| Research Background Polycystic ovary syndrome(PCOS)represents one of the most common endocrine and metabolic disorders occurred in women of reproductive age.PCOS is characterized by anovulation,rare ovulation,excessive androgen,or polycystic ovary morphology.PCOS is closely related to obesity.At present,the key pathophysiological mechanisms that mediate PCOS have not been elucidated,and there are currently no effective drugs for PCOS.Adipocyte fatty acid binding protein(A-FABP),the member in the fatty acid binding protein family,is a metabolic regulatory factor mainly secreted by adipocytes and macrophages.AFABP is involved in intracellular fatty acid signaling and is closely associated with to a variety of metabolic and inflammation-related diseases.A-FABP levels in peripheral blood were significantly improved in PCOS patients than healthy women.At present,there is no report on the pathophysiological function and molecular mechanism of A-FABP in regulating PCOS.Fufang Zhenzhu Tiaozhi Recipe(FTZ)is a traditional Chinese medicine formula created by a team led by Professor Jiao Guo,the academic leader of this research team.Previous studies have shown that FTZ have the effects of ameliorating insulin resistance,lowering blood glucose,and anti-inflammatory effects.The role and mechanism of FTZ intervention in PCOS is still unclear.It is of great scientific significance to investigate the function of A-FABP modulating the PCOS in mouse model to reveal the pathophysiological mechanism of PCOS and the therapeutic effectiveness of FTZ in mouse model of PCOS.Objectives 1.To induce mouse model of POCS by the combination strategy of high-fat diet and continuous infusion of letrozole;2.To investigate the role of A-FABP in the pathogenesis of PCOS by the comparison of phenotypes in the morphology,function of ovary,and systemic metabolism in functional homeostasis between A-FABP knockout and wild type mice;3.To investigate the effects of A-FABP pharmacological inhibitors on experimental PCOS in mice;4.To explore the effects of FTZ on PCOS mouse model.Methods 1.The induction of PCOS mouse model with a combination strategy of high-fat diet and letrozole Female,4-week-old wild type(WT)C57BL/6J mice were fed by high-fat diet(HFD)and subcutaneously implanted with a custom-made continuous-release pellet containing letrozole(50 ?g/day)for 5 weeks to induce PCOS mouse model.During the modeling process,body weight was measured weekly;vaginal smears were collected daily to evaluate the estrous cycle by vaginal cytology method;H&E staining was used to evaluate ovarian histopathological changes.Androgen and estrogen levels in serum were detected by ELISA to evaluate mouse endocrinology functions.To evaluate the phenotype of glucose and lipid metabolism in mice,fasting blood glucose,glucose tolerance test,weekly body weight,and body composition were detected.2.The investigation of pathophysiological role of A-FABP in the development of PCOS Based on the loss-of-function strategy,the characteristics of PCOS phenotype between A-FABP knockout mice and wild type(WT)mice were compared.Four-week-old female A-FABP KO and WT mice were induced into PCOS mouse model following the protocol established in Part 1.The same evaluation parameter as in Part 1 were used to identify the phenotypes of A-FABP KO and WT mice to reveal the pathophysiological role of A-FABP in the pathogenesis of PCOS.3.The exploration of the effect of A-FABP inhibitor on experimental PCOS in mice Four-week-old,female A-FABP KO and WT mice were subjected to induce PCOS following the protocol established in Part 1.During the modeling process,A-FABP inhibitor(BMS309403)was used to intervene the PCOS model of WT mice(15mg/kg·weight/day,dissolved in 4% Tween 80)by oral gavage for 35 consecutive days.An equal volume of 4% Tween 80(vehicle)were administrated to the control group by the same way.The same set of parameters as in Part 1 was used to evaluate the intervention effect of BMS309403 on PCOS.4.The investigation of the therapeutic effect of FTZ on PCOS model in mice Four-week-old,female A-FABP KO and WT mice were induced into PCOS mouse model following the protocol established in Part 1.During the modeling period,FTZ(2.892g/kg·weight/day)was used to treat the PCOS WT mice model by oral gavage continuously for 35 days.An equal volume of dd H2 O were administrated to the control group by same way.The same parameter as in Part 1 was used to evaluate the effect of FTZ on PCOS.Results 1.The mouse model induced by the combination strategy of high-fat diet and letrozole recapitulated the key clinical characteristics of PCOS The mouse model induced by the combination strategy of high-fat diet and letrozole exhibited the key clinical characteristics of PCOS,including irregular estrous cycles,pathological changes in ovarian tissue,increased cystic follicles,decreased corpus luteum.,which is similar to the phenotypes of anovulatory,rare ovulation,and polycystic ovary morphology in PCOS patients.In addition,the mouse model showed significantly reduced estrogen,and an increased trend of androgen,which are partially consistent with the endocrine disorders of PCOS.In addition,feeding with by feeding high-fat diet,mice has the phenotype of obesity and impaired glucose tolerance,which is consistent with the clinal features.(n=6,more details are shown in Chapter 3).2.The enhanced A-FABP in ovary was specifically occurred in granulosa cells in mouse model of PCOS Western blotting analysis revealed that A-FABP protein levels were significantly higher in PCOS mouse ovary compared with controls.Immunohistochemical and immunofluorescence analysis of ovary tissue with anti-AFABP antibody demonstrated that AFABP signal specifically expressed in ovarian granulosa cells(n=6,more details are shown in Chapter 4).3.A-FABP deficiency significantly alleviated phenotype in mouse model of PCOS In comparison to wild type control mice,A-FABP knockout(A-FABP KO)mice exhibited significantly alleviated morphological changes in the ovaries in the mouse model of PCOS including decreased cystic follicles and increased corpus luteum.Moreover,A-FABP KO mice showed significant improvement in PCOS-associated disruption of estrous cycle, reduction of estrogen level and alleviation of glucose intolerance in mice(n=6,more details are shown in Chapter 5).4.The treatment with A-FABP inhibitor significantly alleviated phenotypes in mouse model of PCOS When compared with WT model mice,BMS309403 treatment resulted in marked improvement in PCOS-associated disruption of morphological changes,recovering estrogen level and significant improvement of glucose tolerance(n=6,more details are shown in Chapter 6).5.FTZ exerted therapeutic benefits in experimental model of PCOS The treatment with FTZ significantly alleviated PCOS-related disruption of estrous cycle,morphological changes,including decreased cystic follicles,increased corpus luteum and antral follicles.In terms of endocrine function,FTZ treatment significantly decreased testosterone and restored circulating levels of estrogen.Furthermore,in terms of endocrine function,FTZ administration significantly alleviated the fasting blood glucose and improved the glucose tolerance.FTZ blocked body weight gain,and body fat mass(n=6,more details are shown in Chapter 7).Conclusions 1.The mouse model of PCOS induced by the combination strategy of HFD and LET can server as a reliable animal model for the investigation of PCOS pathogenesis,as it mimicked the main pathological characteristics of PCOS and several metabolic phenotypes.2.The expression of A-FABP were significantly increased in PCOS ovarian granulosa cell.3.Both genetic deletion and pharmacological inhibitor-mediated blockade of A-FABP significantly improved experimental model of PCOS.4.FTZ exerted therapeutic benefits in experimental model of PCOS.In summary,the data in the present study reveal that A-FABP functions as a key mediator for the pathogenesis of experimental model of PCOS in mice.Thus,our research supports that A-FABP as a novel potential target for the development of novel drugs for the treatment of PCOS. |
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