Effect of Mitochondrial Function of Ovarian Granulosa Cells on In Vitro Fertilization and Embryo Transfer Outcomes in Obese Polycystic Ovary Syndrome Patients

Objective:To investigate the effect of abnormal ovarian granulosa cell metabolism on in vitro fertilization and embryo transfer(IVF-ET)outcomes in obese polycystic ovary syndrome(PCOS)patients.Methods:Patients with PCOS who met the study criteria were screened according to the inclusion criteria.A total of 32 patients with obese PCOS were recruited into the study group,and 39 patients with non-obese PCOS were recruited into the control group.The general data(age,body mass index,and years of infertility),insulin resistance index(HOMA-IR),follicle-stimulating hormone(FSH),luteinizing hormone(LH),granulosa cell mitochondrial function,and IVF-ET outcome of patients in the study group and control group were retrospectively analyzed.Results:The differences in age and years of infertility between the study group and the control group were insignificant(P>0.05),and the body mass index(BMI)of the study group and control group was 30.5±1.24 kg/m2 and 22.3±1.12 kg/m2,respectively,in which the difference was statistically significant(P<0.05);the HOMA-IR of the study group was significantly higher than that of the control group(P<0.05);the reactive oxygen species(ROS)in the study group was significantly higher than that in the control group(P<0.05),and the ATP content in the study group was significantly lower than that in the control group(P<0.05);comparing the FSH and LH levels between the two groups,the difference was not statistically significant(P>0.05);the rate of IVF-ET failure was significantly higher in the study group than in the control group.Conclusion:PCOS is a complex endocrine disorder,and obesity is one of the independent risk factors for the development of PCOS.

Effects of Qigongwan on Wnt/β-catenin Signaling Pathway in Rats with Polycystic Ovary syndrome

[Objectives] To explore the therapeutic effect and mechanism of Qigongwan on PCOS model rats by detecting the changes in sex hormone levels in rats with polycystic ovary syndrome (PCOS), and observing the effects of ovarian pathological morphological changes, apoptosis and expression of Wnt/β-β catenin signaling pathway protein. [Methods] Ten of 40 female SD rats were randomly selected as the normal group, and the other 30 rats were treated with letrozole combined with high-fat diet to establish the PCOS rat model. After successful modeling, the model group was randomly divided into Qigongwan group, positive Daying-35 (Ethinylestradiol and Cyproterone Acetate Tablets) group and model group, with 10 rats in each group. Qigongwan group was given 14.7 g/(kg·d) by gavage, Daying-35 group was given 0.21 mg/(kg·d) by oral gavage, and normal group and model group were given the same amount of distilled water, and the intervention lasted for 21 d. ELISA method was used to detect the levels of hormones such as follicle-stimulating hormone (FSH), luteinizing hormone (LH), testosterone (T), estradiol (E 2) and progesterone (P) in serum. HE staining was used to observe the pathological morphological changes of ovarian tissues;TUNEL staining was used to observe apoptosis of ovarian tissue granule cells;the expression of Wnt, β-catenin protein in rat ovarian tissue was detected by immunohistochemistry. [Results] (i) Compared with the model group, Qigongwan group and Daying-35 group could significantly increase serum E 2 and P levels, significantly reduce serum T levels ( P <0.01), significantly reduce serum LH levels and LH/FSH ratio ( P <0.01), and increase serum FSH levels ( P <0.05) in different degrees. (ii)The results of HE staining showed that compared with the model group, Qigongwan and Daying-35 groups could improve follicular development and reduce atretic follicles in different degrees. Compared with Daying-35 group, the number of GC layers in Qigongwan group was significantly increased. (iii) The results of TUNEL staining showed that compared with the model group, the rate of TUNEL-positive cells in the Qigongwan group and Daying-35 group decreased significantly ( P <0.01). (iv) The immunohistochemical results showed that compared with the model group, the expression levels of wnt and β-catenin in the Qigongwan group and the Daying-35 group increased in different degrees ( P <0.05), and the expression range increased. [Conclusions] Qigongwan can regulate the secretion level of sex hormones such as FSH and LH, improve the pathological damage of ovarian tissue, and inhibit apoptosis of ovarian granule cells, and its mechanism may be related to the activation of Wnt/β-catenin signaling pathway.

Expression of Leptin Long-form Receptor mRNA in Luteinized Granulosa Cells of Obese Women with Polycystic Ovary Syndrome

To investigate the expression of mRNA of leptin long-form receptor （OB-Rb） in luteinized granulosa ceils of obese women with polycystic ovary syndrome （PCOS）, and to determine the role of leptin in the physiopathology of PCOS, luteinized granulosa cells were collected from the follicle fluid of 10 obese women who met the diagnostic criteria for PCOS and their BMI was equal to or greater than 25 kg/m^2, and at the same time, granulosa cells were collected from 10 normal women undergoing IVF-ET who served as the control group. Some luteinized granulosa cells were taken from normal women for in-vitro culture, into which human leptin of different concentrations was added （0, 10, 100 and 1000 ng/mL）. After stimulation with leptin for 48 h, RT-PCR was employed for the detection of the expression of OB-RLInRNA in the luteinized granulosa cells. Our results showed that the level of OB-RLmRNA in luteinized granulosa cells of obese PCOS women was higher than those in the control （P〈0.05）. In luteinized granulosa cells cultured in vitro and stimulated by human leptin for 48 h, the level of OB-RLmRNA was higher than those without leptin stimulation （P〈0.01）, and when leptin concentration was at 100 ng/mL, and the level of OB-RLmRNA reached a peak, It is concluded that in obese PCOS women, the level of serum leptin is increased, which promotes the expression of OB-RL in luteinized granulosa cells and increases the sensitivity of the granulosa cells to leptin. Leptin may contribute to anovulation in obese women with PCOS.

