本研究通过调节Hippo信号通路观察白藜芦醇(Res)对卵巢低反应(POR)小鼠卵巢功能的保护作用,探讨Res抑制卵巢细胞凋亡的潜在机制。雌性小鼠有规律的发情周期,随机分为空白组,一个模型组,以及低剂量和高剂量Res组(20和40mg·kg〜(-1)),每组20只小鼠。空白组接受等体积的0.9%生理盐水灌胃,模型组和Res组给予50mg·kg〜(-1)雷公藤多苷(GTW)混悬液,灌胃两周诱导模型。建模后,低、高剂量Res组连续灌胃给药2周,空白组和模型组灌胃等体积的0.9%生理盐水。在治疗结束后的第二天,在所有组中诱导排卵。最后,每组随机抽取12只雌性小鼠,其余八只雌性小鼠与雄性小鼠以1∶1的比例饲养。使用阴道细胞学涂片观察小鼠发情周期的变化。排卵卵的数量,卵巢湿重,卵巢指数,并测量小鼠的妊娠率。抗苗勒管激素(AMH)的水平,卵泡刺激素(FSH),雌二醇(E_2),采用酶联免疫吸附试验(ELISA)测定血清中黄体生成素(LH)含量。采用苏木精-伊红(HE)染色和末端脱氧核苷酸转移酶dUTP缺口末端标记(TUNEL)染色观察卵巢组织形态和卵巢细胞凋亡,分别。免疫组织化学(IHC)检测Yes相关蛋白(YAP)1和PDZ结合基序转录共激活因子(TAZ)的蛋白表达水平,而哺乳动物无菌20样激酶(MST)1/2、大肿瘤抑制因子(LATS)1/2、YAP1、TAZ、B细胞淋巴瘤-2(Bcl-2),Westernblot检测Bcl-2相关X蛋白(Bax)。结果表明,与空白组相比,模型组小鼠动情周期破坏率增加,正常发育的卵巢卵泡数量减少,卵巢卵泡阻塞的数量增加,卵巢颗粒细胞凋亡增加,排卵减少,降低卵巢湿重和卵巢指数,血清FSH和LH水平升高,AMH和E_2水平降低,卵巢组织中YAP1和TAZ蛋白表达水平降低,MST1/2、LATS1/2和Bax蛋白的相对表达水平增加,YAP1,TAZ的相对表达水平降低,和Bcl-2蛋白。此外,共安置后,每窝的胚胎数量显着减少。与模型组相比,低剂量和高剂量Res组小鼠的动情周期破坏率降低,卵泡的数量和形态都有不同程度的改善,减少卵巢卵泡阻塞的数量,改善卵巢颗粒细胞凋亡,排卵增加,卵巢湿重和卵巢指数升高,血清FSH和LH水平降低,AMH和E_2水平升高,卵巢组织中YAP1和TAZ蛋白表达水平升高,MST1/2、LATS1/2和Bax蛋白的相对表达水平降低,和增加YAP1,TAZ的相对表达水平,和Bcl-2蛋白。此外,共安置后,每窝的胚胎数量都有不同程度的增加。总之,Res能有效抑制小鼠卵巢细胞凋亡,提高卵巢反应性。其机制可能与Hippo通路中关键分子的调控有关。
This study observed the protective effect of resveratrol(Res) on ovarian function in poor ovarian response(POR) mice by regulating the Hippo signaling pathway and explored the potential mechanism of Res in inhibiting ovarian cell apoptosis. Female mice with regular estrous cycles were randomly divided into a blank group, a model group, and low-and high-dose Res groups(20 and 40 mg·kg~(-1)), with 20 mice in each group. The blank group received an equal volume of 0.9% saline solution by gavage, while the model group and Res groups received suspension of glycosides of Triptergium wilfordii(GTW) at 50 mg·kg~(-1) by gavage for two weeks to induce the model. After modeling, the low-and high-dose Res groups were continuously treated with drugs by gavage for two weeks, while the blank group and the model group received an equal volume of 0.9% saline solution by gavage. Ovulation was induced in all groups on the day following the end of treatment. Finally, 12 female mice were randomly selected from each group, and the remaining eight female mice were co-housed with male mice at a ratio of 1∶1. Changes in the estrous cycle of mice were observed using vaginal cytology smears. The number of ovulated eggs, ovarian wet weight, ovarian index, and pregnancy rate of mice were measured. The le-vels of anti-Mullerian hormone(AMH), follicle-stimulating hormone(FSH), estradiol(E_2), and luteinizing hormone(LH) in serum were determined using enzyme-linked immunosorbent assay(ELISA). Ovarian tissue morphology and ovarian cell apoptosis were observed using hematoxylin-eosin(HE) staining and terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL) staining, respectively. The protein expression levels of yes-associated protein(YAP) 1 and transcriptional coactivator with PDZ-binding motif(TAZ) were detected by immunohistochemistry(IHC), while the changes in protein expression levels of mammalian sterile 20-like kinase(MST) 1/2, large tumor suppressor(LATS) 1/2, YAP1, TAZ, B-cell lymphoma-2(Bcl-2), and Bcl-2 associated X protein(Bax) were determined by Western blot. The results showed that compared with the blank group, the model group had an increased rate of estrous cycle disruption in mice, a decreased number of normally developing ovarian follicles, an increased number of blocked ovarian follicles, increased ovarian granulosa cell apoptosis, decreased ovulation, reduced ovarian wet weight and ovarian index, increased serum FSH and LH levels, decreased AMH and E_2 levels, decreased protein expression levels of YAP1 and TAZ in ovarian tissues, increased relative expression levels of MST1/2, LATS1/2, and Bax proteins, and decreased relative expression levels of YAP1, TAZ, and Bcl-2 proteins. Additionally, the number of embryos per litter significantly decreased after co-housing. Compared with the model group, the low-and high-dose Res groups exhibited reduced estrous cycle disruption rates in mice, varying degrees of improvement in the number and morphology of ovarian follicles, reduced numbers of blocked ovarian follicles, improved ovarian granulosa cell apoptosis, increased ovulation, elevated ovarian wet weight and ovarian index, decreased serum FSH and LH levels, increased AMH and E_2 levels, elevated protein expression levels of YAP1 and TAZ in ovarian tissues, decreased relative expression levels of MST1/2, LATS1/2, and Bax proteins, and increased relative expression levels of YAP1, TAZ, and Bcl-2 proteins. Furthermore, the number of embryos per litter increased to varying degrees after co-housing. In conclusion, Res effectively inhibits ovarian cell apoptosis in mice and improves ovarian responsiveness. Its mechanism may be related to the regulation of key molecules in the Hippo pathway.