| Osteoporosis is a disease of high bone remodeling, with an imbalance of bone resorption over bone formation, resulting in decreased bone mineral density and disruption of bone microarchitecture. In the United State alone, 10 million persons already have osteoporosis, and 18 million more have low bone mass. China has the largest population in the world and it is the only country in the world with close to 200 million people aged 60 years or over. Increased aging populations are always accompanied with the increased incidence of osteoporosis. So, the WHO has identified osteoporosis as a major public health concern. Postmenopausal osteoporosis(PMOP) is a kind of osteoporosis, and due to the function of ovary loss and the reduction of secreted estrogen with aging. It is affecting the health of the elderly, shortened life spans, and even increased burden of country, the family’s financial and human resources. Therefore, it has far-reaching significance in effective prevention and treatment of PMOP development.Estrogen, derived from ovarian endometrial cells and ovarian granulosa cells, is the female hormone that generated by adrenal cortex, ovary, and the placenta. During follicle development, estrogen was converted to estradiol(E2), which causes physiological metabolism effect in the stimulation of granule cells. Along with the age growth and the impact of external factors, ovarian function decline, resulting in the sharp decline in the amount of E2, which causes a series of estrogen deficiency diseases, such as menopausal syndrome. Studies reported that Estrogen has role in promoting the retention of sodium and water, the development of uterine and ovarian, and the deposition of bone calcium and eburnation. Therefore, estrogen replacement therapy is the main treatment for PMOP in clinical practice. However, as the increased cognitive of estrogen found that the treatment schemes might increase the breast cancer and cardiovascular and cerebrovascular diseases risk, so Women’s Health Association and the US Food and drug administration put forward the re-evaluation the clinical value of estrogen replacement therapy. Therefore, it is imperative to find new solutions to postmenopausal osteoporosis especially dietary plan.Estrogen regulates bone metabolism and bone turnover in vivo, which is mainly mediated by osteoblast(OB) activity, activated by estrogen receptor(ER). ER is a protein molecular in target organ cells. Estrogen can exert its biological effects trough specifically binding with ER. Estrogen is divided into two classic nuclear receptors(ER alpha and ER beta, respectively), with similar structure and high homologous. However the functions of the two receptors are not the same due to the different affinities with their ligands. Although recent studies most focus on the effects of ER alpha in bone metabolism, there is some microarray studies showed that the ER beta also played an important role in regulating gene expression of bone metabolism.Soybean isoflavones(SI) is a kind of typical phytoestrogens and its molecular structure is similar to estrogen. SI can bind to the estrogen receptor with weak estrogenic effects, and do not carry the side effect of estrogen. Study indicates that the clinical effectiveness of SI is closely related to its intestinal ultimate metabolites, equol. Equol(Eq), molecular formula C15H14O3, is stable and its structure and bioactivity are similar with estrogen in vivo. In addition, Eq has stronger effects than its precursor, daidzein. Subjects who can produce Eq is called Eq producers(EP+), otherwise is called not Eq producers(EP-). Equol have three forms, S-Eq, R-Eq and the racemate. Although the two isomers, S-Eq and R-Eq, both have estrogenic activities, their affinities are not the same. Studies reported that S-Eq, the only isomer, can be metabolized by human, and has strong affinity with ER beta receptor. Nevertheless, R-Eq has weak affinity with ER alpha receptor and much weaker affinity with ER beta receptor.OPG/RANK/RANKL signal system is a major breakthrough for understanding the role of OB and osteoclast(OC) in regulating bone metabolism. Studies found that cytokines and hormones could directly or indirectly regulate the osteoprotegerin(osteoprotegerin, OPG), osteoclast differentiation factor(RANKL), and the relations between them and receptor activator of nuclear factor-κB(receptor activator of NF-κB, RANK). The proportions of RANKL and OPG expression are the key determinants in bone resorption or formation in bone tissue microenvironment. OPG is secreted by osteoblasts and can competitively bind to RANKL, resulting in inhibiting osteoclast formation with blocking the RANK-RANKL binding sites in osteoclast cell. OPG/RANK/RANKL system directly or indirectly affected the senile and postmenopausal osteoporosis.Therefore, in this study, we used postmenopausal osteoporosis model to study the effects of Eq on preventing