| Background Polycystic ovary syndrome is one of the common endocrine and metabolic disorders in women.The main signs and symptoms including oligo-ovulation,androgen excess and polycystic ovarian morphology.PCOS not only affects women's normal fertility,but also has metabolic abnormalities such as insulin resistance,diabetes and hyperlipidemia.Therefore,the treatment for PCOS should not only consider improving the patients' fertility function,but also pay attention to the long-term complications caused by metabolic abnormalities.Clinically,oral contraceptives(OC)are commonly used to regularize the menstrual cycle and improve hyperandrogenism,but at the same time use of OC does increase the risk of insulin resistance,worsening the disease process.For patients with fertility requirements,clomiphene citrate(CC)administration is considered the preferred treatment to induce ovulation in PCOS women.Despite treatment with CC,a variable percentage of PCOS women still does not achieve a pregnancy or with high risk of miscarriage.Therefore,it is necessary to explore more ideal treatment methods to improve the endocrine and metabolic abnormalities of PCOS patients from different approaches.Soy isoflavones are common phytoestrogens,which have certain effects on the regulation of female endocrine and glycolipid metabolism.In endocrine regulation,it can bind and activate estrogen receptor,regulating 17?-estradiol biosynthesis and metabolism and reducing steroid hormone levels.At the same time,soy isoflavones also have the inhibitory effect of aromatase,binding to the active site of aromatase in a different orientation from that in the estrogen receptor,inhibiting the enzyme activity and affecting the transformation of androgens to estrogen.The metabolic regulation of soy isoflavones mainly includes reducing insulin resistance(IR),decreasing cholesterol, and the anti-inflammatory and antioxidant effects.The current research focuses on the beneficial effect of soy isoflavones,but the effective dose standard has not been reported.Objective In this study,the PCOS rat model was constructed by the aromatase inhibitor letrozole.We observed the effects of different doses of soybean isoflavones on blood glucose,sex hormone,inflammatory state and ovarian tissue morphology in PCOS model rats,as well as detected the expressions of ovarian androgen receptor(AR)and insulin-like growth factor-1(IGF-1).And proposed the possible mechanism of action of soy isoflavones,which providing a reference for the safe dose and possible mechanism of action of soy isoflavones in the clinical treatment of PCOS.Methods 1.65 female SD rats were randomly divided into Na Cl group(N=13)and model group(N=52).The model group treated with 1.0mg/kg/d letrozole for 21 days to build rat PCOS model,while the Na Cl group treated with 1ml/100g/d Na Cl.2.Vaginal smear was performed on all rats daily to observe the changes of estrus cycle.3.The fasting blood glucose concentration of the rats after modeling was tested by the ACCU-CHEK Active.4.After modeled,trail vein blood samples were collected and the serum was separated by centrifugation.Serum testosterone(T),estradiol(E2),luteinizing hormone(LH),follicle-stimulating hormone(FSH),anti-Mullerian hormone(AMH),Interleukin-1?(IL-1?)and Interleukin-18(IL-18)were determined by enzyme linked immunosorbent assay(ELISA).5.The successful model rats were randomly assigned into model control(PCOS)group,metformin group,low-dose(150mg/kg)soy isoflavones group and highdose(250mg/kg)soy isoflavones group,with 13 rats in each group.The treatment lasted for 21 d,during which both the Na Cl control group and the model control group were given 1ml/100 g normal saline by gavage.6.At the end of the experiment,all the rats were subjected to abdominal aortic puncture for blood collection.The rats were sacrificed by cervical dislocation,and bilateral ovaries were taken and weighed.7.After blood centrifugation,serum levels of sex hormones and inflammatory factors were determined by ELISA.8.The histological changes of ovary were observed by Hematoxylin-eosin staining.9.The expression of IGF-1,AR,AKT in ovarian tissue was detected by quantitative Real-time PCR and western blot(WB).Results 1.After modeling,PCOS rats gained weight(P<0.05)and increased fasting glucose(P<0.05).Compared with the control group,the serum levels of T,LH,AMH and LH/FSH were increased,with statistically significant differences(P<0.05),while the levels of E2 and FSH were decreased.Serum inflammatory cytokines IL-1?,IL-18 expression is significantly higher than control group(P<0.05);Compared with the control group,PCOS rats had cystic changes in ovarian structure,oocytes disappeared,luteal number decreased,and the number of granulosa cells in the follicles decreased,which proved that the PCOS rat model was successfully constructed.2.By comparing the effects of two doses of soy isoflavones on PCOS symptoms,it was found that both doses of soy isoflavones could effectively restore the estrus cycle of rats,reduce the weight of PCOS rats,and reduce the fasting blood glucose concentration.Serum levels of T,LH,LH/FSH,AMH,IL-1?,and IL-18 decreased after intervention with two doses of soy isoflavones(P<0.05),while E2 and FSH increased(P<0.05).3.Compared with the low-dose(150mg/kg)of soy isoflavones,the high-dose(250mg/kg)of soy isoflavones had more obvious effect on reducing serum LH,IL-1? and IL-18(P<0.05).There was no statistically significant difference between two doses of soy isoflavones in improving serum T,E2,LH/FSH and AMH,restoring estrus cycle,reducing body weight and reducing fasting blood glucose(P>0.05).4.Compared with the control group,the expression of IGF-1 and AR genes in ovarian tissues of PCOS rats was significantly increased(P<0.05).The results of quantitative real-time PCR showed that both doses could reduce the expression of IGF-1(P<0.05),and the down-regulation effect of low-dose on IGF-1 was more than that of high-dose group(P<0.05).The expressions of AR and AKT was downregulated by 150mg/kg soy isoflavones,while 250mg/kg soy isoflavones upregulated the expression of AR and AKT abnormally(P<0.05).5.Both soy isoflavone and metformin could restore the estrus cycle and ovarian morphology of PCOS rats,reduce the weight and the fasting glucose,decreased the serum levels of T,LH,LH/FSH,AMH,IL-1?,IL-18,and increased the level of E2 and FSH.6.Compared with metformin,soy isoflavones increased serum E2,T and AMH more significantly(P<0.05),while 150mg/kg soy isoflavones decreased LH less than metformin(P<0.05).There was no significant difference between metformin and soy isoflavones in the improvement of fasting glucose,FSH and LH/FSH(P>0.05).After intervention with two doses of soy isoflavones respectively,the inflammatory factors IL-1? and IL-18 were significantly decreased,with statistically significant differences(P<0.05).7.Quantitative real-time PCR results showed that metformin could down-regulate the expression of IGF-1 and AR in ovary of PCOS rats(vs.PCOS,P<0.05).Compared with the metformin group,there was no difference in the regulatory effect of 150mg/kg soy isoflavones on IGF-1 and AR(P>0.05),and the expressions of IGF-1 and AR were abnormally increased after intervention with 250mg/kg soy isoflavones(P<0.05).Conclusion 1.PCOS rat model was successfully established by continuous induction of 1mg/kg/d aromatase inhibitor letrozole for 21 days.2.Low-dose soy isoflavones could decrease androgen levels in PCOS model rats and improve hyperandrogenemia,while high-dose soy isoflavone did not significantly improve the hyperandrogenemia.3.Soy isoflavones have the effect similar to metformin,showing the effect of insulin sensitizer.The effect of high-dose soy isoflavones on improving insulin resistance was better than that of low-dose,but the decrease of androgens was not obvious.4.Soy isoflavones can affect the levels of androgen and insulin growth factor in PCOS rats,and the mechanism may be related to decreasing the expression of IGF-1,inhibiting the AKT pathway activation,and down-regulating the expression of AR. |
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