| Objectives:To establish mice model with premature ovarian failure (POF) induced by cisplatin intraperitoneal injection, observe the immune system and reproductive system effects for mouses with POF by different doses of Bushentianjing, investigate therapeutic effect and mechanism of action for Bushentianjing.Methods:Hundred thirty-five Kunming female mices were randomly divided into normal control group, model group, prednisone group, estrogen group, high dose Chinese medicine group (H group), Middle dose Chinese medicine group (M group), low dose Chinese medicine group (L group). All mices were injected cisplatin to establish POF model, except for normal control group. H group, M group and L group were given Bushentianjing decoction by intragastric administration. Prednisone group were given prednisone by subcutaneous injection. Estrogen group were given diethylstilbestrol dipropionate by subcutaneous injection. Normal control group and model group were given Sodium Chloride by intragastric administration. The ovarian function for mice was determined by vaginal smear. Weight data for weight, spleen, ovary, thymus were recorded. The ovarian histopathology was tested by using HE staining and ultrastructural pathology. Serum estradiol (E2) and follicle stimulating hormone (FSH) were determinated by chemiluminescence. The ovarian cell apoptosis was determined by flow cytometry. The peripheral blood T cell subsets (CD3+, CD4+,CD8+) and natural killer cell (NKT cell) were detected by SAP. The mice anti-ovarian antibodies (AOAb) was tested by ELISA. The ovarian granulosa cell apoptosis regulators Bcl-2 and Fas-L, expressed in cytoplasm and cell membrane, were determinated by immunohistochemistry and analyzed by computer.Results:1. Body weight of mice after cisplatin intraperitoneal injection decreased significantly, and gradually increased after treatment, especial in the M group and the estrogen group. Compared with the normal control group, vaginal smears for model mices were showed signs with estrogen deficiency; for model mices, the levels of E2 were significantly increased (P<0.01), but FSH decreased (P<0.01). Compared with the model group, the levels of E2 were significantly higher (P<0.05) and the levels of FSH were significantly lower (P<0.01) in the M group and the estrogen group. The E2 and FSH levels for the prednisone group, the H group and the estrogen group were similar (P>0.05).2. The percentages of CD3+ was similar for all groups (P>0.05). The levels of T-lymphocyte subsets in the model group were quite different from the normal control group, with much less the percentages of CD4+ and the CD4+/CD8+ ratios, and much more the percentages of CD8+. After treatment for treated groups, the percentages of CD4+CD8+ and the CD4+/CD8+ ratios were increased, but the percentages of CD8+ were increased, especial in the prednisone group, the H group and the estrogen group. The levels of T-lymphocyte subsets in the prednisone group, the H group and the estrogen group were similar.3. Compared with the control group, the NKTC were significantly increased for all model animals (P<0.01). Compared with the model group, the NKTC for the treated groups were significantly decreased, especial in the prednisone group, the H group and the estrogen group (P<0.01). The NKTC were significantly lower in the M group than those in the estrogen group (P<0.01).4. Compared with the control group, the AoAb were significantly increased for all model animals, especial in the model group, the prednisone group, H group, M group,L group (P<0.01). Compared with the model group, the AoAb in all treatment groups were decrased, especial in the estrogen group (P<0.01) and the M group(P<0.05). Compared with the estrogen group, the AoAb in the M group were not different.5. The brown particles for Bcl-2 and Fas-L expressions were shown in ovarian granulosa cell cytoplasm and cell membrane. Compared with the control group, the Bcl-2 expressions were significantly decreased for all model animals, especial in the model group. Compared with the model group, the Bcl-2 expressions in all treatment groups were more positive (P<0.01). Compared with the estrogen group, the Bcl-2 expressions in the M group were not different (P>0.05), the H group and the L group were significantly less (P<0.05). The Bcl-2 expressions in all Chinese medicine groups were significantly less than those in the prednisone group (P<0.01). In contrast, the expressions for Fas-L were inhanced for mices which were injected by cisplatin, especial in the model group (P<0.01). Compared with the model group, the expressions for Fas-L were lower in the treated groups. The expressions for Fas-L in the H group and the estrogen group were similar (P>0.05). The expressions for Fas-L were significantly lower in the H group than those in the groups which were treated by Chinese medicine.6. Compared with the control group, the ratios of ovarian cell apoptosis were significantly increased for all model animals (P<0.01). Compared with the model group, the ratios of ovarian cell apoptosis in the treatment groups were significantly decreased, especial in the prednisone group, the H group and the estrogen group (P<0.05). The ratios of ovarian cell apoptosis in the prednisone group, the H group and the estrogen group were similar (P>0.05).7. Compared with the normal control group, the spleen index for all groups did not change significantly, but the thymic index for the model group and the treatment groups were lower (P<0.01) and the ovarian index for the model group were significantly decreased (P<0.05). Compared with the model group, the thymic index and ovarian index were increased, especial the thymic index for the H group (P<0.01) and the ovarian index for the estrogen group (P<0.05). the thymic index and the ovarian index for the H group (P<0.01) and the estrogen group were similar (P>0.05).8. Compared with the normal control group, pathological observation of the model mices had different degrees of bilateral ovarian atrophy, and were pale. Light microscope after HE staining was observed that ovarian volume reduced significantly, ovarian tissue "fibrosis" pattern presented, stromal hyperplasia, ovarian follicles grow and mature follicles decreased significantly, follicles unmatured, atretic follicles increased. Compared with the model group, pathological observation of the treated mices were shown mature follicles increased, atretic follicles decreased and interstitial fibrosis reduced. Ultrastructural observation for the ovary in the model group were shown that apoptotic cells within the follicle, narrow and fuzzy transparent, smaller follicular granulosa cells with disorganized and sizes, the zona pellucida increased, granule cells connections widened, and organelles reduction obvious swelling of mitochondria and cristae blurred. Ultrastructural observation for all treatment groups were improved.Conclusions:1. The production mouse model with premature ovarian failure was combined with disease and syndrome,which was characterized by the changes of immunity in cellular immune and ovarian autoimmunity and genes in ovarian apoptosis.2. Bushentianjing decoction can regulate reproductive endocrine and cellular immune, down-regulating the AOAb and the FAS-L expression in granulosa cell membrane, enhancing the BCL-2 expression in granulosa cell membrane, decreasing ovarian cell apoptosis.3. Bushentianjing decoction can regulate apoptosis genes for ovarian granulosa cell, enhance their disease resistance and self-repair capacity by multi-channel and multi-target.4. The middle dose of Bushentianjing decoction had the side best dose effects for therapy, which can replace estrogen and glucocorticoid on the experiment.5. Chinese medicine Bushentianjing decoction can treat POF, which may be approach to achieve therapeutic effect through the Chinese medicine kidney-Brain-Chong and Ren-uterus and the modern medicine under the hypothalamus-pituitary-ovarian-immune network.
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