木豆叶提取物对人的类成骨细胞TE85成骨功能和体外破骨细胞分化的影响

Effects of the extracts of Cajanus cajan L. on cell functions in human osteoblast-like TE85 cells and the derivation of osteoclast-like cells

木豆素是从天然植物木豆叶中提取的单体化合物,结构类似乙烯雌酚。本文观察木豆素及4种木豆叶提取物总成分(32-1,35-1,35-2和35-3)对人的类成骨细胞HOS TE85成骨功能、间质矿化及体外破骨细胞分化的影响。以木豆叶提取物作用于细胞48 h,观察细胞增殖、3H-脯氨酸掺入量及碱性磷酸酶活性;用抗酒石酸酸性磷酸酶(TRAP)染色法观察体外破骨细胞形成;作用于细胞18 d,用茜素红染色法观察细胞间质矿化。结果表明,作用于细胞48 h后,木豆素可促进细胞增殖,在1×10-8g.mL-1时细胞数增加57.7%,3H-脯氨酸掺入量增加98.5%。35-1和35-3还可明显增加碱性磷酸酶活性。长时程作用后,32-1和35-3可明显增加成骨细胞间质矿化程度。木豆素(1×10-7g.mL-1)对破骨细胞形成有明显的抑制作用,抑制率为22.8%,32-1(1×10-7g.mL-1)的抑制率为37.9%。木豆素及木豆叶提取物都具有刺激成骨细胞骨形成、促进细胞间质矿化及抑制破骨细胞形成的活性,提示木豆素在骨细胞上有类雌激素样作用,具有抗骨质疏松新药的研究前景。

The cajanine （longistylin A-2-carboxylic acid） is isolated and identified from extracts of Cajanus cajan L. （ECC） , which structure is similar to diethylstilbestrol. The regulation properties of the cajanine and other four extracts of Cajanus cajan L. （32-1 , 35-1 , 35-2, and 35-3） were tested in human osteoblast-like （HOS） TE85 cells and marrow-derived osteoclast-like cells. By using MTT assay to test the change of cell proliferation, ^3H-proline incorporation to investigate the formation of collagen, and by measuring alkaline phosphatase （ALP） activity, bone formation in HOS TE85 cell was evaluated after pretreated for 48 hours. Bone marrow cells were cultured to examine the derivation of osteoclast cells （OLCs）, which were stained with tartrate-resistant acid phosphatase （TRAP）. The long term effect （pretreated for 18 days） on promoting mineralized bone-like tissue formation was tested by Alizarin red S staining in HOS TE85 cells. After the treatment with cajanine （1 × 10^-8 g ·mL^-1） for 48 hours, cell number increased significantly （57.7%）. a H-Proline incorporation also statistically increased （98.5%） in those cells. Significant change of ALP activity was also found （P 〈0.01 ） in 35-1 and 35-3 treated cells （they were 66.2% and 82.4% in the concentration of 1 × 10^-8 g ·mL^-1, respectively）. The long term （ 18 days） effects of 32-1 and 35-3 on promoting mineralized bone-like tissue formation in HOS TE85 cell were obvious. There were much more red blots over the field of vision compared with that of control group. After the treatment of cajanine, derived-osteoclast cells appeared later and much less compared with control. The inhibition of cajanine was 22.8% while it was 37.9% in 32-1 treated cells in the dose of 1 × 10^-7 g ·mL^-1 It is obvious that cajanine and ECCs promoted the osteoblast cells proliferation and mineralized bone-like tissue formation in HOS TE85 cells, while inhibited derivation of osteoclast cells. All of these suggested that cajanine has the estrogen-like action on osteoblast and osteoclast, which could be developed as anti-osteoporosis drugs.