Molecular Mechanism Of Crocus Acid And Tanshinone IIA In Improving Polycystic Ovary Syndrome

BACKGROUND:Polycystic ovary syndrome(PCOS)is one of the most common endocrine disorders in reproductive women with a prevalence of 6-21%,according to the current diagnostic criteria and population surveys,PCOS account for about 80%of cases of anovulatory infertility.It is mainly characterized by anovulation,hyperandrogenism and polycystic ovarian morphology with the major clinical manifestations including menstrual disorder,infertility,obesity,dyslipidemia,and hirsutism.However,the treatment regimens are confused,and long-term complications are also lack of reasonable prevention and treatment measures.Therefore,it is imperative to find a safe and effective drug for the treatment of PCOS.Saffron,belonging to an Iris family plants,is a traditional Chinese medicine for the therapy of primary dysmenorrhea,premenstrual tension and other gynecological diseases.Its primary component,Crocetin,is a polyunsaturated conjugated acid,it has a variety of curative activities including alleviating insulin resistance,anti-oxidative stress,anti-inflammation and so on.At present,a number of studies have shown that insulin resistance and abnormal glucose metabolism are one of the pathogenesis of PCOS.Local insulin resistance in the ovaries of PCOS patients leads to ovarian dysfunction and endocrine abnormalities.In addition,as another causative agent of PCOS,local inflammation can also affect ovarian follicle growth and development in PCOS,leading to a decline in ovarian function.Salvia miltiorrhiza is dried roots and rhizomes of Lamiaceae,it is reported to treating irregular menstruation and dysmenorrhea.Salvia miltiorrhiza has been used to treat acne caused by hyperandrogen in the body and improve the fertility of patients with PCOS.Tanshinone ⅡA is one of the main component of Salvia miltiorrhiza.Due to its quinoid structure,it is easily oxidized and reduced,so it has function of antiinflammation and anti-oxidation.It also has functions of immune regulation,treatment of cardiovascular disease and neuroprotection.Recent studies have found that Tanshinone ⅡA as a phytoestrogen,it is expected to treat cardiovascular disease by hormone replacement therapy because it is similar to the chemical structure of 17βestradiol,which can bind to estrogen receptor,through the extracellular signalregulatory kinase pathway,Tanshinone ⅡA not only inhibit the inflammation and oxidative stress in the body but also improve the state of cardiovascular disease in postmenopausal women.Thus,we presume that Crocetin and tanshinone ⅡA have potential therapeutic effects on PCOS and explore its possible mechanism.Part Ⅰ The mechanism of Crocetin treatment of DHT-induced PCOS miceOBJECTIVE:To observe the therapeutic effect of Crocetin on polycystic ovary syndrome(PCOS)induced by dihydrotestosterone(DHT)in mice and explore its possible mechanism.METHODS:Pregnant dams were subcutaneously(s.c.)injected with DHT from gestation day(GD)16 to GD18,and female offspring were analyzed as the prenatally DHT-treated mice models of PCOS.50%of eight weeks old female offspring in PCOS group were treated with Crocetin daily for 4 weeks by oral gavage,which were analyzed as Crocetin treatment group,the another part were analyzed as model group.The body weight and estrous cycle were examined during the treatment.Sixteen weeks later,the mice were sacrificed after getting orbital blood and the hypothalami and ovaries were obtained.HE staining was used to observe the pathological change of ovarian tissue.The levels of serum estradiol(E2),testosterone(T),progesterone(P4),luteinizing hormone(LH)and follicle-stimulating hormone(FSH)were quantified using ELISA kit.Immunohistochemical,western blot and quantitative Real-time PCR methods were used to detect the expression of kisspeptin in anteroventral periventricular(AVPV),arcuate nucleus(ARC)and the expression of GnRH in the preoptic area(POA).Seven weeks old ovariectomized females were subcutaneously(s.c.)injected E2 or its solvent(corn oil)and treated with crocetin by oral gavage for 4 consecutive weeks.Which were analyzed as control group,OVX group,OVX+Crocetin group,OVX+E2 group and OVX+E2+Crocetin group.RESULTS:1)Compared with the control group,the ratio of ovary to body weight of in PCOS-mice was increased by 22.56%(P<0.05)and PCOS-mice exhibited anobvious prolongation of estrous cycle.2)Multiple enlarged