| Objectives: Polycystic ovary syndrome(PCOS)is a clinically common reproductive endocrine and metabolic disease,mainly including reproductive dysfunction,excess androgen and metabolic disorders.PCOS is widespread and has a great impact on women’s reproduction and future health,but its etiology and pathogenesis are still unclear.Abnormalities of the hypothalamic-pituitary-ovarian(HPO)axis play an important role in the occurrence and development of PCOS.Elevated basal levels of luteinizing hormone(LH)and increased LH pulse frequency and amplitude have been observed in PCOS patients and PCOS animal models,and are closely related to decreased fertility.Elevated LH can promote the synthesis of androgens in ovarian thecal cells,leading to hyperandrogenism and arrested follicular development.It can damage the synthesis of estrogen and FSH,inhibit the growth of follicle and ovulation.It can also promote the secretion of insulin-like growth factor-1 in the ovary,promote the binding of LH to thecal cells and the synthesis of androgens,and ultimately promote the formation and progression of polycystic ovaries in PCOS.The pulsatile secretion of Gn RH in animal models is consistent with the LH pulse,so the frequency and amplitude of the LH pulse in PCOS patients may be related to the impaired Gn RH pulse in the hypothalamus and the abnormal activation of the HPO axis,resulting in impaired negative feedback of gonadal hormones.The secretion of GnRH is regulated by many upstream neural and endocrine factors,among which kisspeptin is a key upstream regulator of Gn RH pulse formation,which may play a role in the neuroendocrine mechanism of PCOS.Letrozole-induced PCOS mice exhibited metabolic abnormalities such as obesity and insulin resistance(IR),as well as reproductive function with polycystic ovarian changes,increased androgen and LH levels,decreased FSH levels,and increased LH pulse frequency amplitude.damage.An animal model of letrozole-induced PCOS exhibits increased expression of kisspeptin in the hypothalamus.SIRT1,as a molecular signal linking energy metabolism and reproductive metabolism,may play an important role in the regulation of kisspeptin.Studies of obese mice with central drug activation of SIRT1 or overexpression of SIRT1 found that the expression of kisspeptin in the hypothalamus was significantly reduced and LH levels were significantly reduced.However,there is no research on the relationship between SIRT1 and kisspeptin in the hypothalamus in PCOS,an animal model with abnormal metabolism and reproductive function.Due to the heterogeneity of clinical manifestations of PCOS patients,individualized treatment should be performed for PCOS patients according to their needs,mainly for symptomatic treatment,including lifestyle intervention,improvement of metabolism,adjustment of menstruation,and promotion of fertility.Insulin sensitizers such as metformin,thiazolidinediones,and glucagon-like peptide-1 receptor agonists(GLP-1RA)have been shown to be effective in improving IR in PCOS patients.After GLP-1RA treatment,the expression of enzymes related to androgen synthesis in the ovarian tissue of PCOS rats was decreased,the apoptosis of granulosa cells in the ovary was reduced,and the blood glucose fluctuation and average blood pressure were significantly improved.The current research on GLP-1RA in the treatment of PCOS mainly focuses on the improvement of metabolic disorders,while the research on LH and FSH secreted by the pituitary is less.Most studies of GLP-1RA in PCOS animal models focus on the effects on peripheral organs,but the effects on LH pulse and hypothalamus have not been studied.GLP-1R is widely expressed in the brain,and is expressed in Gn RH neurons and kisspeptin neurons in the hypothalamus.Therefore,the purpose of this study 1.To retrospectively observe the effect of GLP-1RA combined with metformin and metformin monotherapy on serum pituitary gonadotropins in overweight and obese patients with PCOS;2.To induce obese PCOS animal model by letrozole and high-fat diet,and analyzed the changes of LH pulse,observed the changes of SIRT1 and kisspeptin expression in the hypothalamus of the obese PCOS animal model,and treated with SIRT1 agonist at the same time to explore whether SIRT1 can regulate LH pulse by affecting the expression of kisspeptin in PCOS;3.We used exenatide to