Apoptosis and Expression of Protein TRAIL in Granulosa Cells of Rats with Polycystic Ovarian Syndrome

The relationship between apoptosis of granulosa cells and follicle development arrest in polycystic ovarian syndrome (PCOS) rats, and the contribution of tumor necrosis factor related apoptosis inducing ligand (TRAIL) in apoptosis of granulosa cells were explored. By using sodium prasterone sulfate rat PCOS model was induced. The apoptosis of granulosa cells in ovaries of rats was observed by TdT-mediated dUTP-biotin nick end-labeling (TUNEL), and the expression of TRAIL protein and mRNA in granulosa cells was detected by using immunhistochemical staining and reverse transcription polymerase chain reaction (RT-PCR) respectively. The apoptotic rate and the expression of protein TRAIL in granulosa cells were significantly higher in antral follicles from the PCOS rats than in those from the control rats (P<0.01, P<0.05). There was no significant difference in apoptotic rate and the expression of TRAIL protein in granulosa cells of preantral follicles between the PCOS rats and the control rats (P>0.05). No apoptosis and the expression of TRAIL protein in granulosa cells of primordial follicles were found in the two groups. The expression of TRAIL mRNA was significantly stronger in granulosa cells from the PCOS rats than in those from the con- trol rats (P<0.01). It was suggested that the apoptotic rate in granulosa cells was significantly higher in antral follicle from the PCOS rats than in those from the control rats. TRAIL played a role in regu- lating the apoptosis of granulosa cells in PCOS rats.

Observation on human ovarian theca cell and granulosa cell interaction in polycystic ovary syndrome

Objective: To investigate the role of human theca cell(TC) and granulo sa cell(GC) interaction and insulin(INS) in steroidogenesis in normal ovarian cy cle and in patients with PCOS. Methods: Ovarian theca and granulosa cells from eleven normal wo men and eight PCOS patients were co-cultured on opposite side of collagen with or without INS. The concentrations of estradiol(E2), progesterone(P) and andro stenedione (A) in the culture medium were examined by ELISA method.Results: When co-cultured with GC, TC in PCOS group produced mo re A and less P than those of normal group. When co-cultured with theca cells, granulosa cells in PCOS group produced more E2 than those of normal group. Add ition of INS increased the difference significantly.Conclusions: The GC and TC interaction from the normal and PCOS ovaries is different. There is a high A and high E2 intraovary loop of PC OS leading to premature arrest of follicle growth and anovulation. Insulin may p lay an important regulatory role.

Effect of Electro-acupuncture on Expression of IRS-1/PI3K/GLUT4 Pathway in Ovarian Granulosa Cells of Infertile Patients with Polycystic Ovary Syndrome-Insulin Resistance of Phlegm-Dampness Syndrome

Objective:To evaluate the effect of electro-acupuncture(EA)in infertile patients with phlegmdampness polycystic ovary syndrome-insulin resistance(PCOS-IR).Methods:Seventy-six PCOS-IR patients who underwnet in vitro fertilization and embryo transfer(IVF-ET)were equally assigned to two groups according to a random digital table:the EA group and the control group,with 38 cases in each group.Before undergoing IVF,the two groups were treated with EA or pseudo-acupuncture,respectively,for 3 menstrual cycles.The intervention was 25 min twice a week until the day of oocyte collection.The selected acupoints were Zhongwan(RN 12),Tianshu(ST 25),Daheng(SP 15),Daimai(GB 26),Qihai(CV 6),Guanyuan(CV 4),and bilateral points including Xuehai(SP 10),Fenglong(ST 40),Zusanli(ST 36),and Yinlingquan(SP 9).Evaluation of phlegm-dampness syndrome score and IR score were carried out before and after treatment.Additionally,the number of oocytes retrieved,transplantable embryo rate,high-quality embryo rate,clinical pregnancy rate and live birth rate were compared between the two groups.Real-time polymerase chain reaction analysis was used to monitor the m RNA expression of the insulin receptor substrate(IRS-1),phosphatidylinositiol 3-kinase(PI3 K)and glucose transport factor 4(GLUT4)in ovarian granulosa cells.Results:EA treatment reduced the phlegm-dampness syndrome score as well as the IR scores compared with the control group(P<0.05).No significant differences in the number of oocytes retrieved and clinical pregnancy rate between the two groups(P>0.05).Moreover,the transplantable embryo rate[49.0%(284/580)vs.41.9%(273/652)],high-quality embryo rate[36.6%(104/284)vs.27.8%(76/273)],and live birth rate[50%(19/38)vs.26.3%(10/38)]in the EA group were significantly higher than in the control group(P<0.05).Gene expression analyses revealed significantly elevated IRS-1,PI3 K and GLUT4 m RNA in ovarian granulosa cells of the EA group compared with the control group(P<0.05).Conclusions:EA may ameliorate the effects of phlegm-dampness syndrome and ovarian IR in PCOS-IR patients.Mechanistically,this effect might be through an upregulation of the IRS-1/PI3 K/GLUT4 signaling pathway,which may result in improved oocyte quality and embryonic development potential.(Registration No.Chi CTR1800015453)

