EZH2在卵巢颗粒细胞氧化应激反应诱导子宫内膜异位症相关不孕中的作用

余晓燕; 王烈宏; 杨惠林; 马英兰; 李燕子

doi:10.16766/j.cnki.issn.1674-4152.002714

EZH2在卵巢颗粒细胞氧化应激反应诱导子宫内膜异位症相关不孕中的作用

doi: 10.16766/j.cnki.issn.1674-4152.002714

青海红十字医院生殖中心，青海西宁 810000

基金项目:

青海省自然科学基金项目 2019-q-051

详细信息

通讯作者:
余晓燕, E-mail: xiaoyanyua1@163.com

中图分类号: R711.71
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出版历程
- 收稿日期: 2021-09-03
- 网络出版日期: 2022-12-30

Role of EZH2 in endometriosis-related infertility induced by oxidative stress in ovarian granulosa cells

Department of Reproductive Center, Qinghai Red Cross Hospital, Xining, Qinghai 810000, China

摘要

摘要: 目的探讨Zeste基因增强子同源物2(EZH2)在卵巢颗粒细胞(GCs)氧化应激反应(OS)诱导的子宫内膜异位症(EM)相关不孕中的作用。方法收集2019年4月—2020年11月青海红十字医院生殖医学科66例深浸润性EM患者和63例输卵管不孕症患者(对照组)的GCs。采用流式细胞术分析GCs中活性氧(ROS)水平。建立EZH2低表达COV434细胞模型，采用H₂O₂处理，通过SA-β-gal测定细胞衰老情况。建立小鼠EM模型，通过腹腔注射EZH2抑制剂DZNep进行治疗，并进行为期6个月的生育力测试。结果与对照组比较，EM组患者慢性盆腔疼痛评分、痛经疼痛评分和血清CA-125抗原均显著增加(均P＜0.001)，并且平均取卵数量、平均成熟卵母细胞数量、平均2 PN胚胎数量、平均优质胚胎数量以及每个胚胎移植周期的临床妊娠率均显著降低(均P＜0.001)。与对照GCs相比，EM患者GCs中的细胞内ROS显著增加(3.62±0.26 vs. 12.25±0.74，P＜0.001)，并且GCs中诱导型一氧化氮合酶(iNOS)和超氧化物歧化酶1(SOD1)蛋白表达显著减少(均P＜0.05)。EZH2敲低显著逆转了H₂O₂诱导的ROS水平、SA-β-Gal活性增加(均P＜0.05)，并下调衰老相关蛋白表达(均P＜0.05)。EM+DZNep组中的子宫内膜异位病变尺寸比EM组显著减小[(433±74) mm³ vs. (1 624±182)mm³, P < 0.001]，并且每次分娩的平均幼崽数明显大于EM组(均P＜0.05)。结论 EZH2参与GCs细胞OS诱导的EM相关不孕。
- Zeste基因增强子同源物2 /
- 卵巢颗粒细胞 /
- 氧化应激 /
- 子宫内膜异位症
Abstract: Objective To explore the role of enhancer of Zeste homologue 2 (EZH2) in ovarian granulosa cells (GCs) and oxidative stress (OS)-induced infertility related to endometriosis (EM). Methods The GCs were collected from 66 patients with deep invasive EM and 63 patients with tubal infertility (control group) in the Department of Reproductive Medicine, Qinghai Red Cross Hospital from April 2019 to November 2020. Flow cytometry was used in analysing the level of ROS in GCs. The COV434 cell model with low EZH2 expression was established in vitro and treated with H₂O₂. Cell senescence was measured by SA-β-gal. A mouse EM model was established, treated with an intraperitoneal injection of the EZH2 inhibitor DZNep and subjected to 6-month fertility test. Results Compared with the control group, the chronic pelvic pain score, dysmenorrhea pain score and serum CA-125 antigen significantly increased in the EM group (all P < 0.001), and the number of cells, average number of 2 PN embryos, average number of high-quality embryos and clinical pregnancy rate per embryo transfer cycle significantly decreased (all P < 0.001). Compared with control GCs, the intracellular ROS in GCs of patients with EM increased significantly (3.62±0.26 vs. 12.25±0.74, P < 0.001), and the expression levels of iNOS and SOD1 in the GCs decreased significantly (all P < 0.05). EZH2 knockdown significantly reversed H₂O₂-induced increase in ROS levels and SA-β-Gal activity (all P < 0.05) and down-regulated the expression of senescence-related proteins (all P < 0.05). The EM lesions in the EM+DZNep group was significantly smaller than those in the EM group [(433±74) mm³ vs. (1 624±182)mm³, P < 0.001], and the mean number of pups per delivery was significantly greater than in the EM group (all P < 0.05). Conclusion EZH2 is involved in EM-related infertility induced by GC cell OS.
- Enhancer of Zeste homologue 2 /
- Ovarian granulosa cells /
- Oxidative stress /
- Endometriosis

