滋肾育胎丸对免疫性不孕模型大鼠卵母细胞生物学行为的调节作用

Regulatory Effect of Zishen Yutai Pill on the Biological Behavior of Oocytes in Immune Infertility Model Rats

目的探讨滋肾育胎丸对抗精子抗体(As Ab)阳性的不孕大鼠卵母细胞的干预作用。方法采用主动免疫建立As Ab阳性的免疫性不孕模型。将100只雌性SD大鼠随机分为空白对照组、模型组及滋肾育胎丸低、中、高剂量组(简称中药低、中、高剂量组),每组20只。造模22天开始灌胃给药,模型组给予5 m L/kg生理盐水灌胃,中药低、中、高剂量组分别给予0.075、0.100、0.150 g/m L滋肾育胎丸灌胃,每天1次,至36天。分别在造模21天及36天采用ELISA法测定As Ab和转化生长因子(transforming growth factor-β,TGF-β)水平。处死大鼠后取卵母细胞,采用实时定量聚合酶链反应(Real-time PCR)及Western blot分别检测TGF-βmRNA及其蛋白表达水平;采用流式细胞学(FCM)和末端终未标记(TUNEL)法测定卵母细胞凋亡率;采用细胞计数-8法(CCK-8)及5-乙炔基-2'脱氧尿嘧啶核苷(5-ethynyl-2'-deoxyuridine,Ed U)检测卵母细胞增殖水平;采用划痕实验及侵袭小室(Transwell)测定卵母细胞迁移与侵袭能力。结果与空白对照组比较,模型组卵母细胞凋亡增加,细胞增殖减少,迁移与侵袭能力减弱,TGF-βmRNA及其蛋白表达水平显著下调(P<0.05);与模型组比较,中药高剂量组卵母细胞凋亡减少,细胞增殖增加,迁移与侵袭能力增强,TGF-βmRNA及其蛋白表达水平显著上调(均P<0.05);且中药高剂量组作用明显优于中药低、中剂量组(P<0.05)。结论应用高剂量滋肾育胎丸干预免疫性不孕大鼠可明显上调其卵母细胞TGF-β水平,改善卵母细胞功能。

Objective To observe the intervention effect of Zishen Yutai Pill （ZYP） on the oocytes of infertile rats with positive anti-sperm antibody （AsAb）. Methods AsAb positive immune infertility model was established using active immunity. Totally 100 female SD rats were randomly divided into the blank control group, the model group, low, middle, and high dose ZYP groups, 20 in each group. Different medications were administered to rats in corresponding groups by gastrogavage from the 22nd day of modeling to the 36th day, once per day. Normal saline （5 mL/kg） was administered to rats in the model group by gastrogavage. ZYP at 0.075, 0. 100, 0. 150 g/mL was respectively administered to rats in low, middle, and high dose ZYP groups. AsAb and transforming growth factor 13 （TGF-13） levels were meas- ured by ELISA. Oocytes were collected after rats were sacrificed, mRNA and protein expression levels were detected using Real-time quantitative polymerase chain reaction （RT-PCR） and Western blot. Oocyte apoptosis rate was determined by flow cytometry （FCM） and transferase-mediated deoxyuridine triphosphate-biotin nick end labeling （TUNEL）. The oocyte proliferation level was detected by cell count- ing kit 8 （CCK-8） and 5-ethynyl-2＇-deoxyuridine （EdU）. Oocyte migration and invasion was determined u-sing invasion chamber （Transwell） and scratch experiment. Results Compared with the blank control group, the apoptosis of oocytes increased, the proliferation of oocytes was reduced, the capabilities of migration and invasion were enhanced, mRNA and protein expression levels of TGF-13 were significantly down-regulated （P 〈0.05）. Compared with the model group, the apoptosis of oocytes was reduced, the proliferation of oocytes was increased, the capabilities of migration and invasion were enhanced, mRNA and protein expression levels of TGF-13 were significantly up-regulated in the high dose ZYP group （P 〈 0.05）. Besides, their effects were obviously superior in the high dose ZYP group than in low and middle dose ZYP groups （P 〈0.05）. Conclusion The application of high dose ZYP could obviously up-regulate the level of TGF-β and improve the function of oocytes.