Effects of donkey-hide glue reinforcing bone oral solution medicated serum on osteoprotegerin and osteoprotegerin ligand mRNA expression of osteoblast in fetal rats / 中国组织工程研究

ABSTRACT

BACKGROUND:The therapeutic effects of donkey-hide glue reinforcing bone oral solution on osteoporosis have been determined, but the exact effective mechanism is to be approached. OBJECTIVE: To investigate the effects of donkey-hide glue reinforcing bone oral solution (DGRBOS) medicated serum on osteoprotegerin (OPG)and its ligand(OPGL)mRNAexpression of osteoblast in fetal rats and explore the molecular mechanism of treating osteoporosis with DGRBOS. DESIGN: A randomized controlled trial. MATERIALS: The experiment was carried out from June 2003 to October 2004 in Bone Metabolic Laboratory of Department of Integrative Chinese and Western Medicine, Affiliated Hospital of Tongji Medical College,Huazhong University of Technology and Science. Totally 30 3-month-oldWistar rats (15 males and 15 females) were randomly divided into 3 groups, I.e. DGRBOS group, estrogen group and control group, with 10 rats in every group. 12 clean newborn SD rats were selected to isolate and cul ture osteoblast. METHODS: ①After intragastric administration for 7 days, medicated serum was prepared respectively from the three groups. ②Skull osteoblast isolated from newborn SD rats was made into single cell suspension, then after digestion and passage, the subcultured osteoblast cell was made into cell suspension. The cultured osteoblasts were divided into 5 groups and given equal volumes of drug liquor. The DGRBOS group was given DGRBOS-medicated serum at the concentration of 100, 500 and 1 000 g/L which was diluted by nutrient solution; the estrogen group was given tibolone-medicated serum of 100 and 1 000 g/L; the control group was givenonly culture fluid. Meanwhile every group was given calf serum (100 g/L) for further culture. ③The osteoblast proliferation was measured by antigenic MTT colorimetric analysis and 3H-TdR penetration method. The in tra-cellular BGP contents were evaluated by radioimmunity .The mRNA expression of OPG and RANKL in osteoblast was analyzed by Rt-PCR. ④ One-way analysis of variance was applied to compare data among groups. MAIN OUTCOME MEASURES: mRNA expression of OPG and PAN KL in osteoblasts from fetal rats after intervention by medicated serum ofDGRBOS or Livial. RESULTS: ①The osteoblast proliferation measured by antigenic MTT colorimetric analysis and 3H-TdR penetration method showed that the proliferation in the DGRBOS group and tibolone group was enhanced moresignificantly than that in the control group (P ＜ 0.05-0.01), and reached maximal effect at the concentration of 500 g/L (P ＜ 0.01), but when the concentration was over 500 g/L, the effect tended to saturate. The medicated serum with all concentrations from DGRBOS and estrogen groups could increase the contents of BGP in osteoblasts (P ＜ 0.05). ②The mRNA expression of OPG reached the peak when the DGRBOS medicated serum was 1 000 g/L, and was obviously higher than that at the concentration of 100 and 500 g/L (P ＜ 0.05). The expression in DGRBOS group at the concentration of 1 000 g/L and in the estrogen group at the concentration of 100 and 1 000 g/L was apparently higher than that of the control group (P ＜ 0.01). ③The mRNAexpression of RANKL was the highest in DGR BOS group with 1 000 g/L concentration, and was markedly lower than that of the concentration of 100 and 500 g/L (P ＜ 0.05). The expression in DGRBOS group at the concentration of 1 000 g/L and in the estrogen group at the concentration of 100 and 1 000 g/L was noticeably lower than that in the control group (P ＜ 0.01).CONCLUSION: ①The DGRBOS could remarkably enhance osteoblast proliferation in dose-dependent and a dose-saturable manner, and the effect was close to that of tibolone. ②Partial mechanism of DGRBOS in treating osteoporosis might be promoting osteoblast proliferation and regulating OPG/RANKL expression.