Lü Xiang, Zhou Ying, Chen Ke-Ming, Zhao Zhi, Zhou Jian, Ma Xiao-Ni
College of Life Science, Guizhou University, Guiyang 550025, China.
Yao Xue Xue Bao. 2013 Mar;48(3):347-51.
This study is to investigate the effect of 8-prenylnaringenin (8-PNG) on osteoclastogensis of bone marrow cells and bone resorption activity of osteoclasts. Osteoclasts were separated from long bone marrow of newborn rabbits and cultured in alpha-MEM containing 10% FBS. 8-PNG was added into culture media at 1 x 10(-7), 1 x 10(-6), 1 x 10(-5) mol xL(-1), separately. 17beta-Estradiol (E2, 1 x 10(-7) mol x L(-7)) was used as positive control. T RAP staining and TRAP activity measurement were performed after 5 days, and the bone resorption pits were analyzed after 7 days. Annexin V staining for the detection of apoptotic osteoclasts was performed after 2, 4, 8, 12, 24, 36 and 48 h separately. The mRNA expression level of TRAP and cathepsin K (CTSK) was measured by real-time RT-PCR. 8-PNG significantly reduced the number of osteoclasts which was TRAP staining positive and with more than three nucleus, the area and number of bone resorption pits decreased obviously in 8-PNG-supplemented groups. The apoptosis rate peaked earlier in the 8-PNG-supplemented groups and the mRNA expression level of TRAP and CTSK decreased significantly. All these inhibitory effects were in a dose dependent manner, the highest effect was obtained by 1 x 10(-5) mol x L(-1) 8-PNG. 8-PNG inhibits bone resorption activity of osteoclasts by inducing osteoclast apoptosis and inhibiting the gene expression and enzyme activity including TRAP and CTSK, and restrains bone marrow cells to osteoclast differentiation.
本研究旨在探讨8-异戊烯基柚皮素（8-PNG）对骨髓细胞破骨细胞生成及破骨细胞骨吸收活性的影响。从新生兔长骨骨髓中分离破骨细胞，并在含10%胎牛血清的α-MEM培养基中培养。分别以1×10⁻⁷、1×10⁻⁶、1×10⁻⁵ mol·L⁻¹的浓度将8-PNG添加到培养基中。17β-雌二醇（E2，1×10⁻⁷ mol·L⁻¹）用作阳性对照。5天后进行抗酒石酸酸性磷酸酶（TRAP）染色及TRAP活性测定，7天后分析骨吸收陷窝。分别在2、4、8、12、24、36和48小时后进行膜联蛋白V染色以检测破骨细胞凋亡。通过实时逆转录聚合酶链反应（RT-PCR）测定TRAP和组织蛋白酶K（CTSK）的mRNA表达水平。8-PNG显著减少了TRAP染色阳性且核数超过三个的破骨细胞数量，在添加8-PNG的组中骨吸收陷窝的面积和数量明显减少。添加8-PNG的组凋亡率更早达到峰值，且TRAP和CTSK的mRNA表达水平显著降低。所有这些抑制作用均呈剂量依赖性，1×10⁻⁵ mol·L⁻¹的8-PNG效果最佳。8-PNG通过诱导破骨细胞凋亡、抑制包括TRAP和CTSK在内的基因表达和酶活性来抑制破骨细胞的骨吸收活性，并抑制骨髓细胞向破骨细胞分化。
