| Aim:To establish animal endometriosis model and pituitary cell model of rat primary culture,and to study the pharmacodynamic action and mechanism of action on endometriosis.Methods:1.The release curve of nomegestrol microballoons in vitro was determine by Release Experiment in vitro;The inhibition on sexual cycle and oulation on SD rat for single administration of nomegestrol microballoons was observed.2.A new ICR mouse model with subcutaneous implants was established by hypodermic injection,and then pathohistologic chanracteristics were observed under the microscope.3.①The pharmacodynamic action of nomegestrol microballoons on rats EMs model was investigated:The model of endometriosis in rats was made by surgically autotransplanting endometrium to the peritoneum.After 3 weeks,the rats were laparotomized to investigate the growth of transplantation.The rats with peritoneal implants were randomly assigned to 5 groups:model control group; R2323 group(1mg/kg.14d);nomegestrol microspheres group(doses of 0.5,2,4 mg/kg.d).After administrations for 3 weeks,the rats were laparotomized again and the volumes of peritoneal implants were mesured.When killing the rats,blood samples in abdominal aortas were collected to measure levels of serum E2,P, using enzymeimmunometric assay(EIMA).The weight of uterus and ovary was recorded for calculating the organ module.Uterus sections were made,then pathohistologic chanracteristics were observed under the microscope.②The pharmacodynamic action of nomegestrol microballoons on mice EMs model was investigated:The model of endometriosis in mice was made by hypodermic injection.After 3 weeks,the mice were laparotomized to investigate the growth of transplantation.The mice with subcutaneous implants were randomly assigned to 7 groups:normal group,model control group;LHRH group(0.02mg/kg.d); nomegestrol microspheres group(doses of 3,6,12,24mg/kg.d).After administrations for 3 weeks,the mice were laparotomized again and the volumes of subcutaneous implants were mesured.When killing the mice,blood samples in abdominal aortas were collected to measure levels of serum FSH,LH,using ELISA.The weight of uterus and ovary was recorded for calculating the organ module.Uterus sections were made,then pathohistologic chanracteristics were observed under the microscope.4.①When killing the mice,IHC(Immunohistochemistry,ABC method) was used to evaluate ERαand PR expression in the uteri and explants of different groups of mice;IHC was used to evaluate ERα,ERβand PR expression in the hypothalamus of different groups of mice;IHC was used to evaluate kiss-1 and GPR54 expression in nucleus arcuatus hypothalami and pituitary of different groups of mice,to study the effect ofvomegestrol on ER,PR,kiss-1 and GPR54 expression.②Rat anterior pituitary was digested with pancreatic enzyme,rat primary pituitary cells were cultured in vitro for 72h and then treated with different concentration of nomegestrol for 49h.The FSH and LH secretion of the cells was induced by GnRH,PMA and high[K+]e for 1h,to study the action of PKC and VSCC in the effect of nomegestrol on the FSH and LH secretion of the cells.③When killing the mice,the mouse brains were gained quickly and stored in liquid nitrogen of -70℃.By the Biospring signal transduction array the difference of protein expression in hypothalamus of EMs model group and nomegestrol 12mg/kg.d group was compared,to study the effect of nomegestrol on the protein expression in Biospring signal transduction array.Results:1.Release profiles of microspheres for one month in vitro showed that accumulated release of 28 days was 85.1%,release 3.04%per day;Release profiles of microspheres for three months in vitro showed that accumulated release of 91 days was 96.5%,release 1.06%per day.Anestrum of SD rat were maintained for 77±1.26 days and 79.67±4.13 days by injecting the 20070109A or 20070109B (80mg/kg.3m).2.Achievement ratio of the model was 83.33%,subcutaneous implants located on muscular layer subcutaneouly,some was in mucous membrane subcutaneouly, looked diaphanous capsular with fluid in it;endomembrane epithelial cell was in the inner wall of the capsular.3.①Achievement ratio of Rat EMs model was 86.9%;Both nomegestrol 2 and 4mg/kg.d decreased the volumes of ectopic endometria,suppression efficient was significant;nomegestrol 0.5,2 and 4mg/kg.d decreased the uterus and ovary organ index significantly;nomegestrol 0.5 and 2mg/kg.d decreased the height of endometrium epithelium and serum E2 level significantly;nomegestrol had no effect on serum P level.②Achievement ratio of mouse EMs model was 82.5%;nomegestrol 6,12 and 24mg/kg.d decreased the volumes of ectopic endometria,suppression efficient was significant;nomegestrol 3,6,12 and 24mg/kg.d decreased the ovary organ index significantly,but not the uterus organ index;nomegestrol had no effect on the height of endometrium epithelium;and serum E2 level significantly; nomegestrol had no effect on serum P level,nomegestrol 6 and 12 mg/kg.d decreased t serum FSH and LH level significantly.4.