Study On The Effect And Its Possible Mechanism Of Soybean Isoflavone On The Osteoporosis In Postmenopausal Women With Different Equol Phenotypes

Osteoporosis is a disease of bone that leads to an increased risk of fracture of lumbar vertebra and hip. In osteoporosis, the bone mineral density (BMD) is reduced, bone microarchitecture is disrupted, and the amount and variety of proteins in bone is altered. It is estimated that 1 in 3 women and 1 in 9 men over the age of 50 worldwide have osteoporosis. As China is the developing country with great number of population, especially the aged group increasing fast, osteoporosis and postmenopausal osteoporosis (PMOP) especially are serious public health problem. For this reason, the research on prevention and cure for PMOP is practical importance for improve health and life quality of post-menopause women.As result approved, a decrease in estrogen levels at menopause leading to a rapid loss of bone mineral density is key cause of PMOP. Treatment with estrogen has been reported to reduce the risk of hip and wrist fractures. Hormone replacement therapy (HRT) is the standard of treatment for maintenance of internal estrogen level and improvement of menopausal syndrome. Though HRT could prevent PMOP and reduce bone fracture, Women's Health Initiative in USA found that HRT also brought risk of breast cancer and cardiovascular diseases to cured persons. FDA in USA recommends that HRT would not be first-line therapy for PMOP and new approach of diet and exercise would be considered to prevent and cure it.In recent year, phytoestrogen and soybean isoflavones (SI) derived from soybean are focused in research and expected to be alternative remedy of HRT. This group of compound is with weak estrogenic effects, which has estrogen agonist and antagonist actions without side-effect by estrogen treatment. Epidemiologic studies found that, there was positive correlation between amount of SI intake and BMD in Asian post-menopause women with high intake of bean product. However, there were conflicted results of effect on PMOP in research with more clinical research approached. In recent explanation, the difference of intervention by SI was relation with capability of metabolism of SI. For example, Equol (Eq) is the final metabolite resulting from the metabolism of daidzein in the intestine, which has higher estrogenic activity in body comparison of genistein, glycitein, dihydrodaidzein. It is reported that approximately 30–50% of adult humans who are"equol producers"and metabolism capability are different between race. Past estimations of effect of SI on prevetion and cure of PMOP were seldom focused on Chinese post-menopause women and without consideration of daidzein-metabolizing phenotypes (DMPs) by intake bean product. For this reason, it is interesting and practical that the intervention effect of SI and its mechanism on PMOP with consideration of DMPs is carried out to solve the increase of PMOP in china now.Basing on the above the analysis, our research focused on influence of biological effect of bean products due to DMPs. There were involved with 152 cases of PMOP and 308 cases of control in our Case–control studies conducted in Chongqing. The risk of PMOP was studied with analysis methods of semi-quantitative food frequency questionnaire on dietary pattern and intake of bean product, judgment of DMPs according to detection of urinary excretion of isoflavone status by HPLC. The correlation betweenrisk of PMOP and level of exposure to SI metabolite and intake of bean product was analyzed by SPSS 13.0. For further analysis of intervention effect of SI according to DMPs, the group of soybean protein isolate(SPI 20g/d, containing 50mgSI) with SI and the group of whey protein (20g/d) without SI were carried out for 6 months. The evaluation of prevention and cure on PMOP by protein isolate with relation of DMPs was done with analysis of metabolism of SI, BMD, Serum lipid and sex hormone level of different groups at before, middle and final phase of the experiment. Moreover, the wistar rat-derived osteoblast cultured inα-MEM supplemented with 20% fetal bovine serum were cured with 10-8 mol/L～10-6mol/L equol and estradiol(E2) respectively, proper amount of estrogen receptor antagonist ICI182780 administered too. Proliferation and differentiation of primary culture of osteoblast influenced by equol was studied by detection of Protein kinase C(PKC) by Western blot, cell proliferation by MTT, activity of alkaline phosphatas (ALP) and activity of Boneglaprotein (BGP) , which mainly concerned the mechanism of prevention and cure of PMOP by Eq at cellular level. Main results1. Population study1. 