and curing PMOP, as well as its effects on the regulating OPG/RANK/RANKL expression in vivo and in vitro. Further, we aimed to reveal the healthy effects of Eq, and open up new strategies and methods in prevention and treatment of PMOP.The experimental results in vitro1. Eq and E2 treatment increase the proliferation of rat osteoblast cells(ROS1728)A range of doses of Eq and E2 treated the rat osteoblast cells. With the increase of concentration, dose effect more obvious, more rapid proliferation.2. Eq and E2 regulate ROS1728 cell apoptosisDifferent concentrations of Eq, E2, agonist(DPN) and inhibitor(TAM) treatment of 48 h in ROS1728 cells showed that the apoptosis of ROS1728 cell in Eq+TAM and E2+TAM groups increased significantly compared to Eq, E2, Eq+DPN, E2+DPN and sham operation groups respectively.3. Eq have the effects on regulating ROS1728 cell cycle.Different concentrations of Eq treated cells 48 h, and showed that cells in G1 phase were relatively stable, detected by flow cytometry. When cells went from G1 phase to S phase, the cells in Eq and Eq+DPN agonist groups went through the restriction point of G1 phase and multiplied so quickly. However the proliferations of cells are slow in TAM and Eq+TAM groups. Meanwhile the percentage of cells in G2 phase decreased obviously in sham group.4. Eq have the effects on regulating the relative gene exression of OPG/RANK/ RANKL.ROS1728 osteoblasts with different concentrations of Eq treatment showed that the mRNA levels of RANK and RANKL decreased significantly, while OPG, ER alpha, ER beta increased markedly compared to control group. These effects indicated that Eq could regulate OPG/RANK/RANKL signaling pathway with binding to ER alpha、ER beta.5. Eq have the effects on regulating the relative protein expression of OPG/RANKL/ RANK.ROS1728 osteoblasts with different concentrations of Eq treatment showed the significant dose effects that OPG, JNK, ER alpha and ER beta protein expressions were increased, while protein expressions of BGP, RANKL and RANK decreased gradually.6. Eq have the effects on protein expressions of OPG/RANKL/RANK with ER beta and ER alpha knocked-down by relative si RNA and the results demonstrated that after knocking-down ER beta, the expressions of RANKL and RANK increased significantly and OPG expression decreased markedly. Nevertheless, the changes of these proteins were not significant with ER alpha knocked-down.The experimental results in vivo1. Postmenopausal osteoporosis disease model of SD rats were established. Female SD were randomized divided into sham operation group(sham group), ovariectomized model group(OVX group), high dose of Eq group, Eq low dose group, and E2 positive control group. The rats were anesthetized by Chloral hydrate, disinfected skin by alcohol. We find out the ovary in the below kidney, put it up, ligate fallopian tube and blood vessel of ovary and then remove the ovary by 2cm of incision along with kidney. Sham operation group were removed fat closed to ovary for control. The survival rates of rats during the period of 8 wks modeling were observed. In order to confirm whether models were successful, bone density and serum estrogen level were measured. After modeling, rats were treated by Eq for 8 wks.2. Morphological detection(HE staining) after Eq supplement showed that the trabecular bone of femoral metaphysis was rich and dense in intervention group and was similar to the sham shape. While in model group, the trabecular bone of femoral metaphysis was sparse and disperse, even lost in some regions.3. The effects of Eq on bone mineral density(BMD) showed that BMD in OVX group was much lower than sham group but higher than model group. So, we speculated Eq have good effects on BMD.4. The effects of Eq on serum biochemical factors formation and absorption of bone metabolism showed that the levels of blood calcium, E2, and 1, 25(OH)2D3 decreased significantly, however serum phosphate, BGP, and PCI increased in OVX group. The effects were completely opposite in sham and Eq groups.5. The effects of Eq on serum biological factors of bone resorption showed that the levels of TRAP、PYD and DPD increased significantly in OVX group, but decreased in sham and Eq groups.6. The effects of Eq on the proportions of serum OPG and RANKL expressions showed that the level of OPG decreased significantly and RANKL increased in OVX group. Moreover, the OPG increased and RANKL decreased after Eq treatment in consistent with the results of PCR.Conclusions: Eq could play an anti-apoptotic role in promoting the proliferation of ROS1728 cells, and maintain the balance of bone turnover with regulating relative gene and protein expressions of OPG/RANK/RANL. Moreover, Eq could prevent osteoporosis with binding to ER beta. In addition, the experiments in vivo also proved the validity and stability of the Eq in preventing PMOP. |
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