intervene in obese PCOS rats,observed the changes of LH pulses and detected the changes of SIRT1 expression in the hypothalamus.possible mechanism of influence.Methods:Part I: We selected patients who were diagnosed with PCOS and treated with GLP-1RA combined with metformin or single-agent metformin who were admitted to the outpatient department of Endocrinology and Metabolic Diseases,Shengjing Hospital Affiliated to China Medical University from September 2019 to August 2021.Patients in the metformin group were treated with metformin 2.0g/ day,and patients in the combination group were treated with GLP-1RA subcutaneous injection on the basis of oral metformin.GLP-1RA included liraglutide,exenatide and benaglutide.Liraglutide 1.2mg was subcutaneously injected once daily,exenatide 10μg was subcutaneously injected twice daily,and benaglutide 0.2mg was subcutaneously injected three times daily.All patients received standard measurements of height,weight and abdominal circumference at baseline,4 weeks of treatment or 12 weeks of treatment,and the levels of fasting blood glucose,fasting insulin and serum sex hormones were measured to observe the effect of two groups of drug treatment on serum pituitary gonadotropin in overweight and obese PCOS patients.Part II: We intervened 6-week-old SD female rats with letrozole carboxymethyl cellulose(CMC)solution by gavage for 4 weeks to induce an obese PCOS rat model,and the rats in the model group were randomly divided into There are three groups: PCOS group,PCOS-DMSO group,PCOS-SRT 1720 group,n=9.Rats in the PCOS-SRT 1720 group were placed in the third ventricle with an Alzet micro-osmotic pump,and the SIRT1 agonist SRT 1720 5 μL(2 μg SRT 1720 dissolved in 5 μL DMSO)was injected every 24 hours through the micro-osmotic pump.Rats in the PCOS-DMSO group were Alzet micro-osmotic pump was placed in the third ventricle,and 5 μL of vehicle DMSO was injected daily through the micro-osmotic pump as a control.The three groups of PCOS rats were gavaged with letrozole solution and fed with high-fat diet during the 3-week intervention period until the rats were killed.The rats in the control group were gavaged with CMC solution and fed with normal chow for 7 weeks.After completing the above operations,four rats in each experimental group were selected under anesthesia through carotid artery catheterization,and 100 μl of blood was collected every 6 minutes for 2 hours.The LH level was measured at each point,and the LH pulse frequency and amplitude were analyzed in each group.The expressions of kisspeptin and SIRT1 in the hypothalamus were detected by Western blotting(WB),Real-time PCR(rt-PCR)and immunohistochemical methods.Part III: Letrozole and high-fat diet-induced obese PCOS model rats were randomly divided into three groups: PCOS group,metformin group(MET group)and exenatide group(EX group),n=9.During the 3-week intervention period,the rats in the three groups continued to be fed with high-fat diet and administered letrozole solution by gavage every day until the rats were sacrificed.The rats in the metformin group were given 300 mg/kg of metformin saline solution by gavage every day;the rats in the exenatide group were given subcutaneous injection of 10 μg/kg of exenatide saline solution twice a day;the rats in the PCOS group Daily subcutaneous injection of the same volume of normal saline as exenatide.After the intervention,the LH pulse and the expressions of GLP-1R,kisspeptin and SIRT1 in the hypothalamus of rats in each group were detected.The successfully modeled rats were randomly divided into two groups: EX-DMSO group and EX-EX 527 group,n=9.During the intervention period,the rats in the two groups continued to be fed with high-fat diet,intragastric administration of letrozole solution,and subcutaneous injection of exenatide saline injection at 10 μg/kg twice a day until the rats were sacrificed.After modeling,both groups of rats were placed in the third ventricle to install Alzet micro-osmotic pump.The EX-DMSO group was injected with 5 μL of vehicle DMSO daily by micro-osmotic pump as control,and the EX-EX 527 group was injected with micro-osmotic pump every 24 hours.The SIRT1 inhibitor EX 527 5 μg(5 μg EX 527 dissolved in 5 μL DMSO)was injected for a total of 3 weeks of intervention.After intervention,the expressions of LH pulse,kisspeptin and SIRT1 in