Expression Of Apelin In Polycystic Ovary Syndrome And Mechanism Of Action In Sd Rat Ovarian Granular Cells

Polycystic ovary syndrome(PCOS) is the most commonendocrinopathy associated with metabolism dysfunction in women ofreproductive age,with the incidence4%-10%,which is a heterogeneousdisease characterized by oligo-ovulation or chronic anovulation,features ofhyperandrogenism and/or biochemical hyperandrogenism,insulin resistanceand polycystic ovarian morphology on ultrasonograph. Compared with thehealthy women, PCOS women with menstrual disorder, infertility,hirsutism and acne have a greater risk for cardiovascular disease, diabetesmellitus, tumorous.Women with PCOS has a serious negative impact onhealth-related quality of life, affecting both physical and psychologicalwell-being. Follicular developmental anomaly is the main cause ofoligomenorrhea or amenorrhea, infertility in reproductive age women withPCOS. The study about follicular developmental failure in PCOS can help usto understand the pathogenesis of PCOS. The numbers of primordialfollicles in ovaries of patients with PCOS and healthy women were similar,while the number of growing and atresia follicles were increased,and the number of over-recruitment follicles recruited into the growing phase fail tomature even result in the appearance of polycystic ovaries, ovulationfailure. Some scholar propose that PCOS is a primarily follicle disease. Inspite of the mechanism of abnormal folliculogenes is unclear, manyresearches showed that alteration of many factors may directly or indirectlyregulation the microenvironment of folliculogenes through endocrine andlocal paracrine/autocrine actions.In anovulatory women with PCOS and obesity undergoing a lifestyleprogram, resume ovulation women lose more body weight and abdominalfat than remain anovulatory women. In addition, this study shows thatconsistent loss of intra-abdominal fat is associated with resumption ofovulation. It is indicated that adipokines correlated with obesity or insulinresistance play important roles in the development of follicles. Recently,some domestic and international researches focuses on various adipokinesgene expression and/or protein function defects in insulin resistance possiblemechanisms of patients with PCOS, while the relationship betweenadipokines and follicular microenvironment in patients with PCOS has notbeen reported yet. Apelin is an endogenous ligand of the orphanG-protein-coupled receptor APJ, which is consisting of77amino acidresidues and exists in multiple molecular forms. Apelin signaling pathwayplays a role in the central and peripheral regulation of the cardiovascularsystem, such as blood pressure, in water and food intake, and possibly in immune function. In2005, studies detected the apelin can secrete by ratand human hypodermic adipose cell. Many studies explained thepathogenesis between apelin and insulin resistance correlated disease,such as2type diabetes mellitus,primarily hypertension,PCOS. Wangstudied that insulin, HOMA-IR and apelin levels in women with PCOS,compared with healthy women；were significantly increased；apelin levelspositively correlated with BMI,WHR, insulin and HOMA-IR, and BMIwas the independent influencing factor for apelin levels changes. G renreported that the apelin level in women with PCOS were significantly highcompared with healthy group, while failed to found the relationshipbetween apelin levels and biochemical indicator in patient withPCOS.Increased apelin levels have been reported in women with PCOS,but not all previous studies.Chang reported that apelin levels were lowerthan that of healthy women and apelin was positively correlated withapolipoprotein A levels in patients with PCOS. All studies indicated that thechanges of apelin levels are associated with obesity and IR,and contribute,in part to pathogenesis of PCOS, while there is no study about themechanism of apelin in pathogenesis for women with PCOS. Our study wasdesigned to explore the apelin level in Chinese patients with PCOS and therole of apelin in lipometabolism,glycometabolism and insulin resistance；to study the apelin express in the rat ovary with PCOS and control group；to future exprole the effect apelin on the cultured rat granulose cells. The study including four chapters, as following:Chapter1Serum apelin levels in women with Polycystic OvarySyndrome[Abstract] Objective This study is designed in order to investigateserum apelin levels between PCOS patients and healthy subjects. MethodsThe basal levels of fasting blood glucose(FBG), insulin (Ins),high-density lipoprotein(HDL), low density lipoprotein(LDL),triglycerides(TG), total cholesterol(TC), follicle-stimulating hormone(FSH), luteinizing hormone (LH), testosterone (T),progestin(P),prolactin(PRL),estrogen(E2) and apelin were measured on fifty women withPCOS, and20healthy women. Subjects with PCOS were further dividedinto two subgroups according to