The aim of the present study was to explore the effect and mechanism of Bushen Huoxue recipe（BHR） on ovarian reserve in mice with premature ovarian failure（POF）. Mice were divided into 3 groups： normal group, mod...The aim of the present study was to explore the effect and mechanism of Bushen Huoxue recipe（BHR） on ovarian reserve in mice with premature ovarian failure（POF）. Mice were divided into 3 groups： normal group, model group and BHR group. Intraperitoneal injection of cyclophosphamide was performed to create the POF model. Primordial follicular（PDF） number, ovarian wet weight, ovarian index, and estrous cycle were analyzed to evaluate the effect of BHR on POF. Meanwhile, the m RNA and protein level of Mouse Vasa Homologue（MVH） in the bone marrow, peripheral blood and ovary were detected, to explore the underlying mechanism of the treatment efficacy of BHR on ovarian reserve. By the time of BHR treatment for 28 days, BHR increased the PDF number and shortened the estrous cycle of POF mice. BHR also decreased the m RNA level of MVH in the bone marrow, and increased m RNA and protein level of MVH in the ovary of POF mice. Our results demonstrated a treatment efficacy of BHR on POF mice, and revealed that BHR might repair the dysfunction of germline stem cells in the bone marrow, and thus to improve the ovarian reserve and enhance the ovarian function of POF mice through neo-oogenesis.展开更多
The effect and underlying mechanism of Bu-Shen-An-Tai recipe on ovarian apoptosis in mice with controlled ovarian hyperstimulation(COH) implantation dysfunction were studied. The COH implantation dysfunction model in ...The effect and underlying mechanism of Bu-Shen-An-Tai recipe on ovarian apoptosis in mice with controlled ovarian hyperstimulation(COH) implantation dysfunction were studied. The COH implantation dysfunction model in mice was established by intraperitoneal injection of 7.5 IU pregnant mare’s serum gonadotrophin(PMSG), followed by 7.5 IU human chorionic gonadotrophin(HCG) 48 h later. Then the female mice were mated with male at a ratio of 2:1 in the same cage at 6:00 p.m. The female mice from normal group were injected intraperitoneally with normal saline and mated at the corresponding time. Day 1 of pregnancy was recorded by examining its vaginal smears at 8:00 a.m. of the next day. Fifty successfully pregnant mice were equally randomly divided into 5 groups: normal control pregnant group(NC), COH implantation dysfunction model group(COH), low dosage of Bu-Shen-An-Tai recipe group(LOW), middle dosage of Bu-Shen-An-Tai recipe group(MID) and high dosage of Bu-Shen-An-Tai recipe group(HIGH). Then from day 1, the mice in different groups were respectively intragastrically given corresponding treatments at 9:00 a.m. for 5 consecutive days. The concentrations of 17β-estradiol(E) and progesterone(P) were determined by radioimmunoassay(RIA). The ultrastructural changes of ovarian tissues were observed by transmission electron microscope(TEM). The histopathological changes of ovarian tissues were observed by HE staining. The number of atretic follicles and pregnant corpus luteum were also recorded. TUNEL was applied to measure apoptotic cells of ovarian tissues. Western blotting was used to detect the protein expression of apoptosis-related factors like