摘要
研究8-异戊烯基柑橘素(8-prenylnaringenin,8-PNG)对骨髓细胞向破骨细胞分化及骨吸收活性的影响。从出生24 h内的青紫兰兔四肢骨髓中分离培养破骨细胞,加入8-PNG(1×10 7、1×10 6和1×10 5mol.L 1),以1×10 7mol.L 117β-雌二醇(17β-estradiol,E2)作为阳性对照。培养5天后进行TRAP染色和TRAP活性检测,7天后进行甲苯胺蓝染色和骨吸收陷窝数量与面积的分析。分别于加药后2、4、8、12、24、36及48 h进行AnnexinV染色,观察破骨细胞凋亡情况;于12、24和36 h提取总RNA,用real-time RT-PCR法检测TRAP(tartrate-resistant acid phosphatase)和CTSK(cathepsin K)的基因表达水平。结果显示,8-PNG(1×10 6和1×10 5mol.L 1)组的TRAP染色阳性细胞即破骨细胞数量明显低于空白对照组(P<0.01),但只有8-PNG(1×10 5mol.L 1)组显著低于E2组(P<0.05);TRAP活性的变化呈相同趋势;骨陷窝数和面积的计量结果亦呈相似变化趋势;E2组使破骨细胞的凋亡高峰大大提前,且凋亡数量最多,其次为8-PNG(1×10 5mol.L 1)组,8-PNG(1×10 7mol.L 1)组与空白对照组比较无差异。8-PNG(1×10 7、1×10 6和1×10 5mol.L 1)可显著抑制TRAP和CTSK的基因表达,且呈剂量依赖性。结果表明,8-PNG能抑制骨髓细胞向破骨细胞分化,并能抑制破骨细胞的骨吸收活性,其机制与诱导破骨细胞凋亡和抑制骨吸收关键酶的基因表达与活性有关。
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 a-MEM containing 10% FBS. 8-PNG was added into culture media at 1 x 10^-7, lxl0^-6, 1x10^ 5 tool.L-l, separately. 17β-Estradiol （E2, 1x10 ^-7 tool.L-l） was used as positive control. TRAP 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 positiveand 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 X10-s mol-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.
出处
《药学学报》
CAS
CSCD
北大核心
2013年第3期347-351,共5页
Acta Pharmaceutica Sinica
基金
甘肃省科技重大专项计划资助项目(092NKDA025)
贵州省国际科技合作计划项目(黔科合外G字[2008]700108)
教育部新世纪优秀支持计划(教技函(2011)95号)
贵州省优秀青年科技人才培养对象专项资金(黔科合人字2011(34)号)
