白藜芦醇对卵巢低反应小鼠保护作用的机制研究

Mechanism of protective effect of resveratrol on poor ovarian response in mice

探讨白藜芦醇(resveratrol,Res)对卵巢低反应(poor ovarian response,POR)小鼠的治疗作用和潜在机制。通过网络药理学收集Res和POR的共有靶点基因,使用基因本体论(Gene Ontology,GO)分析和京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)通路富集分析筛选关键的信号通路,并通过动物实验验证。将筛选后动情周期规律的小鼠随机分成正常组、POR组以及Res低、高剂量组。正常组给予等体积的0.9%氯化钠溶液灌胃,POR组以及Res低、高剂量(20、40 mg·kg^(-1))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、高剂量组小鼠给予连续灌胃治疗2周,正常组和POR组给予等体积的0.9%氯化钠溶液灌胃。治疗结束次日各组小鼠开始促排卵并完成取材。采用阴道脱落细胞涂片观察小鼠的动情周期变化,检测小鼠的获卵数、卵巢湿重与卵巢指数,通过酶联免疫吸附剂测定(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;通过苏木素-伊红(hematoxylin-eosin,HE)染色和脱氧核苷酸转移酶dUTP末端标记(terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling,TUNEL)染色法观察卵巢组织形态和卵巢颗粒细胞凋亡情况;通过Western blot法检测磷脂酰肌醇3-激酶(phosphoinositide 3-kinase,PI3K)、蛋白激酶B(protein kinase B,AKT)、叉头盒O(forkhead box O,FOXO)3a、缺氧诱导因子(hypoxia-inducible factor,HIF)-1α、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。最终收集到Res和POR交集靶点222个;GO富集分析发现,Res-POR参与的生物过程(biological process)主要是氧化应激反应。KEGG富集分析发现,Res可以通过PI3K/AKT、HIF-1、FOXO等信号通路参与POR治疗。动物实验表明,与正常组比较,POR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且形态较差,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HIF-1α、FOXO3a和Bax蛋白表达水平升高,PI3K、AKT和Bcl-2蛋白表达水平降低;与POR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,卵泡闭锁数量降低,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提高,血清中FSH和LH含量降低、AMH和E_(2)含量升高,卵巢组织HIF-1α、FOXO3a和Bax蛋白表达水平降低,PI3K、AKT和Bcl-2蛋白表达水平升高。综上所述,Res可以抑制小鼠卵巢细胞凋亡,改善卵巢反应下降,其机制可能与调节PI3K/AKT/FOXO3a和HIF-1α通路中的关键分子有关。

This study aims to investigate the therapeutic effects and potential mechanisms of resveratrol(Res) on poor ovarian response(POR) in mice.The common target genes shared by Res and POR were predicted by network pharmacology,used for Gene Ontology(GO) annotation and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment,and then validated by animal experiments.The mice with regular estrous cycle after screening were randomized into normal,POR,and low-and high-dose(20 and 40 mg·kg~(-1),respectively) Res groups.The normal group was administrated with an equal volume of 0.9% sodium chloride solution by gavage,and the mice in other groups with tripterygium glycosides suspension(50 mg·kg~(-1)) by gavage for 2 weeks.After the modeling,the mice in low-and high-dose Res groups were treated with Res by gavage for 2 weeks,and the mice in normal and POR groups with an equal volume of 0.9% sodium chloride solution by gavage.Ovulation induction and sample collection were carried out on the day following the end of treatment.Vaginal smears were collected for observation of the changes in the estrous cycle,the counting of retrieved oocytes,and the measurement of ovarian wet weight and ovarian index.The enzyme-linked immunosorbent assay(ELISA) was employed to measure the levels of anti-mullerian hormone(AMH),follicle-stimulating hormone(FSH),estradiol(E_2),and luteinizing hormone(LH) in the serum.The ovarian tissue morphology and granulosa cell apoptosis were observed by hematoxylin-eosin(HE) staining and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling(TUNEL),respectively.Western blot was employed to determine the protein levels of phosphatidylinositol 3-kinase(PI3K),protein kinase B(AKT),forkhead box O(FOXO) 3a,hypoxia-inducible factor(HIF)-1α,B-cell lymphoma-2(Bcl-2),and Bcl-2-associated X protein(Bax).A total of 222 common targets shared by Res and POR were collected.GO annotation indicated that these targets were mainly involved in oxidative stress response.KEGG enrichment analysis revealed that Res can intervene in POR via PI3K/AKT,HIF-1,and FOXO signaling pathways.Animal experiments showed that the model group had higher rate of estrous cycle disorders,lower number and poorer morphology of normally developed follicles at all levels,more atretic follicles,higher apoptosis of ovarian granulosa cells,lower number of retrieved oocytes,lower ovarian wet weight and ovarian index,higher serum levels of FSH and LH,lower levels of AMH and E_2,higher expression levels of HIF-1α,FOXO3a and Bax,and lower expression levels of PI3K,AKT,and Bcl-2 in the ovarian tissue than the normal group.Compared with the POR group,low-and high-dose Res decreased the rate of estrous cycle disorders,improved the follicle number and morphology,reduced atretic follicles,promoted the apoptosis of ovarian granulosa cells,increased retrieved oocytes,ovarian wet weight and ovarian index,and lowered serum FSH and LH levels.Moreover,Res down-regulated the expression levels of HIF-1α,FOXO3a and Bax,and up-regulated the expression levels of PI3K,AKT and Bcl-2 in the ovarian tissue.In summary,Res can inhibit apoptosis and mitigate poor ovarian response in mice by regulating the PI3K/AKT/FOXO3a and HIF-1α pathways.