启宫丸对多囊卵巢综合征大鼠WNT/β-catenin信号通路的影响

目的本实验通过检测启宫丸对多囊卵巢综合征大鼠性激素水平变化,观察卵巢病理组织形态改变、凋亡状况及Wnt/β-连环蛋白(β-catenin)信号通路蛋白表达的影响,探究启宫丸对多囊卵巢综合征模型大鼠的治疗作用及机制。方法40只SD雌性大鼠,随机选取10只作为正常组,其余30只采用来曲唑联合高脂饲料法制备多囊卵巢综合征大鼠模型。造模成功后,将造模组随机分为中药启宫丸组、阳性药达英-35组、模型组,每组各10只。启宫丸组给予14.7 g/(kg·d)灌胃,达英-35组给予0.21 mg/(kg·d)口服灌胃,正常组和模型组给予等量蒸馏水,干预持续21天。ELISA法检测血清中促卵泡刺激素(follicle-stimulating hormone,FSH)、促黄体生成素(luteinizing hormone,LH)、睾酮(testosterone、T)、雌二醇(estradiol,E 2)及孕酮(progesterone,P)等激素水平;HE染色观察卵巢组织病理形态变化;TUNEL染色观察卵巢组织颗粒细胞凋亡情况;免疫组化法检测大鼠卵巢组织Wnt、β-catenin蛋白的表达。结果(1)与模型组相比,启宫丸组及达英-35组血清E 2及P水平显著升高,血清T水平显著降低(P<0.01),血清LH水平及LH/FSH比值显著降低(P<0.01),以及可不同程度升高血清FSH水平(P<0.05)。(2)HE染色结果显示:与模型组相比,启宫丸组达英-35组可不同程度改善卵泡发育,减少闭锁卵泡。与达英-35组相比,启功丸组GC层数明显增加。(3)TUNEL染色结果显示:与模型组相比,启宫丸组和达英-35组TUNEL阳性细胞率明显下降(P<0.01)。(4)免疫组化结果显示:与模型组相比启宫丸组和达英-35组wnt及β-catenin表达水平均有不同程度上升(P<0.05),表达范围增加。结论启宫丸可调节FSH、LH等性激素分泌水平,改善卵巢组织病理损伤,抑制卵巢颗粒细胞凋亡,其机制可能与激活Wnt/β-catenin信号通路有关。

微RNA-93-5p对多囊卵巢综合征患者卵巢颗粒细胞增殖的影响及机制

目的探讨血浆微RNA(miR)-93-5p对多囊卵巢综合征(PCOS)患者卵巢颗粒细胞增殖的影响及其可能机制。方法选择2022年1月至2023年1月焦作市人民医院收治的120例育龄期PCOS患者为PCOS组,选择同期在本院体检的18例育龄期健康女性为对照组。收集2组受试者的年龄、体质量和身高,计算体质量指数(BMI)。2组受试者均在自然月经周期的卵泡期或黄体酮诱导的撤退性出血期采集空腹静脉血,采用电化学法测定血清黄体生成素(LH)、促卵泡激素(FSH)、总睾酮(TT)、抗米勒管激素(AMH)、天冬氨酸转氨酶(AST)、丙氨酸转氨酶(ALT)、空腹胰岛素(FINS)、空腹血糖(FBG)水平,并计算胰岛素抵抗稳态模型(HOMA-IR);采用实时荧光定量聚合酶链反应(qRT-PCR)检测2组受试者血浆中miR-93-5p表达水平。取对数生长期人卵巢颗粒细胞KGN,以每孔3×10^(5)个细胞接种至6孔板中,随机分为miR-93-5p mimics组、LY294002+miR-93-5p组、AZD5363+miR-93-5p组、阴性对照(NC)组、空白对照组。miR-93-5p组KGN细胞转染miR-93-5p mimics;LY294002+miR-93-5p组KGN细胞转染miR-93-5p mimics,并于转染前1 h给予50 mmol·L^(-1) LY294002处理;AZD5363+miR-93-5p组KGN细胞转染miR-93-5p mimics,并于转染前1 h给予50μmol·L^(-1) AZD5363处理;NC组KGN细胞转染阴性对照质粒;空白对照组KGN细胞不做任何处理。应用qRT-PCR法检测各组KGN细胞中miR-93-5p表达,细胞计数试剂盒-8试验检测各组KGN细胞增殖情况。结果PCOS组与对照组受试者的年龄及FSH、ALT、AST水平比较差异无统计学意义(P>0.05);PCOS组患者的BMI、TT、AMH、LH、FINS、FBG、HOMA-IR显著高于对照组(P<0.05)。PCOS组患者血浆中miR-93-5p相对表达量显著高于对照组(t=-5.549,P<0.001)。miR-93-5p与TT、FINS、HOMA-IR呈中度正相关(r=0.434、0.622、0.586,P<0.001),与FBG和LH呈低度正相关(r=0.398、0.398,P<0.001)。ROC曲线显示,血浆miR-93-5p诊断PCOS的最佳临界值为1.380,曲线下面积为0.906(95%置信区间:0.839~0.973,P<0.001),敏感度为0.858,特异度为0.833,约登指数为0.691。miR-93-5p mimics组、LY294002+miR-93-5p组、AZD5363+miR-93-5p组KGN细胞中miR-93-5p相对表达量均显著高于空白对照组和NC组(P<0.05)。培养24、48、72 h时,miR-93-5p mimics组、LY294002+miR-93-5p组、AZD5363+miR-93-5p组KGN细胞的增殖能力显著高于空白对照组和NC组(P<0.05);LY294002+miR-93-5p组、AZD5363+miR-93-5p组细胞的增殖能力显著低于miR-93-5p mimics组(P<0.05)。结论PCOS患者血浆miR-93-5p呈过表达,miR-93-5p可能通过基于调控磷脂酰肌醇-3-激酶/蛋白激B信号通路介导PCOS卵巢颗粒细胞增殖来参与PCOS的发生发展,血浆miR-93-5p水平对PCOS有一定的诊断价值。

