Red clover(Trifolium pratense L.) is a perennial herb belonging to the legume Trifolium. The inflorescence and flowers contain abundant isoflavones, which have effects like plant estrogen. Studies have shown that total isoflavones from T. pratense L. can improve the symptoms of menopausal, has therapeutic effect on osteoporosis, and can significantly improve the sex hormone level in female aged rats. Biochanin A, biochanin B, genistein and daidzein are the four major isoflavones in T. pratense L. Studies have showed that these four components contributed to bone formation. But the chemical structure of these four compounds is not the same, the four of which has the high activity of promoting bone formation and its mechanism of action, there is no research at present.MC3T3-E1 cell is osteoblast-like cell line established from C57BL/6 mice parietal cell, which undergoes 3 stages in bone formation:proliferation, differentiation and calcification, and it is often used as a cellular model in the study of bone metabolism in the world. Osteoblast-like cell culture in vitro is a simple and direct method for screening and evaluating the effects of medicine for treatment of osteoporosis. Proliferation, differentiation, mineralization and osteocalcin production were used as index to investigate and compare the effects of total isoflavones from T. pratense L., serum containing the total isoflavones from T. pratense L., biochanin A, biochanin B, genistein and daidzein on MC3T3-E1 cell cultured in vitro. In order to clarify the mechanisms and the material foundation of red clover treatment osteoporosis, further at the mRNA and protein levels their effects on the expression of osteogenic related factors and cell signaling pathway were analyzed and compared.The main contents and findings were as follows:1. The proliferation of MC3T3-E1 osteoblastic cell was determined by MTT method, and the activities of alkaline phosphatase and osteocalcin were determined by ELISA. Different concentrations of total isoflavones from T. Pretense L., serum containing the total isoflavones from T. Pretense L., biochanin A, biochanin B, genistein and daidzein could significantly promote the proliferation, increase the activities of alkaline phosphatase and osteocalcin accumulation of MC3T3-E1 osteoblastic cell, and the best concentrations were 1.5 μg/mL,5%,1×10-7 mol/L, 1×10-7 mol/L, 1×10-7 mol/L and 1×10-7 mol/L, respectively. The proliferation rate of MC3T3-E1 cell treated by biochanin B (1×10-7 mol/L) or genistein (1×10-7 mol/L) for 96 h was significantly higher than that by biochanin A (1×10-7 mol/L) or daidzein (1×10-7 mol/L) (*p<0.05). The activity of alkaline phosphatase of MC3T3-E1 cell treated by biochanin B(1×10-6 or 1×10-5 mol/L) for 3 d was significantly higher than that by the other medicine(*p<0.05); the activity of alkaline phosphatase of MC3T3-E1 cell treated by genistein (1×10-10 or 1×10-9mol/L) for 3 d was significantly higher than that by the other medicine(*p<0.05). The osteocalcin secretion in MC3T3-E1 cell treated by biochanin B or daidzein for 3 d was significantly higher than that by biochanin A (*p<0.05).2. The mineralized nodules were observed by Alizarin red staining method.Total isoflavones from T. pretense L. (1.5 μg/mL), serum containing the total isoflavones from T. pretense L. (5%), biochanin B (1×10-7 mol/L), genistein (1×10-7 mol/L) and daidzein (1×10-7 mol/L) could significantly promote the formation of mineralized nodules in MC3T3-E1 cell at 6th day,12th and 18th day, and biochanin A (1×10-7 mol/L) at 12th and 18th day. The number of mineralized nodules in MC3T3-E1 cell treated by biochanin B, genistein and daidzein was higher than that by biochanin A at 6th day (*p<0.05).3. The expression of osteogenic related factor mRNA was analyzed by RT-PCR. Quantitative RT-PCR analysis revealed that the expression levels of OPG、β-catenin、 Lrp-5、Lrp-6、Frizzled、Dvll、ER-a、ER-β and Osterix were significantly increased by total isoflavones fromt. pretense L.(1.5 μg/mL), and the expression level of RANKL was significantly decreased by total isoflavones from t. pretense L.(1.5 μg/mL); the expression levels of OPG、β-catenin、Lrp-5、Lrp-6、Frizzled、Dvll、ER-α、ER-β and Osterix were significantly increased by Biochanin B(1×10-7 mol/L); the expression levels of OPG、Lrp-5、Lrp-6、Frizzled、Dvll、ER-α、ER-β and Osterix were significantly increased by Genistein(1×10-7 mol/L), and the expression level of RANKL was significantly decreased by Genistein(1×10-7 mol/L); Genistein(1×10-7 mol/L) was significantly better than Biochanin B(1×10-7 mol/L) in promoting the expression of OPG、Lrp-5、Lrp-6、Dvll、Frizzled、ER-β and Osterix(*p<0.05), as well as decreasing the expression of RANKL(*p<0.05); Biochanin B(1×10-7 mol/L) was better than Genistein(1×10-7 mol/L) in promoting the expression of β-catenin and ER-a(*p<0.05).4. The expression of osteogenic related factors in protein level was determined by Western-blotting. Western-blotting analysis revealed that the expression levels of OPG、β-catenin、ER-β、BMP-2、P38 and ERK were significantly increased by total isoflavones from T. pretense L.(1.5 μg/mL), and the expression level of RANKL was significantly decreased by total isoflavones from T. pretense L.(1.5 μg/mL);Biochanin B(1×10-7 mol/L) increased the expression levels of β-catenin、ER-α、BMP-2、P38 and P-ERK, and decreased the expression level of RANKL significantly(*p<0.05); Genistein(1×10-7 mol/L) increased the expression level of β-catenin、BMP-2、P38、 ERK and P-ERK(*p<0.05).Conclusion:The total isoflavones from T. pretense L., serum containing the total isoflavones from T. Pretense L., biochanin A, biochanin B, genistein and daidzein could significantly promote the proliferation, differentiation and mineralization of MC3T3-E1 cell, and the effects of biochanin B and genistein were better than that of biochanin A and daidzein. The total isoflavones from T. Pretense L. could promote osteogenesis by regulating the expression of osteogenic factors in BMP, Wnt/p-catenin, OPG/RANKL/RANK, estrogen and MAPK signaling pathway; Biochanin B could promote osteogenesis by regulating the expression of osteogenic factors in BMP. Wnt/β-catenin. estrogen and MAPK signaling pathway; Genistein could promote osteogenesis by regulating the expression of osteogenic factors in BMP. Wnt/β-catenin and MAPK signaling pathway. The total isoflavones from T. Pretense L., biochanin B and genistein have the potential to develop new medicines for osteoporosis. This project preliminaryly clarified the mechanisms and the material foundation of red clover treatment osteoporosis, and provided certain theory basis for the development of osteoporosis medicines. |