| Part 1 Expression and Significance of Apelin 13 in Serum of PCOS PatientsBackground and objectivePolycystic ovary syndrome(PCOS)is considered to be one of the most common metabolic syndrome among women.Obesity,as one of the most important risk factors for IR,can aggravate the degree of IR in the body of PCOS patients.About 50%-70%of PCOS patients are reported to be associated with obesity.Obesity and IR related fat factors have a certain effect on the follicular development of PCOS patients.At present,the research on the relationship between adipokines and PCOS is mostly focused on the effect of abnormal adipose factor expression or protein dysfunction on glucose and lipid metabolism,but whether adipokines affect follicular development in PCOS patients is very limited.Apelin is the natural ligand of the isolated G protein coupled receptor APJ.It is a fat factor,widely exists in the body,and plays a role in obesity related metabolic diseases such as T2DM,hypertension and PCOS.The research on the relationship between Apelin and PCOS is still in the exploratory stage.There are still differences in its clinical findings.However,further studies are needed to confirm whether Apelin participates in the occurrence of PCOS.This chapter mainly discusses the expression and significance of Apelin 13 in serum of PCOS patients.Methods98 PCOS patients were collected from August 2014 to July 2015 in the gynecologic outpatient department of our hospital.In the same period,45 cases of Health(menstrual law)women in the medical check-up outpatient department of our hospital were collected as the control group.PCOS patients according to whether obesity(body mass index BMI = 25kg/m2)were divided into 56 cases of obesity group and non obesity group 42 cases.The level of serum Apelinl3 and the difference of sex hormone,blood sugar and blood lipid were compared between the PCOS group and the control group.At the same time,the level of Apelinl3 and the difference of sex hormone,blood sugar and blood lipid were compared between the obese PCOS group and the obese PCOS group.The correlation between serum Apelinl3 level and clinical index in PCOS patients was analyzed.ResultsThe levels of BMI,LH,T,Ins,HOMA-IR and TG in the PCOS group were significantly higher than those in the control group(P<0.05),and the levels of BMI,WHR,Ins,BMI and gamma in the obese group were significantly higher than those in the non obese group.In group PCOS,the concentration of serum Apelinl3 was significantly higher than the control group(P<0.05),PCOS patients with obesity group serum Apelinl3 concentration was significantly higher than that in the non obese group(P<0.05),correlation analysis showed that the PCOS levels of serum Apelinl3 and BMI,WHR,Ins,FBG and HOMA-IR were positively correlated,negatively correlated with HDL.ConclusionCompared with the healthy women of childbearing age,the serum Apelin13 of PCOS patients was highly expressed,and the obese PCOS patients were more expressed.The serum Apelin 13 concentration in PCOS patients is positively correlated with BMI,WHR,Ins,HOMA-IR and FBG,and has a negative correlation with HDL.It indicates that Apelin13 plays an important role in the pathogenesis and clinical manifestations of PCOS.Part 2 Expression of Apelinl3/APJ System in the Ovary of PCOS RatBackground and objectiveAdipose tissue is an important endocrine and paracrine organ,Apelin is a new adipokine discovered in recent years.It is involved in the regulation of the physiological and metabolic cycle control process,it has no characteristics of adipocytokines in general,is the main regulatory role in the cardiovascular system and involved in homeostasis.PCOS is the main manifestation of metabolic disorders,so Apelin plays an important role in the development and development of PCOS.At present,the mechanism of abnormal development of PCOS follicles has not been clearly explained.It has been found that its mechanism may be related to local endocrine regulation of multiple cytokines.Apelin13 is an important member of Apelin,it as a cardiovascular active peptide,can promote the proliferation of vascular smooth muscle cells,endothelial cell proliferation and migration and other biological effects,it is also a new fat factor,plays an important role in the occurrence