多囊卵巢综合征患者卵巢颗粒细胞miR-144-3p及其靶基因PTEN的表达及作用研究

目的探讨多囊卵巢综合征患者(polycystic ovary syndrome,PCOS)卵巢颗粒细胞miR-144-3p及其靶基因PTEN的表达及作用。方法选取2020年10月至2022年3月在淮安市妇幼保健院生殖医学科行IVF/ICSI治疗的20例PCOS患者作为研究对象即为PCOS组,选取行IVF/ICSI治疗的输卵管因素或男方因素患者20例作为对照组。收集两组患者颗粒细胞,并分析颗粒细胞miR-144-3p的表达情况。利用Tar-getScan数据库预测miR-144-3p的靶基因,并采用双荧光素酶活性检测验证靶基因。复苏人卵巢颗粒细胞(ovarian granulosa cells,KGN),转染并建立miR-144-3p模拟物组、模拟物阴性对照组、miR-144-3p抑制物组、抑制物阴性对照组、si-PTEN和siRNA-NC组。采用流式细胞仪检测各组的细胞凋亡情况,RT-PCR及蛋白质印迹技术检测颗粒细胞中miR-144-3p、PTEN mRNA及蛋白的表达情况。结果RT-PCR结果显示P-COS组miR-144-3p表达水平显著低于对照组(P<0.05),流式细胞仪检测结果显示miR-144-3p mimic组的颗粒细胞凋亡百分比显著低于mimic-NC组(P<0.05),miR-144-3p inhibitor组颗粒细胞凋亡水平显著高于inhibitor-NC组(P<0.05)。生物信息学预测miR-144-3p的靶基因为PTEN,荧光素酶结合实验证实miR-144-3p能特异结合PTEN-3′UTR并下调PTEN基因表达。RT-PCR测定结果显示PCOS组PTEN mRNA表达显著高于对照组(P<0.05),且与miR-144-3p表达水平呈负相关(r=-0.91,P<0.001)。qRT-PCR及蛋白质印迹检测结果显示与mimic NC组相比,miR-144-3p mimic组中PTEN mRNA及蛋白表达量显著低于mimic NC组(P<0.05)。miR-144-3p inhibitor组PTEN mRNA及蛋白表达量显著高于inhibitor NC组(P<0.05)。流式细胞仪检测结果显示si-PTEN组颗粒细胞凋亡水平显著低于siRNA-NC组(P<0.05),而miR-144-3p inhibitor+si-PTEN组的颗粒细胞凋亡比例显著低于miR-144-3p inhibitor组(P<0.05)。结论PCOS患者卵巢颗粒细胞miR-144-3p表达水平显著降低,而PTEN基因表达水平显著增高,进而促进PCOS患者卵巢颗粒细胞凋亡。

