补肾填精方对卵巢早衰模型小鼠作用机理的实验研究
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摘要
目的：通过腹腔注射顺铂建立小鼠卵巢早衰模型,观察中药补肾填精方不同剂量对卵巢早衰模型小鼠免疫系统及生殖系统的影响,探讨其治疗效果和作用机制。
方法：将135只雌性昆明小鼠随机分为正常对照组(简称正常组)模型组、强的松组、雌激素组、中药高剂量组(简称高剂量组)、中药中剂量组(简称中剂量组)、中药低剂量组(简称低剂量组),除正常对照组外均采用连续腹腔注射顺铂7天建立卵巢早衰模型并阴道细胞涂片判定小鼠雌激素水平；造模结束后第4天开始灌胃,连续30天,中药高、中、低剂量组分别给予不同浓度的补肾填精方水煎剂灌胃,强的松组、雌激素组分别给予强的松和乙底酚水溶液灌胃,正常对照组和模型组生理盐水灌胃；灌胃结束第2天处死小鼠,称量体重和脾、卵巢、胸腺的重量,卵巢组织采用HE染色病理观察和超微病理观察,化学发光法测定血清雌激素(E2)、促卵泡成熟素(FSH),流式细胞术检测卵巢细胞凋亡率,SAP法检测外周血T细胞亚群(CD3+、CD4+、CD8+)和自然杀伤细胞(NKT cell), ELISA法检测小鼠抗卵巢抗体(AOAb),免疫组化法检测卵巢颗粒细胞凋亡调控因子Bcl-2和FasL在细胞浆和细胞膜的表达,并进行图像分析。
结果：1.造模后的小鼠体重均较正常组明显降低,治疗后逐渐回升,以中剂量组和雌激素组最为明显；与正常组比较,模型小鼠的阴道细胞涂片呈雌激素缺乏征象；模型组FSH水平显著升高(P<0.01), E2的水平低于正常组(P<0.01)；与模型组比较,中剂量组、雌激素组E2升高(P<0.05),FSH显著降低(P<0.01)；中剂量组和雌激素组以及强的松组相比较,E2和FSH均无显著性差异(P>0.05)。
2.各组CD3+细胞水平比较,模型组和各治疗组无显著性差异；模型组外周血CD4+、CD8+细胞水平及其比值与正常组比较有显著性差异,CD4+细胞水平降低,CD8+细胞水平明显升高,CD4+/CD8+明显降低；治疗后各组CD4+细胞水平均有上升、CD8+细胞水平均有下降、CD4+/CD8+比值明显升高,和模型组比较,以中剂量组和雌激素组、强的松组最为明显；中剂量组和雌激素组以及强的松组比较,无明显差异。
3.与正常组比较,各组均出现NKTC上升情况,有极显著性差异(P<0.01)；与模型组比较,各治疗组NKTC下降,以中剂量组、雌激素组和强的松组最为显著(P<0.01)；与雌激素组比较,中剂量组NKTC下降明显(P<0.01)。
4.模型组和各治疗组AOAb含量均有增高,和正常组相比,模型组、高、低剂量组和强的松组有显著差异(P<0.01)；与模型组比较,各治疗组有不同程度的降低,其中雌激素组差异最为显著(P<0.01),中剂量组有显著性差异(P<0.05)；和雌激素组比较,中剂量组无显著性差异。
5.卵巢颗粒细胞胞浆和细胞膜上均可见Bcl-2和FasL表达的棕黄色颗粒。和正常组相比较,造模后各组Bcl-2均表达减弱,而以模型组最明显,几乎不表达；和模型组比较,各治疗组均可见Bcl-2阳性表达,但弱于正常组,差异显著(P<0.01)；中药各组和雌激素组比较,中剂量组Bcl-2表达强度无差异(P>0.05),高、低剂量组Bcl-2表达减弱,有显著性差异(P<0.05)；中药各组和强的松组比较有差异显著,Bcl-2表达弱于强的松组(P<0.01)。与Bcl-2表达强度结果相反,和正常组比较,模型组和各治疗组FasL均表达增强,模型组呈现极强表达现象,(P<0.01)；和模型组比较,各治疗组均可见FasL阳性表达减弱,有极显著差异,(P<0.01)；和雌激素组比较,中剂量组无差异(P>0.05),中药各组之间比较,以中剂量组FasL表达最弱(P<0.01)。
6.与正常组比较,模型动物均出现卵巢细胞凋亡率上升,有显著性差异(P<0.01)；和模型组比较,各治疗组卵巢细胞凋亡率下降,以中剂量组、雌激素组、强的松组最为明显(P<0.05)；中剂量组和雌激素组以及强的松组比较,无显著性差异(P>0.05)。
