Research On Pathomechanism Of Premature Ovarian Failure Induced By Chemotherapy And Regulation Effect Of Genestin On Premature Ovarian Failure

BackgroundPremature ovarian failure (POF) is a common endocrine illness of Gynecology clinically, which is characterized by cession of menstruation before or at the age of 40 with elevated gonadotrophins and low estrogen levels. In recent years, the incidence of carcinoma has an increasing tendency, and there are more and more patients suffering from ovarian deficiency and infertility caused by chemotherapy which becomes one of the important etiological factors of POF. It is reported that nephrotoxicity and hepatotoxicity are commonly encountered when undertaking chemotherapy, which induce liver-kidney yin deficiency, and then deficiency fire disturbs, which lead to disorder of Chong and Ren Meridians. Subsequently, deficiency of resource of Qi and blood will occurred. The gonad fails to get enough nourishment, which brings about ovarian deficiency. What's more, it may lead to amenorrhea and infertility. The specific etiological factor is not clear yet. Hormone replacement therapy(HRT) is used to prevent and treat this disease clinically. However, according to a large amount of reports, HRT can increase the risk of suffering from estrogen-independed tumor. It is one of the difficult problems of the prevention and treatment of POF to look for safe and effective herb medicines. ObjectiveOn the base of establishing the animal model of chemotherapy damaged POF, by exploring the therapeutic effect of genistein on POF and its mechanism, and revealing the target point of molecular pharmacology of herb monomer, finding the character of gene expression in pathogenic mechanism of POF and therapeutic effect of genistein, and then drawing the bioinformatics profile of its net regulation, Chinese medical estrogen receptor regulator theory will be completed. MethodThe animal model of POF induced by chemotherapy damage is established by using ICR mice injected i.p. with CDDP. The animal experiment is provided to research POF systematically. Primary culture of granulosa cell from the ovaries of rats is provided the cellular research method to further research of POF. By using the techniques of immunohistochemistry, electronmicroscope, protein immunoblotting, flow cytometry, gene chip and analysis of bioinformatics , pathomechanism of POF and the effective pathway and the target point of GEN are researched systematically.Result1. The establishment of the animal model of chemotherapy damaged POF and its evaluationICR mice were i.p. injected daily with cisplatin at doses of 3.0mg/kg body weight daily for 7days.This method can imitate the pathogenetic progress of POF induced by the chemotherapy damaged. The biological behavior character change of the mice is consonant with the clinical manifestation of POF, and its syndrome belongs to liver and kidney yin deficiency. Serum levels of ovarian hormones are decreased and serum levels of gonadotrophins are increased. Histology of the ovary shows that development of preantral follicle stopped, with the number of atretic follicle increased and local hyperplasia of stroma which have change of fribrosis. CDDP damages ovarian function by two following ways:①damaging DNA directly;②oxidative damage. This model has high achievement ratio, short time of establishment and good result of evaluation.2. The effect of GEN on chemotherapy damaged POF and its mechanismGEN can improve the biological situation of the animal obviously with increasing weight, intermittent ovulation and estrous cycle's restoring to nearly normal situation. Serum levels of estrogen and progesterone restore to nearly normal levels. With the increasing dose of GEN, the change of progesterone is obvious. The result of immunohistochemistry of estrogen receptor (ER) and progesterone receptor (PR) shows that GEN can up-regulate the expression of ER and PR, and the expression of PR is increased more markedly. GEN can reduce the content of MDA in the ovary, and elevate ovarian total antioxidative capacity (T-AOC). Also, GEN can down-regulate the expression the MMP-2, MMP-9, TGF-βand LH correspondingly, which are good for reconstruction of ovarian structure and maturation of follicles and ovulation. After GEN is taken, cellular organs of granulosa cells are rich. Golgi complexes and smooth endoplasmic reticulum become more. There are more Golgi complexes, rough endoplasmic reticulums and mitochondria in interstitial gland cells. Ribosome is also rich in interstitial gland cells.GEN may improve ovarian function by three following ways:①having stronge anti-oxidative damage, restoring damaged DNA , activating the activity of the endogenous protease in granulose cells and then reconstructing the molecular base of synthesis and secretion of hormone②increasing the expression of ER and PR, raising the sensitivity of the receptor, and then achieving self correction are exerted to ovarian function characterized by increased expression of PR③regulating the balance between the formation and degradation of the extracellular matrix, improving the function situation of the granulosa cells and follicles, and promoting differentiation and maturation of the follicles.3. Regulating mechanism of GEN to the function of CDDP affected granulosa cells of ovary.CDDP can inhibit the proliferation of granulosa cells of ovary. Its effecting stage to the cell cycle is S stage, and it can arrest cell cycle to the G1-M stage. In this stage, the number of apoptotic cell increased evidently. GEN is against damaging effect of CDDP, and it can improve the differentiation and proliferation of the cells to make the blockage of the cell cycle disappear. This effect is related to the dose of GEN. The best time of affecting is 24 hour. GEN will rise the amount of Ca2+ in granulosa cells and inhibit the death of them.4. Differential gene expression profile of pathogenic progress of POF and therapeutic effects of genisteinAnalysis of Gene chip shows that CDDP can up-regulate 137 genes expression, and down-regulate 110 genes expression, which are related with synthesis of DNA, energy metabolism crossing membrane, activation of cellular nuclear factor. CDDP also has an inhibiting effect on functional expression of ovarian cells, and then result in imbalance of net regulation of some genes. GEN can up-regulate expression of 22 genes and down-regulate expression of 38 genes, which are closely related with synthesis of hormone ,repair of damaged DNA and energy metabolism. GEN puts an important effect on establishment of key pathway of cellular signal conduction partly and net regulation.ConclusionUsing the theory and technique of morphology, molecular biology and bioinformatics, pathologic processes of POF are displayed systematically by establishing this animal model. CDDP mainly affects the ovarian function by inhibiting the synthesis of DNA and oxidative damage. GEN can improve cell cycle and activate ER and PR, ameliorate the ovary function, and inhibit the death of granulaosa cells, which are the basis for ovarian reconstruction and ovulation. Therefor, GEN is an ideal medicine for treating POF.