follicles were observed in the ovaries,the number of atretic cyst-like follicles was increased by 138.74%(P<0.05),whereas the numbers of large antral follicles,preovulatory follicles and corpus luteum were reduced by 38.80%,56.35%(P<0.01),63.77%(P<0.05)respectively.3)The levels of E2,P4 and FSH in serum were reduced by 40.99%,56.91%(P<0.05),38.80%(P<0.01)respectively,while the levels of T and LH were increased by 43.23%(P<0.05),148.46%respectively;4)The expression of hypothalamic kisspeptin in AVPV and GnRH in POA was reduced,whereas the expression of kisspeptin in ARC was increased.However,the DHT-induced malfunction could be reversed by Crocetin partly;5)Compared with the control group,GnRH and AVPV-Kiss1 mRNA in OVX+Crocetin+E2 group mice increased by 83.55%(P<0.05)and 155.55%(P<0.01),respectively.CONCLUSION:Crocetin can improved the PCOS in mice via increasing AVPVkisspeptin and reducing ARC-kisspeptin expression.Part Ⅱ The mechanism of tanshinone ⅡA treatment of E2induced PCOS miceOBJECTIVE:To observe the therapeutic effect of tanshinone ⅡA on Estradiol(E2)induced polycystic ovarian syndrome(PCOS)mice and to explore its mechanism.MEDTHODS:PCOS model of female offspring was induced by subcutaneous injection of E2 into female newborn mice.50%of eight-week-old female offsprings in PCOS group were treated with Tanshinone ⅡA for 4 weeks by oral gavage,which were analyzed as Tanshinone ⅡA treatment group,the another part were analyzed as model group.The body weight and estrus cycle were measured during the administration period.After about 16 weeks of age,the mice were killed by blood sampling and the hypothalamus and ovaries were removed.HE staining was used to observe the pathological changes of ovarian tissue;the level of Estradiol(E2),testosterone(T),progesterone(P4),Follicle-stimulating hormone(FSH)and luteinizing hormone(LH)in serum were detected by ELISA kit.The expression of FSHR and Aromatase in the ovary were measured by qPCR.After granule cells of control group,model group and treatment group mice were treated with FSH(100 IU/L)for 10 min.,the intracellular cAMP concentration was measured by cAMP ELISA kit.The FSHR protein level in granulosa cells was detected by Western Blot and FSHR\Aromatase mRNA levels was measured by qPCR.RESULTS:1)Compared with the control group,the ratio of ovary to body weight in PCOS mice was increased by 31.54%(P<0.05),and DHT-treated mice exhibited an obvious prolongation of diestrus;2)Large vacuoles appeared in the ovary,and the numbers of atretic cyst-like follicles increased by 159.86%(P<0.01),The number of large antral follicles,preovulatory follicles,and corpus luteum decreased by 65.67%,58.80%,and 89.59%(P<0.01),respectively.After administration of Tanshinone ⅡA,large antral follicles,preovulatory follicles,and corpus luteum of E2-treated mice recovered to normal levels of 80.05%,61.32%and 56.32%(P<0.05),respectively,and the number of atretic cyst-like follicles decreased by 56.53%(P<0.01);3)The levels of E2 and P4 decreased by 44.74%and 23.41%(P<0.05),respectively,while there was no significant change in T,LH,and FSH levels;4)The expression levels of FSHR and aromatase in ovaries decreased by 39.18%and 41.62%(P<0.05),respectively.After FSH stimulated granulosa cells of mouse ovary in vitro.The cAMP concentration in mice in the model group was 44.86%(P<0.05)of control group mice.The cAMP concentration in ovarian granulosa cells of Tanshinone ⅡA treated mice is 91.66%(P<0.05)of the control group mice.The FSHR and aromatase mRNA levels in the granulosa cells of the model group decreased to 40.18%(P<0.01)and 42.62%(P<0.05);while those in the treatment group returned to 96.31%and 102.24%(P<0.05),respectively.CONCLUSION:Tanshinone ⅡA can improved the PCOS in mice by down-regulating the expression of FSHR and aromatase in the ovary.In summary,the major contribution of the present study are listed as follows:1.Crocetin can improve the pathological symptoms of mice with polycystic ovary syndrome,such as polycystic ovary and hormone secretion disorders,while Crocetin increases the activity of the HPO axis,and enhance Kisspeptin expression of Kisspeptin neurons in AVPV,inhibit Kisspeptin expression of Kisspeptin neurons in ARC.2.Tanshinone ⅡA attenuates estradiol-induced polycystic ovarian syndrome in mice via revising FSHR and Aromatase expression in ovary,it also restores the ovarian fertility of PCOS mice and regulates the secretion of sex hormones.