多囊卵巢综合征患者程序性细胞死亡因子4表达及睾酮与胚胎质量相关性研究

目的:探讨控制性超促排卵(COH)中多囊卵巢综合征(PCOS)患者卵巢颗粒细胞中程序性细胞死亡因子4(PDCD4)表达及睾酮的临床意义。方法:选取于枣庄市妇幼保健院生殖遗传中心行体外受精-胚胎移植(IVF-ET)助孕的COH周期PCOS患者(28例)和非PCOS患者(40例),经阴道超声引导下取卵后,收集卵泡液及卵丘周围颗粒细胞,提取RNA,逆转录后应用实时定量PCR检测PDCD4表达情况,分析PDCD4表达及睾酮与胚胎质量的相关性。结果:PCOS组和非PCOS组患者的年龄、不孕年限、体质量指数(BMI)、基础FSH、E_(2)、PRL和睾酮比较,差异均无统计学意义(P>0.05)。两组的正常受精率、卵裂率、优胚率、可用胚率和卵母细胞利用率比较,差异均无统计学意义(P>0.05)。PCOS组的PDCD4表达略高于非PCOS组,但差异无统计学意义(P>0.05)。低可用胚组的PDCD4表达高于正常可用胚组(P=0.04)。高睾酮水平组的正常受精率(P=0.00007)和卵母细胞利用率(P=0.038)低于低睾酮水平组。结论:COH中卵巢颗粒细胞PDCD4表达增高与低可用胚率相关,睾酮水平与受精率和卵母细胞利用率密切相关,睾酮水平升高,受精率和卵母细胞利用率降低。

苍附导痰汤对肥胖型PCOS大鼠内分泌激素及miRNA-16和PDCD-4表达的影响

目的:探讨苍附导痰汤对肥胖型多囊卵巢综合征(PCOS)大鼠内分泌激素及微小RNA(miRNA)-16和程序性细胞死亡因子4(PDCD-4)表达的影响。方法:将50只SPF级雌性SD大鼠随机分为5组,每组10只。阳性对照组大鼠灌胃格华止(0.43 g/kg),低剂量组大鼠灌胃低剂量苍附导痰汤(1.42 g/kg),高剂量组大鼠灌胃高剂量苍附导痰汤(5.68 g/kg),模型组和正常组大鼠灌胃等量生理盐水,各组均持续给药14 d。比较各组大鼠体质量,内分泌激素水平,miRNA-16和PDCD-4 mRNA表达,以及PDCD-4蛋白表达。结果:模型组、阳性对照组、低剂量组和高剂量组大鼠体质量均高于正常组(P<0.05);阳性对照组、低剂量组和高剂量组大鼠体质量均低于模型组(P<0.05),且呈剂量依赖性。模型组、阳性对照组、低剂量组和高剂量组大鼠血清E_(2)均水平低于正常组,而血清T、FSH和LH水平均高于正常组(P<0.05);阳性对照组、低剂量组和高剂量组大鼠血清E_(2)水平均高于模型组,而血清T、FSH和LH水平均低于模型组(P<0.05),且呈剂量依赖性。模型组、阳性对照组、低剂量组和高剂量组大鼠卵巢miRNA-16表达均低于正常组,而PDCD-4 mRNA表达高于正常组(P<0.05);阳性对照组、低剂量组和高剂量组大鼠卵巢miRNA-16表达均高于模型组,而PDCD-4 mRNA表达低于模型组(P<0.05),且呈剂量依赖性。模型组、阳性对照组、低剂量组和高剂量组大鼠卵巢PDCD-4蛋白相对表达量均高于正常组(P<0.05);阳性对照组、低剂量组和高剂量组大鼠卵巢PDCD-4蛋白相对表达量均低于模型组(P<0.05),且呈剂量依赖性。结论:苍附导痰汤可调节肥胖型PCOS大鼠内分泌激素水平,其机制可能与上调miRNA-16表达及下调PDCD-4表达有关。

脐带间充质干细胞治疗多囊卵巢综合征的作用及机制

背景:目前对多囊卵巢综合征的治疗大多是超指征用药,对其治疗仍面临很大的挑战。研究证明人脐带间充质干细胞可修复卵巢功能,但鲜有研究报道其对多囊卵巢综合征的治疗作用。目的:观察人脐带间充质干细胞对多囊卵巢综合征的治疗作用,并初探线粒体自噬与人脐带间充质干细胞改善多囊卵巢综合征症状的相关性。方法:C57BL/6J小鼠皮下连续注射20 d脱氢表雄酮构建多囊卵巢综合征模型,通过尾静脉注射人脐带间充质干细胞(2×10^(6)),治疗后连续10 d取阴道内分泌物检测小鼠的发情周期。治疗后2周,采用ELISA检测小鼠外周血性激素水平,包括黄体生成素和卵泡刺激素;苏木精-伊红染色进行卵巢组织病理学评估,最后通过透射电镜观察卵巢组织中线粒体自噬反应。结果与结论:(1)经人脐带间充质干细胞治疗后,多囊卵巢综合征小鼠卵巢中出现了处在原始卵泡、初级卵泡、次级卵泡等各阶段的卵泡,而且可见黄体组织,说明小鼠排卵功能明显改善;(2)经人脐带间充质干细胞治疗后,多囊卵巢综合征小鼠性激素水平恢复至正常;(3)未经治疗的多囊卵巢综合征小鼠长期处在动情期,缺乏动情间期和动情前期,经过人脐带间充质干细胞治疗的多囊卵巢综合征小鼠动情周期恢复到正常水平;(4)经人脐带间充质干细胞治疗后,多囊卵巢综合征小鼠卵巢组织中的线粒体自噬明显减少;(5)结果表明,人脐带间充质干细胞可有效改善多囊卵巢综合征小鼠内分泌紊乱,促进排卵,这可能与抑制线粒体自噬相关。

