补肾健脾活血方对成骨细胞增殖分化及BMP-2、Smad5影响的研究

Study on Effect of Bushen Jianpi Huoxue Prescription on Proliferation and Differentiation of Osteoblasts as well as BMP-2 and Smad5

目的:研究中药复方补肾健脾活血方对大鼠成骨细胞增殖分化及骨形成蛋白-2 (BMP-2)、Smad5表达的影响。方法:将60只3月龄雌性SD大鼠随机分为空白组、补肾健脾活血方组、阿仑膦酸钠组,每组20只。制备对应的含药血清后分别干预成骨细胞,采用细胞计数法(CCK-8)检测成骨细胞增殖活性,碱性磷酸酶染色(ALP)检测成骨细胞分化能力,实时荧光定量PCR (RT-qPCR)检测BMP-2、Smad5 m RNA表达,蛋白免疫印迹法检测BMP-2、Smad5蛋白表达。结果:干预12 h、24 h、48 h时,各组细胞活性逐渐增强,干预48 h后各组细胞活性均降低。干预48 h时,与空白组、阿伦磷酸钠组比较,补肾健脾活血方组细胞活性最强(P<0.05)。补肾健脾活血方组成骨细胞ALP染色最深,明显深于空白组和阿仑膦酸钠组。与空白组比较,补肾健脾活血方组BMP-2、Smad5蛋白及mRNA表达升高(P<0.05);阿仑膦酸钠组BMP-2 mRNA表达降低(P<0.05)。与阿仑膦酸钠组比较,补肾健脾活血方组BMP-2、Smad5蛋白及mRNA表达升高(P<0.05)。结论:中药复方补肾健脾活血方可以促进大鼠成骨细胞增殖分化,提高BMP-2、Smad5 mRNA和蛋白表达。

Objective: To study the effect of Chinese herbal compounds of Bushen Jianpi Huoxue prescription on proliferation and differentiation of osteoblasts as well as expressions of bone morphogenetic protein-2(BMP-2) and Smad5.Methods:A total of 60 three-month-old female SD rats were randomly divided into the blank group,the Bushen Jianpi Huoxue prescription group and the alendronate sodium group,20 cases in each group. Osteoblasts were intervened after the preparation of the corresponding medicated serum:the proliferation activity of osteoblasts was detected by Cell Counting Kit-8(CCK-8), the differentiation ability of osteoblasts was detected by alkaline phosphatase(ALP), the mRNA expressions of BMP-2 and Smad5 were detected by real-time quantitative PCR(RT-qPCR),and the protein expressions of BMP-2 and Smad5 were detected by Western blot. Results:When the intervention was 12,24 and 48 hours,the cell activity in each group was increased gradually: after intervention for 48 hours, the cell activity in each group was decreased. During 48 hours of treatment, compared with those in the blank group and the alendronate sodium group, the cell activity in the Bushen Jianpi Huoxue prescription group was the highest(P<0.05). The ALP staining of Bushen Jianpi Huoxue prescription group was the deepest,which was significantly deeper than those in the blank group and the alendronate sodium group.Compared with those in the blank group,the protein and m RNA expressions of BMP-2 and Smad5 in the Bushen Jianpi Huoxue prescription group were increased(P<0.05):the expression of BMP-2 mRNA in the alendronate sodium group was decreased(P<0.05). Compared with those in the alendronate sodium group,the protein and mRNA expressions of BMP-2 and Smad5 in the Bushen Jianpi Huoxue prescription group were increased(P<0.05). Conclusion:Chinese herbal compounds of Bushen Jianpi Huoxue prescription can promote the proliferation and differentiation of rat osteoblasts,and increase the mRNA and protein expressions of BMP-2 and Smad5.