of IR is related to obesity,T2DM,hypertension and metabolic syndrome.At present,the conclusion of the study of abnormal follicle development in PCOS patients is not the same,and its mechanism is still in the exploratory stage.Is there a high expression of Apelinl3 in the serum of PCOS patients?What is the mechanism for the development of follicle?Is there a high expression of Apelinl3 and its receptor APJ in the ovaries of PCOS patients?Therefore,in this chapter,we established the PCOS rat model to detect the expression of Apelin13 and its receptor APJmRNA and protein in rat ovary,so as to explore the role of Apelin13/APJ in the occurrence of PCOS.Methods40 SD female rats of 21 day old clean grade were divided into the observation group and the control group according to the random number table method,each 20.In the observation group,the PCOS model was established by gavage of letrozole,and the control group was treated with any treatment.The ovarian morphology and body weight,ovarian weight,ovarian weight/weight ratio,sex hormones,glucose tolerance and estrous cycle in two groups were compared.The expression of Apelinl 3 and APJmRNA and protein in ovarian tissue of rats were monitored.ResultsHE staining showed that the ovarian structure of PCOS rats was disorder,there were multiple cystic dilated follicles under the capsule,and there were no oocytes in some follicles,and corpus luteum was rare.The granular cell layer was reduced,usually 2-3 layers.The ovaries of normal rats have follicles at different stages of development.There are many corpus luteum,the number of granular cells can reach 8-9 layers,and the arrangement is close and neat,and the shape is complete.The weight ratio of the observation group,the weight of the ovary and the weight/weight of the ovary were significantly higher than that of the control group(P<0.05).Vaginal smear and methylene blue staining showed that the estrus cycle and estrous cycle were 4-5 days in the control group,while the estrous cycle changed in the PCOS group after fourteenth days.Vaginal smears showed almost all white cells and were in estrus,indicating anovulation.The levels of LH and T in the observation group were significantly higher than those in the control group,E2 was significantly lower than that in the control group(P<0.05),and the 60min and 120min in the observation group were significantly higher than those in the control group(P<0.001).The ISI of the observation group was significantly lower than that of the control group,and the HOMA-IR was significantly higher than that of the control group(P<0.001).Apelin13 and its receptor APJ were expressed in the follicle intima,granulosa cells and ovarian stroma.The expression of Apelinl3 and APJ protein in the ovary of the observation group was significantly higher than that of the control group(P<0.05),and the expression of Apelin13 and its receptor APJ mRNA in PCOS rats was significantly higher than that in the control group(P<0.001).ConclusionLetrozole gavage induced PCOS rat model has the characteristics of ovarian morphology,reproductive endocrine disorders and glucose metabolism disorders,which is similar to the typical characteristics of PCOS patients.Both PCOS rats and normal rats showed Apelinl3 and APJ expression in the follicle intima and granulosa cells.Compared with normal rat ovarian tissue,the expression of Apelin13/APJ gene and protein was significantly up-regulated in PCOS rat ovary,and the abnormal up-regulated expression may play a role in the abnormal proliferation of PCOS follicles.Part 3 The Regulation of Apelin13 on the Signaling Pathway of Rat Ovarian DevelopmentBackground and objectiveIn PCOS ovarian tissue,the phosphorylation of insulin receptor weakens and the insulin signaling pathway in granulosa cells is impaired.The signaling pathway of phosphatidylinositol 3-kinase(P13K)is closely related to the intracellular sugar metabolism,and its downstream molecules are mainly serine threonine protein kinase(AKT).Reduction of phosphorylation of P13K/AKT signaling pathway is an important feature of IR in PCOS patients.It is an intracellular insulin pathway and plays an important role in cell proliferation,differentiation and apoptosis.Transcription factor FoxO1 is an important member of Fox family.It plays a role