多囊卵巢综合征病理机制中的颗粒细胞自噬

多囊卵巢综合征(polycystic ovary syndrome,PCOS)是育龄期女性最常见的生殖内分泌紊乱性疾病之一,病理机制尚未阐明。自噬参与卵泡发育的各个阶段,其中卵泡颗粒细胞自噬相对活跃。通过维持颗粒细胞自噬的稳态支持卵巢功能、调控卵泡发育,最终影响胚胎质量及妊娠结局。自噬相关基因和蛋白的异常表达可激活不同的自噬通路,抑制卵泡颗粒细胞发育、干扰内分泌稳态,参与PCOS的病理过程,并影响其预后。综述参与颗粒细胞自噬的关键分子及其在PCOS发生发展中的作用,以期为研究PCOS发生机制及探索临床治疗提供参考。

毛兰素对多囊卵巢综合征大鼠卵巢颗粒细胞凋亡的影响及机制

目的 探究毛兰素(ERI)对多囊卵巢综合征(PCOS)大鼠卵巢颗粒细胞凋亡的影响及机制。方法 通过皮下注射脱氢表雄酮构建PCOS大鼠模型,将造模成功的大鼠随机分为PCOS组,ERI低、中、高剂量组(10、20、40 mg/kg)和ERI高剂量+维替泊芬组(40 mg/kg ERI+10 mg/kg维替泊芬),每组10只;另取10只正常大鼠作为正常组。各给药组大鼠灌胃相应剂量的ERI和/或腹腔注射维替泊芬,PCOS组和正常组大鼠灌胃等体积的1%二甲基亚砜,每日1次,连续6周。给药结束后,检测各组大鼠体重和空腹血糖(FPG),血清中雌二醇(E2)、睾酮(T)、卵泡刺激素(FSH)、黄体生成素(LH)水平,观察卵巢组织形态学变化,分析卵巢组织细胞凋亡情况,检测卵巢组织中凋亡相关蛋白[B细胞淋巴瘤2(Bcl-2)、Bcl-2相关X蛋白(Bax)、胱天蛋白酶3(Caspase-3)]和HippoYAP信号通路相关蛋白[大肿瘤抑制激酶1(LATS1)、磷酸化LATS1(p-LATS1)、Yes相关蛋白(YAP)、磷酸化YAP(p-YAP)、转录共激活子因子PDZ结合基序(TAZ)]表达水平。结果 与PCOS组比较,ERI低、中、高剂量组大鼠卵巢多囊特征减轻,闭锁卵泡数量减少,颗粒细胞层增厚,体重、FPG水平、T水平、LH水平、LH/FSH、囊性卵泡数量、细胞凋亡指数和Bax、Caspase-3、p-LATS1、pYAP蛋白表达水平均显著降低或减少(P<0.05),黄体数量、E2水平和Bcl-2、LATS1、YAP、TAZ蛋白表达水平均显著升高或增多(P<0.05)。与ERI高剂量组比较,ERI高剂量+维替泊芬组大鼠的上述指标变化均被抑制(P<0.05)。结论 ERI能促进PCOS大鼠卵巢颗粒细胞增殖,调节性激素水平,其作用机制可能与抑制Hippo-YAP信号通路有关。

多囊卵巢综合征患者卵巢颗粒细胞胰岛素抵抗的相关信号通路

胰岛素抵抗(insulin resistance,IR)与多囊卵巢综合征(polycystic ovary syndrome,PCOS)的发生发展有关,而与IR相关的胰岛素信号通路异常会引起卵巢局部代谢紊乱,进而影响卵泡发育及卵子质量。PCOS卵巢颗粒细胞中胰岛素作用的经典通路,包括磷脂酰肌醇3激酶(phosphoinositide 3-kinase,PI3K)/蛋白激酶B(protein kinase B,Akt)通路和丝裂原激活的蛋白激酶(mitogen-activated protein kinase,MAPK)/细胞外信号调节激酶(extracellular signal-regulated kinase,ERK)通路;与炎症相关的通路包括Toll样受体(Toll-like receptor,TLR)/核因子κB(nuclear factor-κB,NF-κB)通路等;氧化应激相关通路包括核转录因子红系2相关因子2(nuclear factor-erythroid 2-related factor 2,Nrf2)/血红素加氧酶-1(heme oxygenase-1,HO-1)途径和GTP酶免疫相关蛋白7(GTPase of immunity-associated protein 7,GIMAP7)/音猬因子(sonic hedgehog,SHH)途径;与脂质代谢相关的通路如激活转录因子4(activating transcription factor 4,ATF4)通路等。明确PCOS患者卵巢颗粒细胞中IR相关信号转导通路,增进PCOS病理生理机制的认识,进一步为PCOS的治疗提供新思路。