Bax, Bcl-2 and cleaved-caspase-3 in ovarian tissue of mice. The results showed that ovarian weight, the concentrations of Eand P, the number of atretic follicles and pregnant corpus luteum, as well as the apoptosis of granulosa cells were significantly increased in the COH group. The ultrastructures of ovarian tissues in the COH group showed that chromatin in granulosa cells was increased, agglutinated, aggregated or crescent-shaped. The focal cavitation and the typical apoptotic bodies could be seen in granulosa cells in the late stage of apoptosis. After the treatment with different doses of Bu-Shen-An-Tai recipe, the ultrastructural changes of ovarian granulosa cells apoptosis were dramatically improved and even disappeared under TEM. Visible mitochondria and mitochondrial cristae were increased and vacuoles were significantly reduced. The lipid dropltes were shown in a circluar or oval shape. The protein expression levels of Bax and cleaved-caspase-3 were decreased, and the expression of Bcl-2 protein was increased after treatment. It was concluded that Bu-Shen-An-Tai recipe can inhibit the apoptosis of ovarian granulosa cells, probably by up-regulating the protein expression of Bcl-2 and down-regulating Bax and cleaved-caspase-3, which contributes to the formation and maintenance of ovarian corpus luteum. It’s helpful to promote the embryonic implantation, to reduce embryo loss and ultimately to improve the success rate of pregnancy.展开更多
目的探索归肾丸对卵巢储备功能低下(DOR)小鼠卵巢Oct-4、MVH及Egr-1的影响。方法将40只C57BL/6J雌性小鼠随机分为正常组、模型组、归肾丸组和脱氢表雄酮(DHEA)组。连续序贯腹腔注射孕马血清(PMSG),人绒毛膜促性腺激素(HCG),前列腺素F2α...目的探索归肾丸对卵巢储备功能低下(DOR)小鼠卵巢Oct-4、MVH及Egr-1的影响。方法将40只C57BL/6J雌性小鼠随机分为正常组、模型组、归肾丸组和脱氢表雄酮(DHEA)组。连续序贯腹腔注射孕马血清(PMSG),人绒毛膜促性腺激素(HCG),前列腺素F2α(PGF2α)产生连续超数排卵,再结合臭氧吸入构建DOR模型。其后归肾丸组给予0.3 m L归肾丸中药灌胃,DHEA组给予0.3 m L DHEA灌胃,而正常组与模型组均给予相同剂量生理盐水灌胃,21天。采用ELISA法检测血清雌二醇(E2)、孕酮(P)和抗苗勒氏管激素(AMH)水平以及Real time-PCR检测Oct-4、MVH、Egr-1 mRNA的变化。结果与模型组比较,各治疗组血清E2、P及AMH水平均明显升高,ER、PR、MVH、Oct-4 mRNA表达均明显升高,差异有统计学意义(P<0.05)。结论归肾丸能改善DOR小鼠Oct-4、MVH、Egr-1 mRNA表达,改善卵巢功能。展开更多
基金supported by the National Natural Science Foundation of China(No.81173396)
文摘The aim of the present study was to explore the effect and mechanism of Bushen Huoxue recipe（BHR） on ovarian reserve in mice with premature ovarian failure（POF）. Mice were divided into 3 groups： normal group, model group and BHR group. Intraperitoneal injection of cyclophosphamide was performed to create the POF model. Primordial follicular（PDF） number, ovarian wet weight, ovarian index, and estrous cycle were analyzed to evaluate the effect of BHR on POF. Meanwhile, the m RNA and protein level of Mouse Vasa Homologue（MVH） in the bone marrow, peripheral blood and ovary were detected, to explore the underlying mechanism of the treatment efficacy of BHR on ovarian reserve. By the time of BHR treatment for 28 days, BHR increased the PDF number and shortened the estrous cycle of POF mice. BHR also decreased the m RNA level of MVH in the bone marrow, and increased m RNA and protein level of MVH in the ovary of POF mice. Our results demonstrated a treatment efficacy of BHR on POF mice, and revealed that BHR might repair the dysfunction of germline stem cells in the bone marrow, and thus to improve the ovarian reserve and enhance the ovarian function of POF mice through neo-oogenesis.
