Xu Bing, Jin Hong-ting, Wang Xiao-feng, Xia Lu-wei, Tong Pei-jian
Zhongguo Gu Shang. 2015 Jun;28(6):553-8.
To observe effects of serum Bushen Huoxue prescription(Chinese characters) on classic Wnt/β-catenin signaling pathways of osteoblasts, and explore mechanism of Bushen Huoxue prescription (Chinese characters) for preventing osteoporosis.
Twenty health female rats were randomly divided into two groups, including Bushen Huoxue (Chinese characters) group and saline group,10 in each group. Bushen Huoxue (Chinese characters) group and Saline group were gavaged Bushen Huoxue and saline every day for 1 week. Bushenhuoxue containing serum and saline containing serum were got according to methods of serum preparation of drug-containing. The osteoblasts was cultured with neonatal rat skull according to Enzyme Consumer Law, and was identified by Wright-Giemsa staining (R-J) and alkaline phosphatase staining (ALP). The third generation of osteoblasts was divided into three groups, including saline group, normal group,Bushen Huoxue (Chinese characters) group. Each group were added to 15% appropriate medium. ALP activity of osteoblasts and osteoblasts proliferation rate were tested, mineralized nodules were observed, the expression of β-catenin, Runx2, Osx mRNA of osteoblasts were tested by RT-PCR.
There was blue granules in cytoplasm, cell nucleus was flint with 1 to 3 nucleoli showed by R-J staining, morphology of osteoblasts were cultured. ALP staining showed cytoplasm with purple granules, the results showed that the cultured cell was osteoblasts. The content of ALP in Bushen Huoxue (Chinese characters)group was (6.272±0.131) ,appreciation rate was (0.81? 0.172), and could significantly improve differentiation and proliferation activity of osteoblasts compared with Saline group (P< 0.01). There were four different size orange nodules, the Maximun nodule was 1.0 x 1.0 cm in Bushen Huoxue (Chinese characters) group after Alizarin red staining, the results showed Bushen Huoxue (Chinese characters)group could obviously improve mineralization of osteoblasts. The expression of mRNA of β-catenin, Runx2 and Osx in Bushen Huoxue (Chinese characters)group were (1.782±0.944), (1.935±0.994) and (1.610±0.811) by RT-PCR,it was significantly increased compared with saline group (P<0.01), but there was no difference between Bushen Huoxue (Chinese characters)group and normal group (P>0.05).
Bushen Huoxue (Chinese characters)group could obviously promote differentiation, proliferation and mineralization of osteoblasts through activation of Wnt, β-catenin signaling pathway. It suggested that the mechanism of action of Bushen Huoxue (O'f f Il.t)particle clould prevent osteoporosis through the activation of Wnt, β-catenin signaling pathway.
观察补肾活血方血清对成骨细胞经典Wnt/β-连环蛋白信号通路的影响，探讨补肾活血方防治骨质疏松症的作用机制。
将20只健康雌性大鼠随机分为两组，即补肾活血方组和生理盐水组，每组10只。补肾活血方组和生理盐水组每天分别灌胃补肾活血方和生理盐水，连续1周。按照含药血清制备方法获取含补肾活血方血清和含生理盐水血清。采用酶消化法用新生大鼠颅骨培养成骨细胞，并用瑞氏-吉姆萨染色（R-J）和碱性磷酸酶染色（ALP）进行鉴定。将第三代成骨细胞分为三组，即生理盐水组、正常组、补肾活血方组。每组加入15%适宜培养基。检测成骨细胞的碱性磷酸酶（ALP）活性、成骨细胞增殖率，观察矿化结节，采用逆转录聚合酶链反应（RT-PCR）检测成骨细胞β-连环蛋白、Runx2、Osx mRNA的表达。
瑞氏-吉姆萨染色显示培养的成骨细胞胞质内有蓝色颗粒，细胞核呈椭圆形，有1～3个核仁。碱性磷酸酶染色显示胞质内有紫色颗粒，结果表明培养的细胞为成骨细胞。补肾活血方组ALP含量为（6.272±0.131），增值率为（0.81±0.172），与生理盐水组比较能显著提高成骨细胞的分化和增殖活性（P<0.01）。茜素红染色后补肾活血方组有4种不同大小的橘红色结节，最大结节为1.0×1.0 cm，结果表明补肾活血方组能明显促进成骨细胞矿化。RT-PCR检测补肾活血方组β-连环蛋白、Runx2和Osx mRNA表达分别为（1.782±0.944）、（1.935±0.994）和（1.610±0.811），与生理盐水组比较显著升高（P<0.01），但与正常组比较差异无统计学意义（P>0.05）。
补肾活血方组可通过激活Wnt、β-连环蛋白信号通路明显促进成骨细胞的分化、增殖和矿化。提示补肾活血方防治骨质疏松症的作用机制可能是通过激活Wnt、β-连环蛋白信号通路实现的。
