Effects And Mechanisms Of Genistein On Polycystic Ovary Syndrome In Mouse

Polycystic ovary syndrome?PCOS?is an endocrine disorder with high heterogeneity among women of uncertain causes and a leading reason of poor fertility.PCOS is characterized by various clinical symptoms,such as infertility,irregular menstrual cycle,hirsutism and/or acne,polycystic changes of ovary,obesity,insulin resistance and metabolic disorders.Many studies have shown that PCOS not only affects reproductive function of women,but also is associated with the increased risk of pregnancy complications and major diseases such as type II diabetes and cardiovascular disease.However,the pathogenesis of PCOS is still not clear.There is still no unified treatment plan and special drugs approved for PCOS.The clinical treatment is symptom-oriented and often with large side effects and heavy burden on PCOS patients.The pathological mechanism and treatment plan of PCOS is still needed more research.It was reported that the clinical symptoms and complications of PCOS may be related to the abnormal androgen level?too high?and insulin resistance,which are two major pathological characteristics of PCOS.Recent studies showed that the increase of oxidative stress level is closely related to the process of PCOS.Since PCOS is a complex disease caused by endocrine and metabolic abnormalities,which often needs long-term systemic regulation,we speculated that Genistein?GEN?,Hydroxysafflor yellow A?HSYA?and Ginsenoside Rb1,which are phytoestrogens and have antioxidant capacity,may have therapeutic potential for PCOS.Therefore,we used DHEA to induce PCOS in mice and subsequently applied GEN,HSYA or Rb1 in PCOS mice to find out the best one.After that,mice ovarian granulosa cells?GCs?were cultured and RT-PCR,Western blot,flow cytometry and promoter mutation were utilized to explore the pathogenesis and treatment strategy of PCOS.The results showed that PCOS mouse model was successfully established.PCOS mice have estrous cycle disorder,polycystic follicles and hormones?T,E₂,P₄,LH,FSH,AMH?secretion disorder.Further analysis showed that the expression of steroid hormone secretion-related genes Star,Hsd3b1 and Cyp11a1 were disturbed as well.GEN has the best therapeutic potential for PCOS mice.GEN restored the irregular estrous cycle,improved cystic follicles,inhibited the abnormal changes of hormones and hormone secretion related genes,which implying GEN plays a therapeutic role through acting as phytoestrogen and regulating the expression of steroid hormone secretion-related genes.Moreover,oxidative stress occurred in PCOS ovaries.But GEN reduced oxidative stress level and elevated antioxidant enzyme activity,which implying that another reason for the therapeutic effect of GEN may be to increase antioxidant capacity of PCOS ovaries.Analysis in mouse ovarian granulosa cells?GCs?showed that GEN restored the secretion of GCs.But the addition of estrogen receptor inhibitor ICI 182780?ICI?inhibited the above effects of GEN,indicating that the protective effect of GEN on GCS secretion was mediated by estrogen receptor?ER?.Further analysis showed that oxidative stress also occurred in DHEA-treated GCs which were inhibited by GEN.However,ICI reversed the effect of GEN,indicating that GEN reduced the oxidative stress level of DHEA-treated GCs through an ER-dependent mechanism as well.In order to further explore the role of antioxidant enzymes and GSH in the effect of GEN,DDC?SOD inhibitor?,3-AT?CAT inhibitor?,MSA?GSH-PX inhibitor?,BSO?GSH synthesis inhibitor?and BCNU?GR inhibitor?were applied in the cells respectively.The results showed that GEN failed to alleviate the oxidative stress in DHEA-treated GCs in the presence of DDC,3-AT,MSA,BSO or BCNU.Moreover,DDC,3-AT,MSA,BSO or BCNU respectively inhibited the improvement of GEN on GCs secretion,implying GSH and antioxidant enzymes?SOD?CAT?GSH-Px and GR?are all crucial in the protection of GEN on DHEA-treated GCs.Besides,our data showed that mitochondrial dysfunction?mitochondrial membrane potential and ATP level were decreased?occurred in DHEA-treated GCs and ICI also significantly inhibited the protective effect of GEN on mitochondrial function,indicating that GEN mediated the mitochondrial dysfunction of GCs in DHEA treatment through ER as well.It is well known that Nrf2 is an important regulator in fighting oxidative stress and is the transcription factor for the ARE-driven genes.Foxo1 was reported to have capability of maintaining oxidative balance by reducing oxidative stress level and elevating antioxidant capability.Our data showed that GEN activated the expression and nuclear translocation of Nrf2.GEN increased the expression of Foxo1 as well.Attentionally,we found that ARE sequences were contained in the promoter regions of Foxo1.pARE-luc,pGL4-Foxo1and pGL4-Foxo1-mutant were applied to investigate if Nrf2 mediated Foxo1 through ARE activation.The results showed that the elevation expression of Foxo1 was abrogated by its mut type,revealing Nrf2activated the expression of Foxo1 to defend oxidative stress via ARE activation.In conclusion,GEN has therapeutic potential for PCOS and ER-Nrf2-Foxo1 was involved in the mechanism.For one thing,GEN ameliorated the hormone secretion and restored the key genes expression in the synthesis of ovary steroid hormones.For another thing,GEN reduced oxidative stress by enhancing antioxidant capacity,improving mitochondrial dysregulation.