| Objective: Polycystic ovary syndrome(PCOS)is a common gynecological endocrine and metabolic disorder among adolescents and childbearing women,which affects the fertility of patients seriously and will cuase long-term complications.In vitro fertilization-embryo transfer showed that ovulation induced by Diane-35 plus metformin could promote better ovarian follicle developmentand ovulation,which will improve the clinical outcome of patients with PCOS.However the mechanism of PCOS follicle development regulated by combination therapy is still not clear.In this study,we constructed a PCOS-IR rat model cured by Diane-35 plus metformin to analyze the changes of reproductive hormones,follicular developmental morphology and energy product metabolism to explore the mechanism of follicular development preliminarily.We discussed the molecular mechanism of ovulation in PCOS cured by combination therapy in order to provide a better clinical evidence for the treatment of PCOS patients with Diane-35 plus metformin.Methods: Female SD rats aged 5 weeks were randomly divided into control group(10 rats)and model group(30 rats),total 40 rats.Rats in the model group were fed for 30 consecutive days by using trazole with high-fat diet.Rats in the control group were given 1% CMC solution daily and fed with normal diet.After models were successfully constructed,these rats were divided into PCOS group(model group)and Diane-35 plus metformin group(combination therapy group).Part I: vaginal smear was used to monitor the changes of estrous cycle in SD rats in each group during modeling and treatment.Reproductive hormones such as fasting insulin,follicle stimulating hormone,luteinizing hormone,testosterone were respectively measured through radio immunoassay method.Ovary paraffin sections stained with hematoxylin-eosin to observe the changes of ovarian tissue structure.The proliferating cell nuclear antigenexpressions were observed through immunohistochemical SP method.The apoptosis of ovarian granulosa cells were observed by using immunohistochemical method.Quantitative real-time PCR was used to analyze the content of PCNA and the expressions of BAX,BCL-2 and CASPASE-3 in apoptosis-related genes.Part II: The contents of pyruvate and lactic acid in the metabolites of ovarian glycolysis were determined by targeted metabolomics based on MRM method.Quantitative analyzed the expressions of GLUT1,MCT2/4,LDH,HK,PFK and PK through qRT-PCR.Results:(1)The estrus cycle disorder occurred when the PCOS model successfully constructed.The levels of reproductive hormones in PCOS rats serum showed LH,T and IR increased significantly.The ovaries showed polycystic changes in PCOS rats and the number of granulosa cell layers decreased or even disappeared.A small amount of corpus luteum were occasionally observed.However,the estrus cycle recovered and follicles at all levels,layers of granulosa cell and a large amount of corpus luteum could be observed after combination therapy.The reproductive hormones were not different from the control group.It suggests that ovary can resume normal ovulation after combination therapy.(2)Immunohistochemistry and qRT-PCR discovered that the proliferation of ovarian granulosa cells was inhibited significantly and granulosa cells apoptosis were obvious in PCOS rats.After combination therapy,the proliferation of ovarian granulosa cells were significantly increased,the expression of ovarian pro-apoptotic factors(BAX,CASPASE-3)was inhibited,and the expression of anti-apoptotic gene(BCl-2)was significantly increased.It reveals combination therapy can promote follicular development.(3)Metabonomics and qRT-PCR discovered that the energy metabolism pathway of PCOS rat ovaries was disordered.The pyruvate increased and the lactic acid decreased significantly.The expression of glycolysis-related rate-limiting enzymes GLUT1 increased significantly while HK decreased.At the same time,the expression of MCT4,LDH,PFK and PKM decreased significantly.After combination therapy,the lactate increased significantly and glycolysis-related rate-limiting enzymes were up-regulated significantly.It reveals that the energy supply of folliculogenesis is mainly through glycolysis pathway.Conclusions: Diane-35 plus metformin can significantly improve the endocrine function of PCOS rats at the ovarian level.It promotes follicular granulosa cell proliferation and inhibits ovarian cell apoptosis.Also it can restore normal ovulation of follicles.Its mechanism is increasing the expression of glycolysis-related rate-limiting enzymes and the level of ovarian lactic acid which is the energy to maintain follicular development. |
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