hypothalamus of rats in each group were detected.Results:Part I: 1.After 4 weeks and 12 weeks of treatment,the body weight,BMI and abdominal circumference of the two groups decreased(P<0.05),and the total testosterone level in the combined group decreased(P<0.05).After 12 weeks of treatment,the FINS and HOMA-IR of the two groups were significantly decreased(P<0.05),and the FPG of the combined group was decreased(P<0.05).2.After 4 weeks of treatment,LH and FSH in the combined group were decreased compared with those before treatment(P<0.05).There was no significant difference in LH,FSH and LH/FSH between the two groups after treatment(P>0.05).3.After 12 weeks of treatment,LH and LH/FSH in the combined group decreased(P<0.05).The LH and LH/FSH in the combined group were lower than those in the metformin group after treatment(P<0.05).Part II: 1.The appearance of the obese PCOS rats was obviously larger than that of the control rats,and the fur was dull.The body weight,serum testosterone and HOMA-IR of the obese PCOS model rats were significantly higher than those of the control group.Vaginal exfoliated cells had been in the estrous phase and the ovary showed polycystic changes.The serum kisspeptin level in the PCOS group increased(P<0.05).2.Compared with the control group,the rats in the PCOS group showed high frequency and amplitude LH pulses.After injection of DMSO into the third ventricle,the LH pulse frequency and amplitude did not change significantly compared with the PCOS group.After the SIRT1 agonist SRT 1720 was injected into the third ventricle,the LH pulse frequency and amplitude were significantly lower than those in the PCOS group(P<0.05).3.The expression of kisspeptin protein and m RNA in the hypothalamus of the PCOS group was significantly higher than that of the control group,and the expression of SIRT1 protein and m RNA in the hypothalamus was significantly lower than that of the control group(P<0.05).After intraventricular injection of SRT 1720,the expression of kisspeptin protein and m RNA in the hypothalamus was lower than that in the PCOS group and the expression of SIRT1 protein and m RNA in the hypothalamus was higher than that in the PCOS group(P < 0.05).Part III: 1.After exenatide and metformin treatment,the body weight of the two groups increased,but the increase rate was significantly lower than that of the PCOS group(P<0.05).2.The serum kisspeptin level and LH/FSH level of rats in EX group decreased(P<0.05),while the FSH level increased(P<0.05).3.There was no significant difference in GLP-1 concentration in cerebrospinal fluid among three groups(P>0.05).The expression of GLP-1R protein in EX group was significantly higher than that in PCOS and MET group(P<0.05).4.The LH pulse amplitude and average LH level of the rats in the EX group were lower than those in the PCOS and MET group(P<0.05);the pulse frequency in the EX group also decreased,but there was no statistical difference among the three groups(P>0.05).5.The expression of kisspeptin protein and m RNA in hypothalamus of EX group was lower than that of PCOS and MET group,and the expression of SIRT1 protein and m RNA in hypothalamus was higher than that of PCOS and MET group(P<0.05).There was no statistical difference between MET and PCOS group(P>0.05).6.In the EX treatment-intracerebroventricular cannulation PCOS model,the amplitude,frequency and average LH value of LH pulses in the EX 527 group were increased compared with those in the DMSO group(P<0.05).7.After intracerebroventricular injection of EX 527,the expression of kisspeptin protein and m RNA in the hypothalamus increased,and the expression of SIRT1 protein and m RNA in the hypothalamus decreased(P<0.05).Conclusion: 1.GLP-1RA combined with metformin may be more effective than metformin monotherapy in reducing LH levels and improving LH/FSH in overweight or obese PCOS patients.2.The expression of SIRT1 in the hypothalamus of obese PCOS rats induced by letrozole and high-fat diet decreased.SIRT1 in the hypothalamus may regulate the LH pulse of obese PCOS rats by affecting the expression of kisspeptin in the hypothalamus.3.GLP-1RA exenatide may improve the abnormal LH pulse secretion in obese PCOS rats by up-regulating the expression of SIRT1 in the hypothalamus,thereby inhibiting the expression of kisspeptin in the hypothalamus. |
PDF Full Text Request