| Endometriosis was a common disease in women and its incidence rate was 10～15% in crowd. The pathogenesy of endometriosis was not definite until now. The therapeutics method of endometriosis were operation and estrin treatment,but its defect were adverse effect and relapse. Neiyixiao was a new traditional chinese drug developed by our team which can treat the endometriosis,it was composed of ligustrazine, ferulic acid and tetrahydropulmatine. The prescription and the preparation technology were submissioned to national technical invention patent.ObjectiveTo explore the effect and the mechanism of Neiyixiao on angiogenesis and estrogen of endometriosis in rats, and it can provide experiments background to clinical.Methods1. The standard of succeed rat model of endometriosisThe rat model of endometriosis was established by surgical implant of the endometrial tissue which belong to its body.The standard of succeed model were based on the volumes,forms and pathology sections of the transplants. It will be the succeed model when it consistent the three standards.①The grafts increased in size,much fluid accumulated to form the transparent saccule appearance,being covered by connective tissue or colic omentum and vascularized in it.and the height of hydrops was more than two millimeter.②We mearsured the volume of grafts.The calculation formula was 0.52×length×width×height,and the volume was more than eight cubic millimeter.③There were endometrial endothelial cells.glandular organ and mesenchymal in grafts being observed with microscope.2.The grouping and administration of the rat model The succeed rats model were grouping by random.it were composed of Model group,Gestrinone 0.5 mg·kg-1·d-1 group,Neiyixiao 0.045 g·kg-1·-d-1 group, Neiyixiao 0.09 g·kg-1·d-1 group, Neiyixiao 0.18 g·kg-1·d-1 group,and every group has ten rats include the Control group.The rats were treated by intragastric administration for 4 weeks,one time every day.3.The effect of Neiyixiao on angiogenesis of endometriosis in rats modelAfter 4 weeks,the rats were sacrificed on its estrus time.We observed the growing condition of the transplants and surgical ablation the transplants.Using the method of hematoxylin and eosin stain to observed the pathology of the transplants. We explored microvessels density (MVD) by the immunohistochemical test as well as the expression of vascular endothelial growth factor (VEGF) protein and endostatin protein in ectopic endometriotic tissue by the immunohistochemical test and western blot. The level of secretion of prostaglandin E2 (PGE2) in the blood serum and ectopic endometriotic tissue was measured by radioimmunoassay (RIA).4.The effect of Neiyixiao on estrogen of endometriosis in rats modelThe rats model of endometriosis and the grouping were in accordance with the method we referred before.every group has sixteen rats.The rats were treated by intragastric administration for 10 weeks.one time every day.After 4 weeks and 10 weeks,the rats were sacrificed on its estrus time,eight rats every time.We surgical ablation the hypothalamus. pituitary, ovary and ectopic endometriotic tissue. We explored the expression of gonadotrop in hormone-releasing hormone (GnRH) in hypothalamus,follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in pituitary,estrogen receptor (ER) in ovary by the immunohistochemical test as well as the expression of ER,heat shock protein 90 (HSP90) and cyclooxygenase-2 (COX-2) in ectopic endometriotic tissue by western blot.The level of estradiol (E2) and progesterone (P) in the blood serum were measured by chemiluminescent immunoassay (CLIA) and enzyme linked immunosorbent assay (Elisa).Results1.The result of the endometriosis rat modelAfter the rats were autotransplanted with endometrial for 4 weeks,we measured the siza of graft using vernier caliper. The result display that the graft was vascularized in it,and much fluid accumulated to form the transparent saccule appearance,the color was clearing or black.The achievement ratio of model was 90.91%. The result of hematoxylin and eosin stain display that in the model group that the epithelial cells submit high prismatical and the nucleus display multilayer cells,but in normal group that epithelial cells submit low prismatical and the nucleus display monolayer cells.2.The effect of Neiyixiao on angiogenesis of endometriosis in rats modelAfter the rats were administer for 4 weeks,we observed the volums of the transplants. Neiyixiao 0.045,0.09,0.18g·kg-1 markedly inhibited the volume of ectopic endometriotic tissue.The results of hematoxylin and eosin stain indicated that that epithelial cells submit low prismatical and the nucleus display monolayer cells.The results of immunohistochemisty and western blot display that the expression of MVD and VEGF protein in model group were significant more than normal group(P<0.01). Neiyixiao 0.09,0.18g·kg-1 reduced MVD and the expression of VEGF protein(P<0.05,P<0.01).The expression of endostatin protein in model group were significant fewer than normal group(P<0.01), Neiyixiao 0.18g·kg-1 advanced the expression of endostatin protein(P<0.01).The results of radioimmunoassay display that the level of secretion of PGE2 in blood serum and ectopia endometrium in model group were significant more than normal group(P<0.01). Neiyixiao 0.09,0.18g·kg-1 reduced the level of secretion of PGE2(P<0.05, P<0.01).3.The effect of Neiyixiao on estrogen of endometriosis in rats modelAfter the rats were administer for 4 weeks, the results of immunohistochemisty display that in the model groups the expression of the GnRH in hypothalamus,the FSH and LH in pituitary,the ER in ovary were significant more than normal group(P<0.01).The results of chemiluminescent immunoassay display that the level of E2 in model group were ignificant more than normal group(P<0.01), Neiyixiao 0.045, 0.09,0.18g·kg-1 reduced the level of secretion of E2(P<0.05, P<0.01).After the rats were administer for 10 weeks, the results of immunohistochemisty display that in the model groups the expression of the GnRH in hypothalamus,the FSH and LH in pituitary,the ER in ovary were significant more than normal group(P<0.01), Neiyixiao 0.09,0.18g·kg-1 significant reduced the expression of the GnRH in hypothalamus,the FSH and LH in pituitary,the ER in ovary(P<0.05, P<0.01).The results of enzyme linked immunosorbent assay display that the level of P in model group were significant lower than normal group, Neiyixiao 0.09,0.18g·kg-1 significant advanced the level of P(P<0.05, P<0.01).The results of immunohistochemisty and western blot display that the expression of ER,HSP90 and COX-2 protein in model group were significant more than normal group(P<0.01). Neiyixiao 0.09.0.18g·kg-1 reduced the expression of ER,HSP90 and COX-2 protein(P<0.05, P<0.01).Conclusion1.Neiyixiao can reduce the degree of angiogenesis of endometriosis in the animal models,and its mechanisms might be associated with the regulation of estrogen and the expression of VEGF and endostatin.2. Neiyixiao can reduce the degree of estrogen of endometriosis in the animal models, and its mechanisms might be associated with the regulation of hypothalamic-pituitary-ovarian axis. |
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