补肾方药有效成分不同比例配伍对成骨细胞增殖和分化的影响

Effects of different compatibility proportion of active ingredients of Kidney Recipe on osteoblast proliferation and differentiation

背景:补肾方药的动物及细胞实验研究提示具有防治骨质疏松及改善骨代谢作用,但补肾方药研究具有相当的复杂性,目前复方中药研究多采用的血清药物代谢动力学的方法却具有局限性,使有效作用成分及药物物质基础尚不确定。目的:通过采用补肾方药有效成分配伍,在不同浓度和时间下,对新生SD大鼠成骨细胞进行干预性培养,并对其增殖与分化情况进行测定,从而确定补肾方药对成骨细胞时效和量效关系,为补肾方药防治骨质疏松症提供实验依据。方法:体外培养原代新生24 h的SD大鼠成骨细胞,采用补肾方药的"补药"和"泻药"有效化学成分不同比例配伍,实验分3组,即补>泻组、补<泻组和对照组,补>泻组、补<泻组分别加入质量浓度10,20,30μg/L的补肾方药无血清培养基干预成骨细胞培养,在培养24,48,72 h分别检测细胞的增殖,在培养48,72 h测定成骨细胞的碱性磷酸酶分泌。结果与结论:质量浓度为10μg/L,在培养48 h,补>泻组具有明显促增殖作用(P<0.05);质量浓度为20μg/L时,各时间点补>泻组、补<泻组无明显差别,但与对照组比较具有明显增殖作用;质量浓度为30μg/L,在培养48 h,补>泻组具有明显促增殖作用最强(P<0.05),72 h时增殖作用减弱;质量浓度为10μg/L,在培养72 h测得补>泻组具有促碱性磷酸酶分泌作用,质量浓度为20μg/L,在培养24 h以补<泻组最明显(P<0.05);质量浓度为30μg/L,在培养72 h补>泻组促明显的促碱性磷酸酶分泌作用最佳(P<0.05)。结果证实,补肾方药有效成分按补>泻组配伍比例对成骨细胞增殖、分化作用最强,且存在时-效及量-效关系。

BACKGROUND： Animal and cell studies have shown that the Kidney Recipe can prevent and treat osteoporosis and improve bone metabolism, but this recipe is complicated. Recent studies on compound Chinese medicine mainly focused on serum drug metabolism and pharmacokinetics, which has limitations, and the effective ingredient and pharmaceutical material basis are uncertain. OBJECTIVE： In the different concentrations and time, by using different compatibility proportion of active ingredients of Kidney Recipe, osteoblasts from neonatal Sprague-Dawley rats were subjected to culture intervention. The proliferation and differentiation of osteoblasts were determined so as to identify time-effect and dose-effect relationship of Kidney Recipe on osteoblasts and to provide experimental evidences for prevention and treatment of osteoporosis.METHODS： Primary neonatal 24-hour osteoblasts of Sprague-Dawley rats were cultured in vitro. Herbs ＂tonic＂ and ＂cathartic＂ active chemical components of different proportion were used. The experiment contained three groups： Tonics Medicine （T） 〉 Cathartic Medicine （C） group, T 〈 C Medicine group and control group. T 〉 C group and T 〈 C group were given 10, 20, 30 pg/L serum-free medium kidney prescription intervention for osteoblast cultures. Cell proliferation was detected at 24, 48 and 72 hours a？ter culture. Alkaline phosphatase secretion of osteoblasts was measured at 48 and 72 hours.RESULTS AND CONCLUSION： At mass concentration of 10 pg/L and 48 hours of culture, a significant promoting effect on the proliferation was found in the T 〉 C group （P 〈 0.05）. At mass concentration of 20 pg/L, no significant difference was detected in the T 〉 C and T 〈 C groups at various time points, but significant difference was found as compared with the control group. At mass concentration of 30 pg/L and 48 hours of culture, a significant promoting effect on the proliferation was visible in the T 〉 C group （P 〈 0.05）, and the proliferation became weak at 72 hours. At mass concentration of 10 pg/L and 72 hours of culture, alkaline phosphatase secretion could be promoted in the T 〉 C group. At mass concentration of 20 pg/L and 24 hours of culture, the promoting effect on alkaline phosphatase secretion was most significant in the T 〈 C group （P 〈 0.05）. At mass concentration of 30 pg/L and 72 hours of culture, the promoting effect on alkaline phosphatase secretion was optimal in the T 〉 C group （P 〈 0.05）. Results confirm that active ingredients of Kidney Recipe compatibility proportion can promote osteoblasts proliferation and differentiation most strongly in the T 〉 C group in the time-effect and the dose-effect relationship.