body mass index (BMI) as BMI≥25kg/m2and BMI<25kg/m2.Results The apelin level of the PCOS groupwith[501.40(98.66)pg/ml]was higher than the controls[416.77(61.07)pg/ml](P<0.001).Both the BMI≥25kg/m2and BMI<25kg/m2women with PCOShad higher apelin levels,compared to the controls(P<0.001).Compared withthe BMI≥25kg/m2patients, the BMI<25kg/m2women with PCOS hadsignificantly lower apelin levels(P<0.05).A positive correlation was foundbetween apelin level and HOMA-IR,BMI and waist: hip ratio(WHR),insulin,glucose in the patients with PCOS((r=0.43,P=0.007；r=0.38,P=0.02；r=0.456,P=0.003；r=0.977,P=0.000；r=0.335,P=0.017),anda negative correlation was found between apelin and HDL(r=-0.456, P=0.005).Logistic analysis showed that apelin levels was significant in thedevelopment of PCOS(P<0.05).Conclusions Our study indicates that apelinin women with PCOS exhibit an increase along with obesity and IR. Furtherstudies are required to clarify the etiology and effects of apelin in womenwith PCOS. Chapter2Expression of apelin/APJ in Polycystic Ovary Syndromeof rats[Abstract] Objective To explore the role of apelin/APJ in thepathogenesis of PCOS.Methods Female SD rats aged21day were dividedinto two groups in pairs. The rats of experimental group were injected S.C.each day with dehydroeplandrosterone(DHEA) and the rats of control groupinjected with oil；Ovarian weight and sex hormone levels in rat blood of bothgroups were determined；Ovarian change was observed by HE staining. Theexpression of apelin and APJ were determined by immunohistochemical-staining in the ovaries； The levels of apelin and APJ mRNA and protein inovary tissue were measured by qRT-PCR and W-B technique. Results (1)The ovarian and body weight of model group were both lower than that ofthe control group(P>0.05),however, the ovarian weight/body weight ofmodel group was higher than that of control group(P<0.05)(.2)Comparedwith the control group the serum of LH and T in model group experiment group were significantly increased (P<0.05).Although there was nosignificant difference of FSH,P and E2between the two groups(P>0.05).(3)There were some early development of little follicles and theabundance of subcaplular ovarian cyst in ovary of model group, which wastypical polycystic ovary morphous,while some different development offollicles and corpus luteum were detected in ovary of control group,without subcaplular ovariancyst.(4)Immunostai-ning of apelin and APJ inthe granulosa cells and theca cells of PCOS rat's ovaries were obviouslystronger than that of normal control (P<0.05, respectively).The levels ofapelin and APJ mRNA and protein in ovary of PCOS were obviously higherthan that of normal control (P<0.05, respectively).Conclusion Thedisruption of apelin/APJ system may be involved in the pathogenesis ofPCOS. Chapter3Effects of apelin on proliferation and apoptosis of theovarian granulosa cells in SD rat[Abstract] Objective To observe effect of apelin on rat ovariangranulosa cells proliferation and apoptosis in vitro. Method primary cultureof SD rat ovarian granulosa cells were cultured with DMEM/F12serum-freemedium without blood serum in control group, low concentration (apelin10-8mol/l) group, high concentration (apelin10-6mol/l) group, and tested by MTT assay and flow cytometry (Annexin/PI)method for the detection ofGC proliferation and apoptosis at12h,24h,48h.Results Apelin10-8mol/lcould significantly promote the proliferation of the GC at48h(P<0.05)andsignificantly inhibit apoptosis of the GC at48h(P<0.05).Conclusion Apelincould promote proliferation and suppress apoptosis of GC in vitro. Chapter4Mechanism of apelin promote proliferation and supressapoptosis in granulosa cells[Abstract] Objective T0investigate mechanism of apelin promoteproliferation and suppress apoptosis in GC. Method (1) The expression ofAkt and pAkt inGC cultured with apelin10-8mol/l at0,5,15,30,60minwas detected by W-B.(2)RNA interference was used to down-regulate theexpression of APJ in rat GC；Rat GC was divided into4groups:A.apelin10-8mol/l group,B.apelin10-8mol/l+APJ-siRNA.C.apelin10-8mol/l+PI-3k inhi--bitor LY294002group, D.apelin10-8mol/l+Akt inhibitor HIM0, theapoptosis rate of GC cultured after48h was detected by MTT；the proteinlevels of Akt,pAkt,Bad,Bax,Foxo3a and Bc1-2were detected by W-B.Results (1) Apelin activated the phosphorylation of Akt kinase at5min,andthe peak at15min,however,the phosphorylation of Akt were blocked byAPJ-siRNA,inhibitor LY294002and HIMO.(2)The effect of apelin10-8mol/l promote proliferation were blocked by APJ-siRNA, inhibitor LY294002and HIMO in GC (P<0.05,respectively).(3)Apelin10-8mol/lincreased Bcl-2protein, decreased bad,foxo3a and bax protein in GC,while,the effect were blocked by APJ-siRNA,inhibitor LY294002andHIMO(P<0.05,respectively).Conclusion These results indicate that theeffect of apelin promote proliferation and suppress apoptosis in GC ismediated via the APJ/PI-3K/Akt signaling pathway.