in cell apoptosis,cell cycle and insulin signaling pathway,and is regulated by P13K/AKT signaling pathway.FoxO1 plays a role in the proliferation of PCOS ovarian granulosa cells.Insulin can promote the proliferation of somatic granulosa cells and inhibit the expression of FoxO1 mRNA in granulosa cells.As a fat factor,Apelinl3 plays an important role in obesity and IR related metabolic diseases.What is the mechanism of the action of Apelin13 on the PCOS rat model and whether it is related to the P13K/AKT signaling pathway.Therefore,this chapter establishes the PCOS model,and Apelin13 and APJ groups were given antagonist intervention respectively,observe the change of PCOS in rat estrous cycle,ovarian morphology,sex hormone and glucose metabolism,to explore the regulatory effect of Apelinl3 on P13K/AKT signaling pathway in ovarian development in rats.Methods44 SD female rats were randomly divided into 14 rats in group PCOS and 30 in the normal group.24 rats in the normal group were randomly divided into Apenlin13 group,Apenlin13+F13A group and normal control group,with 8 rats in each group.In addition,8 groups of PCOS were taken as the PCOS control group.Observe the estrous cycle and the physical and sexual hormone and ovarian weight,fasting blood glucose,insulin,insulin sensitivity and IR.RT-PCR was used to detect PI3K,AKT and FoxOl mRNA in ovarian tissue.WB was used to detect the protein expression of AKT,FoxO1,p-AKT1 and p-FoxO1.ResultsThe estrous cycle of the PCOS control group was in the estrus interval.In group Apenlin13,the estrus cycle began to lengthen 10 days after giving Apenlinl3 intervention.After 21 days of intervention,estrus interval was irregular,most of them were in estrus.21 days after the intervention of Apenlinl3 and F13A in group Apenlin13+F13A,some of the estrous cycles were prolonged and occasionally in the estrous interval.PCOS control group,Apenlinl3 group,Apenlin13+F13A group of rats body weight and ovary weight,serum LH,FSH and T,blood glucose,fasting insulin and HOMA-IR were significantly higher than those in normal control group(P<0.05),ISI decreased significantly;PCOS control group,Apenlin13 group and ovarian weight in two groups of rats weight,serum LH,FSH and T blood glucose,fasting insulin,and HOMA-IR had no significant difference(P>0.05),body weight and ovary of rats in the Apenlin13+F13A group,LH,FSH,ISI weight was significantly lower than that of PCOS group and Apenlinl3 group(P<0.05).The ovarian granulosa cells in the normal control group,the PCOS control group,the Apenlin13 group and the Apenlin13+F13A group had FoxO1 expression.The normal control group was mainly expressed in granulosa cell cytoplasm,PCOS control group and Apenlin13 group were mainly expressed in granulosa cell nucleus,and Apenlin13+F13A group was expressed in granular cytoplasm and nucleus.The expression of AKT,FoxO1,p-AKT and p-FoxO1 protein in all 4 groups of ovarian tissues was expressed.The AKT and FoxO1 protein levels in Apenlinl3 group and PCOS control group were all higher than those in normal control group,while those in Apenlin13+F13A group were lower than those in Apenlinl3 group and PCOS control group,but there was no significant difference in AKT expression among all groups(P>0.05).The expression of p-AKT in the ovarian tissue of group PCOS and group Apenlin13 was significantly higher than that in the normal control group(P<0.05).The expression of p-AKT in the ovarian tissue of the Apenlinl3+F13A group was significantly lower than that in the PCOS group and the Apenlin13 group(P<0.05).The expression of p-ATKp-FoxO1 protein in PCOS control group and Apenlin13 group was significantly higher than that in the normal control group(P<0.05),while that in Apenlinl3+F13A group was significantly lower than that in PCOS control group and Apenlinl3 group(P<0.05).ConclusionThe high expression of Apelinl3 may activate the P13K/AKT signaling pathway,inhibit the expression of apoptosis gene expression,promote the proliferation of rat ovarian granulosa cell apoptosis;the activation of P13K/AKT signaling pathway may be appropriate to increase the level of insulin,caused by IR;also induce fatty acid synthesis increased weight,which may be one of the mechanisms of PCOS. |
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