基金supported by the National Natural Sciences Foundation of China(No.81473494)
文摘The effect and underlying mechanism of Bu-Shen-An-Tai recipe on ovarian apoptosis in mice with controlled ovarian hyperstimulation(COH) implantation dysfunction were studied. The COH implantation dysfunction model in mice was established by intraperitoneal injection of 7.5 IU pregnant mare’s serum gonadotrophin(PMSG), followed by 7.5 IU human chorionic gonadotrophin(HCG) 48 h later. Then the female mice were mated with male at a ratio of 2:1 in the same cage at 6:00 p.m. The female mice from normal group were injected intraperitoneally with normal saline and mated at the corresponding time. Day 1 of pregnancy was recorded by examining its vaginal smears at 8:00 a.m. of the next day. Fifty successfully pregnant mice were equally randomly divided into 5 groups: normal control pregnant group(NC), COH implantation dysfunction model group(COH), low dosage of Bu-Shen-An-Tai recipe group(LOW), middle dosage of Bu-Shen-An-Tai recipe group(MID) and high dosage of Bu-Shen-An-Tai recipe group(HIGH). Then from day 1, the mice in different groups were respectively intragastrically given corresponding treatments at 9:00 a.m. for 5 consecutive days. The concentrations of 17β-estradiol(E) and progesterone(P) were determined by radioimmunoassay(RIA). The ultrastructural changes of ovarian tissues were observed by transmission electron microscope(TEM). The histopathological changes of ovarian tissues were observed by HE staining. The number of atretic follicles and pregnant corpus luteum were also recorded. TUNEL was applied to measure apoptotic cells of ovarian tissues. Western blotting was used to detect the protein expression of apoptosis-related factors like Bax, Bcl-2 and cleaved-caspase-3 in ovarian tissue of mice. The results showed that ovarian weight, the concentrations of Eand P, the number of atretic follicles and pregnant corpus luteum, as well as the apoptosis of granulosa cells were significantly increased in the COH group. The ultrastructures of ovarian tissues in the COH group showed that chromatin in granulosa cells was increased, agglutinated, aggregated or crescent-shaped. The focal cavitation and the typical apoptotic bodies could be seen in granulosa cells in the late stage of apoptosis. After the treatment with different doses of Bu-Shen-An-Tai recipe, the ultrastructural changes of ovarian granulosa cells apoptosis were dramatically improved and even disappeared under TEM. Visible mitochondria and mitochondrial cristae were increased and vacuoles were significantly reduced. The lipid dropltes were shown in a circluar or oval shape. The protein expression levels of Bax and cleaved-caspase-3 were decreased, and the expression of Bcl-2 protein was increased after treatment. It was concluded that Bu-Shen-An-Tai recipe can inhibit the apoptosis of ovarian granulosa cells, probably by up-regulating the protein expression of Bcl-2 and down-regulating Bax and cleaved-caspase-3, which contributes to the formation and maintenance of ovarian corpus luteum. It’s helpful to promote the embryonic implantation, to reduce embryo loss and ultimately to improve the success rate of pregnancy.
文摘目的探索归肾丸对卵巢储备功能低下(DOR)小鼠卵巢Oct-4、MVH及Egr-1的影响。方法将40只C57BL/6J雌性小鼠随机分为正常组、模型组、归肾丸组和脱氢表雄酮(DHEA)组。连续序贯腹腔注射孕马血清(PMSG),人绒毛膜促性腺激素(HCG),前列腺素F2α(PGF2α)产生连续超数排卵,再结合臭氧吸入构建DOR模型。其后归肾丸组给予0.3 m L归肾丸中药灌胃,DHEA组给予0.3 m L DHEA灌胃,而正常组与模型组均给予相同剂量生理盐水灌胃,21天。采用ELISA法检测血清雌二醇(E2)、孕酮(P)和抗苗勒氏管激素(AMH)水平以及Real time-PCR检测Oct-4、MVH、Egr-1 mRNA的变化。结果与模型组比较,各治疗组血清E2、P及AMH水平均明显升高,ER、PR、MVH、Oct-4 mRNA表达均明显升高,差异有统计学意义(P<0.05)。结论归肾丸能改善DOR小鼠Oct-4、MVH、Egr-1 mRNA表达,改善卵巢功能。
