摘要
为探讨淫羊藿黄酮苷及其代谢产物对骨形成和骨吸收的调控作用 ,利用酶水解法和酸水解法分别制备了淫羊藿黄酮苷的 2种代谢产物。采用鼠颅盖骨机械法分离和体外培养获得成骨细胞 ,采用新生兔长干骨机械分离法获得破骨细胞 ,采用VD3诱导鼠骨髓的造血干细胞分化成破骨样的多核巨细胞。实验组培养液为 1 0 %胎牛血清 +αMEM培养液 +不同浓度淫羊藿黄酮苷及代谢产物 ,对照组不加药。分别用四甲基偶氮唑盐和3H TdR法检测成骨细胞的增生和碱性磷酸酶 ,用骨钙素衡量成骨细胞的分化状态 ,用Actin ring染色观察破骨细胞形态变化 ,用图像分析仪分析体外骨吸收陷窝面积 ,用抗酒石酸酸性磷酸酶染色观察OCL的形成 ,用RT PCR法检测和破骨样细胞形成有关的分子机制。发现 :淫羊藿黄酮苷及其代谢产物对大鼠成骨细胞的增生有明显的刺激作用 ,并能够促进成骨细胞合成分泌碱性磷酸酶 ,但对成骨细胞晚期分化指标骨钙素无显著作用。同时 ,使破骨细胞Actin ring回缩 ,从而使骨吸收陷窝面积减少。此外 ,淫羊藿黄酮苷及其代谢产物通过影响成骨细胞中RANKL和OPG的表达而达到抑制破骨样细胞形成。以上结果说明 ,淫羊藿黄酮苷及其代谢产物一方面抑制破骨样细胞形成和成熟的破骨细胞对骨基质的吸收 。
The major and active constituents of the genus Epimedium are flavonoids, which can prevent bon e loss in ovariectonized rats. Icarrin is a flavonoid isolated from Herba Epimed ii. By enzymatic and acidic hydrolysis of icarrin, two metabolites (icariside Ⅱ and icaritin) can be obtained. Here we report the effects of icarrin and its tw o metabolites on the bone formation and bone resorption in vitro.Three cell models were used. Primary osteoblastic cells were isolated from neona tal Wistar rat calvarias. Icarrin and its two metabolites increased the viabilit y of osteoblasts significantly. Besides, alkaline phosphatase (ALP) activity and osteocalcin (OC) synthesis of osteoblasts also increased, as well as the ALP, O C and collagen mRNA expression. Since ALP and OC are phenotypic markers for earl y-stage differentiated osteoblasts and terminally differentiated osteoblasts re spectively, our findings indicate that Icarrin and its metabolites stimulate ost eoblasts differentiation at various stages (from osteoprogenitors to terminally differentiated osteoblasts). The effects of Icarrin and its metabolites on osteoclast-like cells were obtain ed by coculturing bone marrow with osteoblasts for 8 days in α-MEM containing 10 -8mol/L 1.25(OH) 2D 3. The osteoclast-like cell formation was estimat ed by staining for tartrate-resistant acid phosphatase (TRACP), a marker enzyme of osteoclast. Icarrin and its metabolites significantly inhibited osteoclast- like cell formation, and elevated the mRNA levels of OPG and RANKL dose-depend e ntly, resulting in enlarged ratio of the two as revealed by RT-PCR. Hence it i s likely that icarrin and its metabolites regulate bone resorption, at least in part, via its effects on OPG and RANKL expression.Mature osteoclasts were isolated from the long bones of neonatal Japanese white rabbits (60-90 g). The surface area of bone resorption lacunae was measured by p hotomicrography and image analysis. Actin microfilaments were detected by bindin g of rhodamine-conjugated phalloidin (R-PHD) to F-actin. Icarrin and its meta bolites showed the potency to withdraw the actin ring, consistent with the inhib ited bone resorption of osteoclasts. In conclusion, icarrin and its metabolites can not only interfere with the diffe rentiation of osteoclast-like cells and inhibit bone resorption ability of matu re osteoclasts, but also promote bone formation.
出处
《首都医科大学学报》
CAS
2005年第1期37-37,共1页
Journal of Capital Medical University
