Effect and underlying mechanism of Bu-Shen-An-Tai recipe on ovarian apoptosis in mice with controlled ovarian hyperstimulation implantation dysfunction
Effect and underlying mechanism of Bu-Shen-An-Tai recipe on ovarian apoptosis in mice with controlled ovarian hyperstimulation implantation dysfunction
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补肾安胎方对控制性卵巢过度刺激着床障碍小鼠卵巢凋亡的影响及机制
DOI：
10.1007/s11596-017-1747-3
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发表时间：
2017-06-01
影响因子：
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通讯作者：
Zhang, Ming-min
中科院分区：
文献类型：
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作者：
Ma, Wen-wen;Xiao, Jing;Zhang, Ming-min
研究补肾安泰方对控制性超排卵(COH)着床障碍小鼠卵巢细胞凋亡的影响及其机制。给小鼠腹腔注射孕马血清促性腺激素(PMSG)7.5IU，48h后注射人绒毛膜促性腺激素(HCG)7.5IU，建立小鼠COH植入功能障碍模型。然后于下午6点在同一笼中以2：1的比例交配雌雄小鼠。正常组雌性小鼠于相应时间腹腔注射生理盐水进行交配。上午8：00通过检查阴道涂片记录怀孕第一天。第二天。将50只妊娠成功的小鼠随机分为5组：正常妊娠组(NC)、COH着床功能障碍模型组(COH)、补肾安泰方低剂量组(Low)、补肾安泰方中剂量组(MID)、补肾安泰方高剂量组(High)。然后从第1天开始，不同组的小鼠分别在上午9点给予相应的治疗。连续5天。用放射免疫法测定17β-雌二醇(E_2)和孕酮(P_4)浓度。用透射电子显微镜观察卵巢组织超微结构的变化。HE染色观察卵巢组织病理变化。同时记录闭锁卵泡数和妊娠黄体数。采用原位末端标记法检测卵巢组织中的凋亡细胞。免疫印迹法检测小鼠卵巢组织中凋亡相关因子Bax、Bcl2和Caspase-3的蛋白表达。结果表明，COH组大鼠卵巢重量、E_2、P_4水平、闭锁卵泡数、妊娠黄体数及颗粒细胞凋亡率均显著增加。COH组卵巢组织超微结构显示颗粒细胞染色质增多、凝集、聚集或新月形。细胞凋亡晚期，颗粒细胞内可见灶性空化和典型的凋亡小体。不同剂量补肾安泰方治疗后，卵巢颗粒细胞凋亡的超微结构改变明显改善，透射电子显微镜下甚至消失。可见线粒体和线粒体脊增多，空泡明显减少。脂滴呈圆形或椭圆形。治疗后Bax和裂解半胱氨酸天冬氨酸蛋白酶-3蛋白表达水平降低，而Bcl-2蛋白表达水平升高。结论补肾安胎方可抑制卵巢颗粒细胞的凋亡，其机制可能是通过上调Bcl2的蛋白表达，下调Bax和Caspase-3的表达，从而促进卵巢黄体的形成和维持。有利于促进胚胎着床，减少胚胎丢失，最终提高妊娠成功率。
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 (E2) and progesterone (P4) 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 E2 and P4, 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.