黄芩苷对多囊卵巢综合征大鼠颗粒细胞增殖和凋亡的影响

目的探讨黄芩苷对多囊卵巢综合征(polycystic ovary syndrome,PCOS)大鼠颗粒细胞增殖和凋亡的影响及对磷脂酰肌醇3-激酶(phosphatidylinositol-3-kinase,PI3K)/蛋白激酶B(protein kinase B,Akt)信号通路的调节作用。方法48只大鼠成功建立PCOS模型,并随机分为模型组、黄芩苷低剂量组、黄芩苷高剂量组和阳性对照组,每组12只,另取12只大鼠作为对照组。黄芩苷低剂量组、黄芩苷高剂量组大鼠分别灌胃25、50 mg∙kg^(−1)黄芩苷,阳性对照组大鼠灌胃0.3392 mg∙kg^(−1)炔雌醇环丙孕酮片(达英-35),对照组和模型组大鼠灌胃等体积生理盐水,每日1次,连续3周。用酶联免疫吸附试验(enzyme linked immuno⁃sorbent assay,ELISA)检测黄体生成素(luteinizing hormone,LH)、卵泡刺激素(follicle stimulating hormone,FSH)、睾酮(testos⁃terone,T)以及雌二醇(estradiol,E2)含量;用HE染色观察卵巢组织病理损伤,细胞增殖检测试剂盒(cell counting kit-8,CCK-8)检测颗粒细胞存活率;用流式细胞仪检测细胞凋亡率,蛋白质印迹法检测PI3K、磷酸化磷脂肌醇3-激酶(phospho PI3K,p-PI3K)、Akt以及p-Akt蛋白相对表达量。结果对照组大鼠可见黄体,未见囊状扩张卵泡;模型组大鼠卵巢体积变大,少见黄体,可见囊状扩张卵泡;黄芩苷低剂量组、黄芩苷高剂量组及阳性对照组大鼠卵巢体积变小,可见黄体,囊状卵泡扩增不明显。与对照组比较,模型组FSH和E2水平、颗粒细胞存活率以及p-PI3K和p-Akt蛋白相对表达量降低,LH、T水平和颗粒细胞凋亡率升高(P<0.05);与模型组比较,黄芩苷低剂量组、黄芩苷高剂量组和阳性对照组FSH、E2水平,颗粒细胞存活率及p-PI3K和p-Akt蛋白相对表达量升高,LH、T水平及颗粒细胞凋亡率降低,其中阳性对照组各指标水平变化最显著,其次是黄芩苷高剂量组(P<0.05)。结论黄芩苷可促进PCOS大鼠卵巢颗粒细胞增殖,抑制凋亡,改善卵巢组织病理损伤,其可能是通过激活PI3K/Akt信号通路发挥作用的。

氧化修饰高密度脂蛋白激活活性氧启动p38信号促使大鼠卵巢颗粒细胞凋亡

背景:多囊卵巢综合征患者体内高密度脂蛋白发生了部分氧化修饰,而氧化修饰高密度脂蛋白与排卵障碍的关系及其机制尚不清楚。目的:探讨氧化修饰高密度脂蛋白对卵巢颗粒细胞凋亡的影响,并分析其可能的作用机制。方法:构建多囊卵巢综合征大鼠模型,取心脏血并分离血清,进一步分离高密度脂蛋白,采用高密度脂蛋白炎症指数、丙二醛水平和脂蛋白琼脂糖凝胶电泳法检验其氧化程度。分离正常大鼠卵巢颗粒细胞,分别给予模型大鼠血清分离的高密度脂蛋白和氧化修饰高密度脂蛋白处理,CCK-8法检测细胞活力,流式细胞术检测细胞凋亡,H2DCF-DA染色检测活性氧生成水平,Western blot检测p38信号活性。卵巢颗粒细胞分别给予活性氧抑制剂N-乙酰半胱氨酸(NAC)和四甲基哌啶(Tempol)和p38抑制剂SB203580预处理后,再给予氧化修饰高密度脂蛋白处理,然后检测细胞凋亡、活性氧生成和p38信号活性。结果与结论:多囊卵巢综合征模型大鼠血清中高密度脂蛋白部分发生氧化修饰。模型大鼠血清分离的高密度脂蛋白和氧化修饰高密度脂蛋白抑制卵巢颗粒细胞活力和促进细胞凋亡(均P<0.05),二者能促进大鼠卵巢颗粒细胞活性氧生成和提高p38活性(均P<0.05)。NAC、Tempol和SB203580均能逆转氧化修饰高密度脂蛋白诱导的卵巢颗粒细胞凋亡(均P<0.05);Tempol、NAC能抑制氧化修饰高密度脂蛋白诱导的p38活性(均P<0.05);SB203580对活性氧的生成没有调节作用(P>0.05)。结果表明:多囊卵巢综合征能促使高密度脂蛋白发生部分氧化修饰,氧化修饰高密度脂蛋白能通过激活活性氧-p38信号通路促使大鼠卵巢颗粒细胞凋亡。

cathpsin D在多囊卵巢综合征卵巢组织中的表达

目的:检测cathepsin D蛋白在多囊卵巢综合征(polycystic ovarian syndrome,PCOS)患者卵巢组织中的表达。方法:采用Western blot免疫印迹法检测卵巢组织中cathepsin D蛋白的表达,并进行半定量分析;免疫组织化学法观察cathepsin D蛋白的分布定位。结果:PCOS组患者卵巢中cathepsin D蛋白表达显著低于对照组(2.06±0.39 vs 4.76±1.43,P<0.05);cathepsin D在卵泡和卵巢间质细胞上均有表达,其中在卵泡颗粒细胞上染色最深,主要定位在胞浆和胞膜。结论:cathepsin D蛋白在PCOS患者卵巢组织中表达水平显著下调,这可能是PCOS卵泡发育异常的机制之一。