1 Investigation of common risk of PMOP: Which is important risk factores for PMOP in Chongqing are family history of hip fracture, low body quality index and fecundity. But, regular exercise, tea and maintenance of proper estrogen level would be contributive to preventing PMOP.1.2 The correlation between occurrence of PMOP and dietary intake of bean product: The intake of bean product or SPI in PMOP group was lower than that in non-PMOP (P<0.05) significantly. The negative correlation between occurrence of PMOP and intake of bean product or SPI was found by regression analysis of Logistic model. Moreover, there were also negative correlation between occurrence of PMOP and intake of Ca, Vit A, Vit D, dark green vegetables and milk in diet, but positive correlation between PMOP and intake of animal protein and cooking oil.1.3 The correlation between metabolism of daidzein and occurrence of PMOP: The rate of equol producer(32.2%) in PMOP group was lower than that(40.3%) in non-PMOP group(P<0.05) significantly, and the same trend was found in the index of Eq,GNT,GLY and DHD between groups. The positive correlation between occurrence of PMOP and urinary excretion of Eq and GNT was found by regression analysis of Logistic model.1.4 The influence of SPI intervention on the levels of endogenous SI metabolites among different Eq phenotypes: As to the EP+ populations, compared with the control group, SPI intervention can significantly increase the levels of Eq, GLY and GNT (P<0.05). While in EP- populations, SPI intervention only promote the levels of GNT moderately.1.5 The influence on BMD of post-menopause women with different DMPs by intervention of SPI: Compared with the control group, the BMD and T scores was increased in PMOP group by SPI intervention, no matter with equol produced or not, but the most significant improvement was found in EP+ (P<0.01). The significant increase of BMD and T scores was found in EP+ (P<0.05), but not in EP- (P>0.05) by SPI in non-PMOP group.1.5 The influence on serum lipid and sex hormone in groups by SPI: In PMOP group, the levels of E2 of EP+ or EP- was increased significantly and the levels of LDL-C and TC was decreased by comparison of that in control group with intervention of SPI, the most significant change of serum lipid found in EP+, but there was no change of TG. The level of HDL-C was increased a little but no significant difference. In non- PMOP group, compared with control group, the amelioration of serum lipid and E2 was only found in EP+2. Vitro experiment2.1 The influence on proliferation of osteoblast by equol: The MTT absorbance value was enhanced by Eq intervention, and the improvement related to prolong exposure to Eq, but no relation with concentration of Eq. Moreover, the effect of Eq was significantly weaker than that of E2 (P<0.05) at the same period in the same concentration. All effects of Eq and E2 were inhibited by ICI128780 antagonist.2.2 The influence of differentiation of osteoblast by equol: The expressions of ALP and BGP of osteoblast were enhanced by Eq intervention, and was positive dose-effect relationship with action time and concentration (P<0.05). Moreover, the effect of Eq was significantly weaker than that of E2 (P<0.05) at the same period in the same concentration. All effects of Eq and E2 were inhibited by ICI128780 antagonist.2.3 The influence on amount of bone collagen by equol by Eq intervention: The hydroxyproline in cell was increased and was positive dose-effect relationship with action time and concentration (P<0.05). Moreover, the effect of Eq was significantly weaker than that of E2 (P<0.05) at the same period in the same concentration. All effects of Eq and E2 were inhibited by ICI128780 antagonist.2.4 The influence on expression of PKCαof osteoblast by equol: The expressions of PKCαof osteoblast was enhanced by Eq intervention, and was positive dose-effect relationship with concentration, but lower effective than that by E2.The decrease of expression of PKC was found by combination with ICI182780 at each concentration of Eq, but higher than that in control group (P>0.05).ConclusionsOn the basis of the above, the correlation between PMOP in Chongqing and common risk factors, intake of bean product and metabolite of SI was studied; moreover, the influence on prevention and cure of PMOP by SI in different DMPs was also carried out. In population-based clinical trial, family history of hip fracture, low body quality index and fecundity is important risk factores for PMOP in Chongqing, regular exercise, tea and maintenance of proper estrogen level would be contributive to preventing PMOP. The intake of bean product, SPI, Ca, Vit A, Vit D, dark vegetables and milk would be contributive to preventing and curing PMOP, but animal protein and cooking oil would increase of risk of PMOP. Equol produced or not would determine the occurrence of PMOP; furthermore the risk of PMOP was decreased with increase of urinary excretion of Eq and GNT. By the intervention of SPI, the improvement of BMD, serum lipid and estrogen level was found in PMOP group, especially greater in EP+. In non-PMOP group, the effect of SPI on those indexes was only found in EP+. With the evidence of increasing proliferation and differentiation of osteoblast and bone matrix, but inhibited by ER antagonist, it is supposed that Eq exerts estrogenic activity and high affinity for estrogen receptor, mediate proliferation and differentiation of cell as estrogen does, which would be related to signal transduction of transmembrane protein PKCα. In our experiment, the important effect of diet on occurrence of PMOP was confirmed. The Equol metabolizing phenotypes may determine the effect of SI on prevention and cure of osteoporosis, the greater effect found in equol producer. So, to prevent PMOP, proper dietary and fertility patterns should be decided by women with intake of bean product, milk, vegetable and fruits, but decrease of intake of animal protein and cooking oil.