To Study The Mechanism And Effects Of Zuogui Pill On Apoptosis Of Rat's Of POF

Objective:To further explore the mechanism and effect of Zuogui Pill on prevention andtreatment of premature ovarian failure(POF) through study the effect of ZuoguiPill on apoptosis ofimmune POF rats. To study the effects of Zuogui Pill onreproductive endocrine and antibody to ovarian ZP (anti-ZP) in immune POF rats.To make progress on a series of research of Zuogui Pill. To provide theexperimental data and some ideas for the theoretical and clinical research.To search the effective and safe Chinese Medicine for the prevention andtreatment of POF.Methods:1. Study literature: Review Sino-Western (Traditional Chinese Medicine andWestern Medicine) about POF with regard to awareness, treatment methods,current research situation and mechanism of progress of Zuogui Pill on POF,especially on the aspect of the immune system research progress and analysisand try to find out the possible mechanism of Zuogui Pill on ajusting theimmune system to prevent and treat the POF by study the literature. Andthen worked out the experimental research program to explore the mechanismof Zuogui Pill on apoptosis of autoimmune Rat's of POF2. Established the animal model of autoimmune premature ovarian failure (POF):The autoimmune POF rat model was induced with active immunization of adultBALB/C rats by subcutaneous multipoint injection a mouse pellucid zone ZP3peptide (Pzp3) No. 330-342 amino acid sequence(NSSSSQFQIHGPR) in completeFreund's adjuvant (CFA) as immunogen. Histopathology of these animal ovaryare similar with human oophoritis of POF.3. The animal of POF were random assigned to Zuogui Pill low-dosage group, middle-dosage group and high-dosagegroup, CFA group, Prednisone group andStilbestrol group. Prednisone and Stilbestrol were used as control drug.4. Estrus was determined by the vaginal cytology. Histopathology of ovary wasstudied by optical microscope.5. Heft the weigh of body, spleen, thymus and ovary by electronic scale andcompare their weigh indices.6. Serum FSH and E₂ were determined with ELISA method.7. Serum CD₄⁺,CD₈⁺,CD₃⁺,CD₄⁺/CD₈⁺ and NK cell were determined with flow cytometer.8. Serum AZAb was determined with ELISA method.9. The expression of Fas-l and Bcl-2 protein were detected by two-stepimmunohistochemical staining method.10. The rate of apoptosis of follicle was determined by TUNEL method.Results:Model group1. Estrous cycle became irregular gradually. Diestrus is extended. Vaginalcell smear present a lot of leukocytes and little epithelium.2. Histopathology of ovary: More Lymphocytes infiltration was found in stromaovary of autoimmune POF rats. Mature follicle was less.3. The level of FSH rose up while that of E₂ lowed down.4. The weigh indices of body descended. The weight indices of thymus and ovarydescended too. The weight indices of spleen was increased.5. CD4⁺,CD8⁺ and CD4⁺/CD8⁺ were decreased. CD₃⁺ and NK cell were increased.6. Serum AZAb was positive and the level of AZAb rose up.7. Bcl-2 protein decreased markedly while Fas-L increase.8. The rate of apoptosis of follicle elevated obviously.Zuogui Pill group1. Estrous cycle became irregular slightly. Diestrus is slight extended. Itis proestrus mainly. Vaginal cell smear present a lot of epithelium withnucleus. It also present some keratinocytes, but less than that ofStilbestrol group, blank group and CFA group. The changes among three dosageof Zuogui Pill group were not significant.2. Histopathology of ovary: Less Lymphocytes infiltration was found in stromaof ovary of autoimmune POF rats. Mature follicle was more than those of model group obviously.3. The level of FSH lowed down while that of E₂ rose up, especially in thelow-dosage group and high-dosage group of Zuogui Pill.4. The weigh indices of body were slight increase than before. The weightindices of thymus and ovary were increase markedly. The weight of spleenwas decreased.5. CD4⁺,CD8⁺ and CD4⁺/CD8⁺ were increase slightly. CD₃⁺ and NK cell were decreasedobviously.6. Serum AZAb was going down.7. Bcl-2 protein elevated markedly while Fas-L decreased.8. The rate of apoptosis of follicle dropt obviously.Conclution:Zuogui Pill could delay the increase of FSH and raise the level of E₂ and inhibitthe produce of AZAb in autoimmune POF rats. It could prohibit the apoptosisof follicle through improving Bcl-2 mRNA expression and improve the autoimmuneoophoritis induced by AZAb. That may be one of the mechanism of Zuogui Pillon prevention and treatment of premature ovarian failure(POF).