of nuclear factor-κB ligand(RANKL).In this study,we found that rutaecarpine(RUT)had the up-regulating OPG activity,and it could significantly increase OPG protein levels in both mouse embryonic osteogenic precursor MC3 T3-E1 and human osteosarcoma U-2 OS cells.Osteoblastogenic differentiation calcified nodules staining results showed that RUT significantly promoted the osteogenic differentiation of MC3 T3-E1 cells.Osteoclastic differentiation tartrate resistant acid phosphatase(TRAP)staining results showed that RUT obviously inhibited the osteoclast differentiation of mouse macrophages RAW264.7 induced by RANKL.In vivo studies showed that low-dose RUT group(5 mg·kg^(-1)·day^(-1))and high-dose RUT group(45 mg·kg^(-1)day^(-1))treatments for 3 months significantly increased bone density in ovariectomized(OVX)rats;calcein double labeling experiment and toluidine blue staining results indicated that low-dose RUT group promoted bone formation and decreased bone loss in vivo;immunohistochemistry results showed that low-dose RUT group increased the expression of OPG in rat femur.All animal procedures were performed in accordance with the regulations of the Institutional Animal Care and Use Committee of Institute of Medicinal Biotechnology,Chinese Academy of Medical Sciences.In summary,this study demonstrated that RUT could up-regulate OPG expression and had promoting osteoblastic differentiation and inhibiting osteoclastic differentiation effects in vitro and in vivo.