7.与正常组比较,各组小鼠脾脏指数变化不明显,模型组和各治疗组胸腺指数均降低,有显著差异(P<0.01)；模型组卵巢指数降低(P<0.05)。与模型组比较,胸腺指数和卵巢指数均升高,中剂量组胸腺指数升高明显(P<0.01),雌激素组卵巢脏器指数升高明显(P<0.05)；中剂量组和雌激素组比较,胸腺指数和卵巢指数均无明显差异(P>0.05)。
8.病理检查发现模型组小鼠双侧卵巢均有不同程度萎缩,颜色苍白,HE染色后光镜下观察可见卵巢体积明显缩小,卵巢组织呈现“纤维化”样改变,间质增生,卵巢中生长卵泡和成熟卵泡数明显减少,几乎无成熟卵泡,闭锁卵泡增加,和正常组比较有明显差异；各治疗组成熟卵泡较模型组增加,闭锁卵泡减少,间质纤维化程度减轻。观察卵巢的超微结构：模型组卵泡内见凋亡细胞；透明带狭窄、模糊；卵泡内颗粒细胞明显变小,排列紊乱,大小不一,与透明带之间距离增大；颗粒细胞之间连接增宽；胞质内细胞器减少,线粒体明显肿胀,嵴模糊不清；各治疗组和模型组比较均有好转。
结论：1.所制作模型为卵巢早衰病证结合小鼠模型,存在免疫系统和生殖系统的改变。
2.补肾填精方水煎剂可使POF小鼠增重,胸腺指数、卵巢指数增加,E2升高、FSH下降,CD4+增高、CD8+细胞下降、增加CD4+/CD8+比值,降低NKTC,下调AOAb,使Bcl-2在卵泡颗粒细胞膜表达增强,FasL表达减弱,减少卵巢细胞凋亡率。
3.补肾填精方治疗POF的可能作用机制是通过调节生殖内分泌、细胞免疫和体液免疫,从多途径、多靶点增强其抗病能力和卵巢的自我修复能力。
4.实验表明,中剂量的补肾填精方水煎剂对POF具有较好的治疗效果,和雌激素具有相似的作用。
5.中药补肾填精方水煎剂具有治疗POF的作用。其途径可能是通过中医肾-脑-冲任-胞宫和现代医学之下丘脑-垂体-卵巢-免疫网络途径而达到治疗作用。
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.
方法：将135只雌性昆明小鼠随机分为正常对照组(简称正常组)模型组、强的松组、雌激素组、中药高剂量组(简称高剂量组)、中药中剂量组(简称中剂量组)、中药低剂量组(简称低剂量组),除正常对照组外均采用连续腹腔注射顺铂7天建立卵巢早衰模型并阴道细胞涂片判定小鼠雌激素水平；造模结束后第4天开始灌胃,连续30天,中药高、中、低剂量组分别给予不同浓度的补肾填精方水煎剂灌胃,强的松组、雌激素组分别给予强的松和乙底酚水溶液灌胃,正常对照组和模型组生理盐水灌胃；灌胃结束第2天处死小鼠,称量体重和脾、卵巢、胸腺的重量,卵巢组织采用HE染色病理观察和超微病理观察,化学发光法测定血清雌激素(E2)、促卵泡成熟素(FSH),流式细胞术检测卵巢细胞凋亡率,SAP法检测外周血T细胞亚群(CD3+、CD4+、CD8+)和自然杀伤细胞(NKT cell), ELISA法检测小鼠抗卵巢抗体(AOAb),免疫组化法检测卵巢颗粒细胞凋亡调控因子Bcl-2和FasL在细胞浆和细胞膜的表达,并进行图像分析。
结果：1.造模后的小鼠体重均较正常组明显降低,治疗后逐渐回升,以中剂量组和雌激素组最为明显；与正常组比较,模型小鼠的阴道细胞涂片呈雌激素缺乏征象；模型组FSH水平显著升高(P<0.01), E2的水平低于正常组(P<0.01)；与模型组比较,中剂量组、雌激素组E2升高(P<0.05),FSH显著降低(P<0.01)；中剂量组和雌激素组以及强的松组相比较,E2和FSH均无显著性差异(P>0.05)。
2.各组CD3+细胞水平比较,模型组和各治疗组无显著性差异；模型组外周血CD4+、CD8+细胞水平及其比值与正常组比较有显著性差异,CD4+细胞水平降低,CD8+细胞水平明显升高,CD4+/CD8+明显降低；治疗后各组CD4+细胞水平均有上升、CD8+细胞水平均有下降、CD4+/CD8+比值明显升高,和模型组比较,以中剂量组和雌激素组、强的松组最为明显；中剂量组和雌激素组以及强的松组比较,无明显差异。