柚皮素下调RIP1-RIP3-MLKL信号通路抑制多囊卵巢综合征大鼠卵巢颗粒细胞凋亡

目的基于受体相互作用蛋白激酶(receptor interacting protein kinase,RIP)1-RIP3-混合谱系激酶结构域样蛋白(mixed lineage kinase domain-like proteins,MLKL)介导的细胞坏死性凋亡途径,探究柚皮素对多囊卵巢综合征(polycystic ovary syndrome,PCOS)大鼠卵巢颗粒细胞凋亡的影响。方法SD大鼠随机分为正常对照组、模型组、柚皮素组、RIP1抑制剂(Nec-1)组、RIP1-RIP3-MLKL坏死信号激活剂(Z-VAD-fmk)组、柚皮素+Z-VAD-fmk组,每组15只。ELISA法检测卵巢组织IL-1β、TNF-α水平;HE法观察卵巢形态;分离卵巢组织颗粒细胞,流式细胞术检测凋亡率及坏死率;免疫组化法检测卵巢组织磷酸化RIP1(p-RIP1)阳性表达;Western blot法检测RIP1-RIP3-MLKL途径相关蛋白表达。结果RIP1特异性抑制剂Nec-1和柚皮素可阻断RIP1磷酸化活化,抑制RIP1-RIP3-MLKL信号通路,并降低PCOS大鼠炎症水平,缓解卵巢颗粒细胞坏死及凋亡(P<0.05)。Z-VAD-fmk可促进RIP1-RIP3-MLKL途径活化,加重卵巢颗粒细胞凋亡,并部分减弱柚皮素的抗卵巢颗粒细胞凋亡作用(P<0.05)。结论柚皮素可能通过阻断RIP1-RIP3-MLKL介导的坏死性凋亡途径活化,抵抗PCOS大鼠卵巢颗粒细胞凋亡。

沉默信息调节因子1/叉头框蛋白O1信号通路调控铁死亡对多囊卵巢综合征大鼠卵巢颗粒细胞的影响实验研究

目的:探讨沉默信息调节因子1(SIRT1)/叉头框蛋白O1(FOXO1)信号通路调控铁死亡对多囊卵巢综合征(PCOS)大鼠卵巢颗粒细胞的影响。方法:将30只雌性SD大鼠分为对照组(皮下注射豆油)和PCOS组(皮下注射脱氢表雄酮)。将对照组和PCOS组大鼠分离得到的卵巢颗粒细胞分为对照组、PCOS组,取一半PCOS组卵巢颗粒细胞为过表达SIRT1组(20μmol/L SIRT1激活剂SRT 2183干预)。RT-qPCR及Western blot检测细胞中SIRT1、FOXO1、溶质载体家族7成员11(SLC7A11)、谷胱甘肽过氧化物酶4(GPX4)、Bcl-2关联X蛋白(Bax)、半胱氨酸蛋白酶-3(Caspase-3)、B淋巴细胞瘤-2(Bcl-2)mRNA及蛋白表达。Hoechst染色检测细胞活性氧(ROS)及线粒体活性氧(mtROS)水平。ELISA检测细胞中谷胱甘肽(GSH)、丙二醛(MDA)、Fe^(2+)、卵泡刺激素(FSH)、睾酮(T)、黄体生成素(LH)水平。流式细胞术检测细胞凋亡情况。结果:与对照组比较,PCOS组卵巢颗粒细胞中SIRT1、FOXO1、SLC7A11、GPX4、Bcl-2 mRNA及蛋白表达和GSH、FSH水平降低,Bax、Caspase-3 mRNA及蛋白表达和MDA、ROS、mtROS、Fe^(2+)、T、LH水平升高,卵巢颗粒细胞凋亡率增加(均P<0.05)。与PCOS组比较,过表达SIRT1组卵巢颗粒细胞中SIRT1、FOXO1、SLC7A11、GPX4、Bcl-2 mRNA及蛋白表达和GSH、FSH水平升高,Bax、Caspase-3 mRNA及蛋白表达和MDA、ROS、mtROS、Fe^(2+)、T、LH水平降低,卵巢颗粒细胞凋亡率减少(均P<0.05)。结论:SIRT1/FOXO1通路在PCOS卵巢颗粒细胞中下调,激活该通路可能通过下调铁死亡抑制PCOS卵巢颗粒细胞凋亡。