①ERαexpression in ectopic endometria was lower than in eutopic endometria, but PR expression had no difference between ectopic endometria and eutopic endometria;nomegestrol 3,6,12 and 24mg/kg.d decreased ERαand PR expression in eutopic endometria.In hypothalamus,nomegestrol 6,12 and 24mg/kg.d selectively decreased ERαand PR expression,all of nomegestrol dose group decreased the ERα/ERβratio,and nomegestrol 6mg/kg.d increased ERβexpression.In nucleus arcuatus hypothalam(ARC), nomegestrol 3,6,12 and 24mg/kg.d decreased Kiss-1 and GPR54 expression significantly.In anterior pituitary,nomegestrol 3,6 and 12mg/kg.d decreased Kiss-1 and GPR54 expression significantly;nomegestrol 24mg/kg.d only decreased Kiss-1 expression significantly.②Compared with the control cells without drug treatment,in pituitary primary culture cells,10-5 and 10-6mol.L-1 nomegestrol inhibited GnRH induced LH release,10-5,10-6,10-8 and 10-10mol.L-1 nomegestrol inhibited GnRH induced FSH release,10-5,10-6,10-8 and 10-10mol.L-1 nomegestrol inhibited PMA induced LH release,10-5,10-6,and 10-10 nomegestrol inhibited PMA induced FSH release,10-5,10-6 and 10-8mol.L-1 nomegestrol inhibited high[K+]e induced LH release,10-5 and 10-6 mol.L-1 nomegestrol inhibited high[K+]e induced FSH release.③Biospring signal transduction array data of hypothalamic showed that, compared with model control group,of the 164 target proteins,nomegestrol 12mg/kg.d up-regulated two proteins and down-regulated 46 proteins.The two up-regulated proteins included Dystrophin and Alkaline Phosphatase.The 46 down-regulated proteins included(1) hormonal related protein,(2) cytokine related protein,(3) angiogenesis related protein,(4) oncogene related protein, (5) apoptosis related protein,(6) others:Cell cycle related protein,neuroscience related protein,adenovirus related protein,tumor marker related protein and some individual proteins.Conclusions:Nomegestrol microspheres delayed release in SD rat.ICR mouse subcutaneous EMs model was simply established and stable,was a successful animal EMs model.Nomegestrol microspheres inhibited ectopic endometria in EMs rat and mouse model by decreasing serum E2,FSH and LH level,decreasing the height of endometrium epithelium,decreasing the uterus and ovary organ index.ERαand PR expression in eutopic endometria was decreased selectively in nomegestrol treated group;nomegestrol could effected on the top section-hypothalamus and pituitary;in hypothalamus,nomegestrol selectively decreased ERαand PR expression,some extent increased ERβexpression,the selective regulation of nomegestrol on ERα,ERβand PR expression related to the mechanism of nomegestrol for its action on Endometriosis,which we first found.Compared with normal group,we first found the Kiss-1 and GPR54 expression in nucleus arcuatus hypothalam(ARC) of mouse EMs model was increased significantly,which related to the pathogenesy of EMs;Nomegestrol could effect on ARC by negative feedback,decreasd Kiss-1 and GPR54 expression in ARC; Inhibition of nomegestrol on Kiss-1-GPR54-GnRH-GnRH receptor-FSH/LH secrtion cascade pathway related to the mechanism of nomegestrol for its action on Endometriosis,which we first found.Nomegestrol decreased not only Kiss-1 and GPR54 expression in pituitary,but also GnRH induced FSH and LH scretion in rat primary pituitary culture cells, dependence of PKC and VCSS,which related to the mechanism of nomegestrol for its action on Endometriosis. Data in array analysis verificated that effects of nomegestrol on pituitary was PKC-related,but not on hypothalamus.The downregulation of Nomegestrol on FSH-b related to the inhibition of Nomegestrol on serum FSH level.PR expression difference related to the mechanism of nomegestrol for its action on Endometriosis, but not androgen receptor.Data in array analysis firstly found that:①Nomegestrol regulated cytokine correlation pathway by inhibiting cytokine related proteins expression such as IL-6, HGF,NF kappa B,which related to the mechanism of nomegestrol for its action on Endometriosis.②Nomegestrol regulated angiogenesis correlation pathway by inhibiting angiogenesis related proteins expression,which related to the mechanism of nomegestrol for its action on Endometriosis.③Nomegestrol regulated oncogene correlation pathway by inhibiting oncogene related proteins expression,which related to the mechanism of nomegestrol for its action on Endometriosis.④Nomegestrol two-way regulated apoptosis correlation pathway by inhibiting or promoting apoptosis related proteins expression,which related to the mechanism of nomegestrol for its action on Endometriosis.Beside these,cell cycle related protein,neuroscience related protein,adenovirus related protein,and tumor marker related protein perhaps related to the mechanism of nomegestrol for its action on Endometriosis.
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