3.与正常组比较,各组均出现NKTC上升情况,有极显著性差异(P<0.01)；与模型组比较,各治疗组NKTC下降,以中剂量组、雌激素组和强的松组最为显著(P<0.01)；与雌激素组比较,中剂量组NKTC下降明显(P<0.01)。
4.模型组和各治疗组AOAb含量均有增高,和正常组相比,模型组、高、低剂量组和强的松组有显著差异(P<0.01)；与模型组比较,各治疗组有不同程度的降低,其中雌激素组差异最为显著(P<0.01),中剂量组有显著性差异(P<0.05)；和雌激素组比较,中剂量组无显著性差异。
5.卵巢颗粒细胞胞浆和细胞膜上均可见Bcl-2和FasL表达的棕黄色颗粒。和正常组相比较,造模后各组Bcl-2均表达减弱,而以模型组最明显,几乎不表达；和模型组比较,各治疗组均可见Bcl-2阳性表达,但弱于正常组,差异显著(P<0.01)；中药各组和雌激素组比较,中剂量组Bcl-2表达强度无差异(P>0.05),高、低剂量组Bcl-2表达减弱,有显著性差异(P<0.05)；中药各组和强的松组比较有差异显著,Bcl-2表达弱于强的松组(P<0.01)。与Bcl-2表达强度结果相反,和正常组比较,模型组和各治疗组FasL均表达增强,模型组呈现极强表达现象,(P<0.01)；和模型组比较,各治疗组均可见FasL阳性表达减弱,有极显著差异,(P<0.01)；和雌激素组比较,中剂量组无差异(P>0.05),中药各组之间比较,以中剂量组FasL表达最弱(P<0.01)。
6.与正常组比较,模型动物均出现卵巢细胞凋亡率上升,有显著性差异(P<0.01)；和模型组比较,各治疗组卵巢细胞凋亡率下降,以中剂量组、雌激素组、强的松组最为明显(P<0.05)；中剂量组和雌激素组以及强的松组比较,无显著性差异(P>0.05)。
7.与正常组比较,各组小鼠脾脏指数变化不明显,模型组和各治疗组胸腺指数均降低,有显著差异(P<0.01)；模型组卵巢指数降低(P<0.05)。与模型组比较,胸腺指数和卵巢指数均升高,中剂量组胸腺指数升高明显(P<0.01),雌激素组卵巢脏器指数升高明显(P<0.05)；中剂量组和雌激素组比较,胸腺指数和卵巢指数均无明显差异(P>0.05)。
8.病理检查发现模型组小鼠双侧卵巢均有不同程度萎缩,颜色苍白,HE染色后光镜下观察可见卵巢体积明显缩小,卵巢组织呈现“纤维化”样改变,间质增生,卵巢中生长卵泡和成熟卵泡数明显减少,几乎无成熟卵泡,闭锁卵泡增加,和正常组比较有明显差异；各治疗组成熟卵泡较模型组增加,闭锁卵泡减少,间质纤维化程度减轻。观察卵巢的超微结构：模型组卵泡内见凋亡细胞；透明带狭窄、模糊；卵泡内颗粒细胞明显变小,排列紊乱,大小不一,与透明带之间距离增大；颗粒细胞之间连接增宽；胞质内细胞器减少,线粒体明显肿胀,嵴模糊不清；各治疗组和模型组比较均有好转。
结论：1.所制作模型为卵巢早衰病证结合小鼠模型,存在免疫系统和生殖系统的改变。
2.补肾填精方水煎剂可使POF小鼠增重,胸腺指数、卵巢指数增加,E2升高、FSH下降,CD4+增高、CD8+细胞下降、增加CD4+/CD8+比值,降低NKTC,下调AOAb,使Bcl-2在卵泡颗粒细胞膜表达增强,FasL表达减弱,减少卵巢细胞凋亡率。
3.补肾填精方治疗POF的可能作用机制是通过调节生殖内分泌、细胞免疫和体液免疫,从多途径、多靶点增强其抗病能力和卵巢的自我修复能力。
4.实验表明,中剂量的补肾填精方水煎剂对POF具有较好的治疗效果,和雌激素具有相似的作用。
5.中药补肾填精方水煎剂具有治疗POF的作用。其途径可能是通过中医肾-脑-冲任-胞宫和现代医学之下丘脑-垂体-卵巢-免疫网络途径而达到治疗作用。
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.
引文
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