补肾调经方和逍遥丸对体外培养小鼠卵泡排卵率及PR-A/HIF-1α表达的影响  

Effects of Bushen Tiaojing Formula and Xiaoyao Pills on ovulation rate and the expression of PR-A/HIF-1αin vitro cultured oocytes

作　　者：刘亚华 杜惠兰 李丽 范丽洁 贺明 段彦苍 LIU Ya-hua;DU Hui-lan;LI Li;FAN Li-jie;HE Ming;DUAN Yan-cang(College of Integrated Chinese and Western Medicine,Hebei University of Chinese Medicine,Shijiazhuang 050200,China;Hebei Collaborative Innovation Center for Reproductive Diseases with Integrated Traditional Chinese and Western Medicine,Shijiazhuang 050200,China;Hebei Key Laboratory of Integrative Traditional Chinese and Western Medicine on Liver-Kidney Patterns,Shijiazhuang 050200,China)

机构地区：[1]河北中医学院中西医结合学院,石家庄050200 [2]河北省中西医结合生殖疾病协同创新中心,石家庄050200 [3]河北省中西医结合肝肾病证研究重点实验室,石家庄050200

出　　处：《中华中医药杂志》2021年第8期5010-5014,共5页China Journal of Traditional Chinese Medicine and Pharmacy

基　　金：国家自然科学基金项目(No.81973893,No.81774359)。

摘　　要：目的:探讨补肾调经方、逍遥丸诱发排卵的作用机制与调控孕激素受体A(PR-A)及其下游乏氧诱导因子1α(HIF-1α)的关系。方法:144只12日龄雌性昆明乳鼠,机械法分离窦前卵泡,随机分为空白组(不加血清)、正常组(加正常大鼠血清)、补肾组(序贯加入补肾调经Ⅱ、Ⅲ号方高剂量大鼠含药血清)、疏肝组(序贯加入逍遥丸高、低剂量大鼠含药血清),体外培养12d,第12天时加入人绒毛膜促性腺激素(hCG)后16h,观察卵泡成活率、窦腔形成率和卵母细胞逸出率。RT-PCR法、细胞免疫荧光染色法检测培养第12天时加入hCG后0、8、12、16h,卵泡和卵丘-卵母细胞复合体(COCs)中PR-A、HIF-1αmRNA及其蛋白的表达。结果:卵泡生长过程中卵泡直径不断增长,见窦腔形成、COCs排出、卵母细胞逸出。补肾组和疏肝组卵泡成活率、窦腔形成率及卵母细胞逸出率较正常组和空白组均升高(P<0.05),补肾组优于疏肝组(P<0.05);加入hCG后,补肾组与疏肝组卵泡和COCs中PR-A、HIF-1αmRNA及蛋白8h、12h、16h表达较正常组和空白组升高(P<0.05),8h疏肝组高于补肾组(P<0.05),12h补肾组高于疏肝组(P<0.05)。补肾组PR-A、HIF-1αmRNA及蛋白的表达高峰高于疏肝组。结论:补肾调经方、逍遥丸诱发排卵的机制与上调PR-A及其下游的HIF-1α的表达,从而有效调节排卵乏氧微环境有关,补肾法优于疏肝法。Objective: To explore the relationship between the mechanism of tonifying ovulation induced by Bushen Tiaojing Formula and Xiaoyao Pills and the regulation of progesterone receptor A(PR-A) and its downstream factor hypoxiainducible factor 1α(HIF-1α). Methods: Preantral follicles were isolated from 144 female 12-day-old healthy Kunming mice mechanically, which were inoculated by blank group(no serum), normal group(normal rats’ serum), Bushen group(Bushen Tiaojing Ⅱ Formula and Bushen Tiaojing Ⅲ Formula high dose serum), Shugan group(Xiaoyao Pills high, low doses serum), respectively. The survival rate of follicles, formation rate of sinus cavities and the escape rate of oocytes were observed after the hCG was added 16 hours. The real-time fluorescent quantitative PCR(RT-PCR) and immunofluorescence staining were used to detect the mRNA and protein expression of PR-A, HIF-1α in follicles and COCs after the hCG was added 0, 8, 12, 16 hours in 12th day. Results: Morphology and structure changed during follicular growth and follicular diameter increased. COCs could be seen in follicles, the sinus cavities conformed and oocyte escaped. Compared with the blank group and the normal group, the survival rate, sinus formation rate and escape rate of oocytes in Bushen group and Shugan group increased(P<0.05), Bushen group was higher than Shugan group(P<0.05). The mRNA and protein expression of PR-A and HIF-1α in each group increased gradually at 0 h, 8 h, 12 h and 16 h after hCG was added. Compared with the blank group and the normal group, Bushen group and Shugan group increased at 8 h, 12 h and 16 h(P<0.05). Shugan group at 8 h was higher than Bushen group(P<0.05), Bushen group at 12 h was higher than Shugan group(P<0.05). The peak of the mRNA and protein expression of PR-A and HIF-1α in Bushen group was higher than that in Shugan group. Conclusion: Both Bushen Tiaojing Formula and Xiaoyao Pills can up-regulate the expression of PR-A and HIF-1α to induce ovulation, which can regulate the progestrone receptor-med

关 键 词：补肾调经方 逍遥丸 排卵 孕激素受体A 乏氧诱导因子1Α 机制 乏氧微环境 排卵障碍性不孕症 

分 类 号：R285.5[医药卫生—中药学]