白藜芦醇调控Hippo信号通路对卵巢低反应小鼠卵巢保护作用的机制研究

Mechanism of resveratrol in protecting ovary in poor ovarian response mice by regulating Hippo signaling pathway

观察白藜芦醇(resveratrol,Res)通过调控Hippo信号通路对卵巢低反应(poor ovarian response,POR)小鼠卵巢功能的保护作用,探析Res抑制卵巢细胞凋亡的可能机制。将筛选后动情周期规律的雌性小鼠随机分成空白组,模型组以及Res低、高剂量组(20、40 mg·kg^(-1)),每组20只。空白组给予等体积0.9%氯化钠溶液灌胃,模型组以及Res低、高剂量组小鼠给予雷公藤多苷混悬液(50 mg·kg^(-1))灌胃2周,造模完成后,Res低、高剂量组小鼠连续灌胃治疗2周,空白组和模型组给予等体积0.9%氯化钠溶液灌胃,于治疗结束次日对各组雌鼠进行促排操作。最后每组随机选取12只雌鼠进行取材,将每组剩余的8只雌鼠与雄鼠1∶1合笼。采用阴道脱落细胞涂片观察小鼠的动情周期变化,检测小鼠获卵数、卵巢湿重、卵巢指数和妊娠率,通过酶联免疫吸附测定(enzyme-linked immunosorbent assay,ELISA)法检测血清中抗缪勒管激素(anti-Mullerian hormone,AMH)、促卵泡生成素(follicle-stimulating hormone,FSH)、雌二醇(estradiol,E_(2))、促黄体生成激素(luteinizing hormone,LH)的含量;通过苏木精-伊红(hematoxylin-eosin,HE)染色和脱氧核苷酸转移酶dUTP末端标记(terminal deoxynucleotidyl transferase dUTP nick end labeling,TUNEL)染色法观察卵巢组织形态和卵巢细胞凋亡情况;通过免疫组化法检测Yes相关蛋白(Yes-associated protein,YAP)1和带有PDZ结合基序的转录共激活因子(transcriptional coactivator with PDZ-binding motif,TAZ)的蛋白表达,通过Western blot法检测哺乳动物Ste-20样激酶(mammalian sterile 20-like kinase,MST)1/2、大型肿瘤抑制因子(large tumor suppressor,LATS)1/2、YAP1、TAZ、B淋巴细胞瘤-2(B-cell lymphoma-2,Bcl-2)、Bcl-2关联X的蛋白(Bcl-2 associated X protein,Bax)蛋白表达水平的变化。结果发现,与空白组比较,模型组小鼠动情周期紊乱率升高,卵巢组织各级正常发育卵泡数量减少,闭锁卵泡增多,卵巢颗粒细胞凋亡量提高,获卵数下降,卵巢湿重与卵巢指数降低,血清中FSH和LH含量升高,AMH和E_(2)含量下降,卵巢组织YAP1、TAZ蛋白表达水平降低,MST1/2、LATS1/2和Bax蛋白相对表达升高,YAP1、TAZ和Bcl-2蛋白相对表达降低,合笼后每胎胚胎数显著降低;与模型组比较,Res低、高剂量组小鼠动情周期紊乱率降低,卵泡数量和形态得到不同程度改善,卵泡闭锁量降低,卵巢颗粒细胞凋亡程度得到改善,获卵数升高,卵巢湿重与卵巢指数升高,血清中FSH和LH含量降低,AMH和E_(2)含量升高,卵巢组织YAP1、TAZ蛋白表达水平升高,MST1/2、LATS1/2和Bax蛋白相对表达降低,YAP1、TAZ和Bcl-2蛋白相对表达升高,合笼后每胎胚胎数不同程度升高。综上,Res可以有效抑制小鼠卵巢细胞凋亡,改善卵巢反应性,其机制可能与调节Hippo通路中的关键分子有关。

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 levels 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.