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图 1 EM患者GCs的OS表达情况

注：A为流式细胞术分析对照组和EM组患者的GCs中ROS水平。与对照组比较，^aP＜0.01。B为Western blotting分析对照组和EM组患者的GCs中OS应激相关蛋白(iNOS、SOD1)表达。

Figure 1. OS expression of GCs in EM patients

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图 2 生物信息学分析研究EM中GCs的病理变化

注：A为RNA-Seq发现2 954个绝对倍数变化>2的基因。B为GSEA分析发现，EM组GCs的基因显著富集在“对氧化应激的细胞反应”中。C为RT-qPCR证实了对照组和EM组GCs与“对氧化应激的细胞反应”相关上调基因的相对mRNA表达水平。与对照组比较，^aP＜0.05，^bP＜0.01。

Figure 2. Bioinformatics analysis was performed to study the pathological changes of GCs in EM

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图 3 EZH2在OS诱导的GCs衰老中的作用

注：A为COV434细胞在H₂O₂以0.5、1.0、1.5 mmol/L浓度处理6 h后，Western blotting分析细胞中EZH2表达及定量分析。B为COV434细胞经0.5 mmol/L H₂O₂处理2、4、6 h后，Western blotting分析细胞中EZH2表达及定量分析。C为流式细胞术检测EZH2敲低对COV434细胞内ROS水平的影响。D为SA-β-Gal染色检测EZH2敲低对COV434细胞衰老的影响。E为Western blotting分析EZH2敲低对COV434细胞内衰老相关蛋白(p15、p27和p-H2AX)表达的影响。与对照组比较，^aP＜0.01；与H₂O₂+si-NC组比较，^bP＜0.01。

Figure 3. Role of EZH2 in OS-induced GCs senescence

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图 4 DZNep对EM诱导的生育力下降的影响

注：A为术后4周，EM组和EM+DZNep组腹腔可见典型的子宫内膜异位样病变及病变大小比较，^aP＜0.001。B为免疫组织化学评估各组卵巢组织中EZH2蛋白水平。C为Western blotting分析卵巢组织中OS应激相关蛋白和衰老相关蛋白表达。D为造模后6个月内小鼠生育能力的变化。与假手术组比较，^aP＜0.01，^cP＜0.05；与EM组比较，^bP＜0.01, ^dP＜0.05。

Figure 4. Effect of DZNep on EM-induced fertility decline

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表 1 2组患者的基线特征和临床结果比较

Table 1. Comparison of baseline characteristics and clinical outcomes between the two groups

组别	例数	年龄(x±s，岁)	BMI (x±s)	不孕时间(x±s，年)	月经周期(x±s，d)	抗苗勒氏管激素(x±s，ng/mL)	窦卵泡计数(x±s，个)	慢性盆腔疼痛评分[M(P₂₅, P₇₅)，分]	痛经疼痛评分[M(P₂₅, P₇₅)，分]
对照组	63	30.82±3.57	21.47±2.61	3.20±2.15	31.37±4.23	4.35±2.29	11.52±3.56	1.0(0.4, 1.8)	1.5(0.3, 2.2)
EM组	66	31.28±3.48	20.99±2.48	3.32±2.30	30.58±3.80	4.01±2.71	10.05±3.32	5.3(2.5, 7.1)	2.6(0.8, 3.6)
统计量		0.927^a	1.074^a	0.837^a	1.322^a	0.926^a	1.458^a	17.263^c	7.948^c
P值		0.460	0.219	0.566	0.125	0.271	0.107	< 0.001	< 0.001

组别	例数	血清CA-125抗原[M(P₂₅, P₇₅)，U/mL]	平均取卵数量[M(P₂₅, P₇₅)，个]	平均成熟卵母细胞数量[M(P₂₅, P₇₅)，个]	平均2 PN胚胎数量(x±s，个)	平均优质胚胎数量(x±s，个)	优质胚胎率(%)	平均移植胚胎数量(x±s，个)	每个胚胎移植周期的临床妊娠率(%)
对照组	63	15.84(6.27, 32.53)	9(2, 12)	7(3, 11)	6.1±3.4	3.1±2.8	50.64	1.1±0.6	74.52
EM组	66	26.54(60.26, 13.27)	5(2, 8)	4(2, 6)	3.7±2.4	1.7±1.5	44.66	1.2±0.8	48.04
统计量		18.415^c	8.542^c	6.082^c	8.471^a	6.182^a	1.608^b	0.488^a	9.263^b
P值		< 0.001	< 0.001	< 0.001	< 0.001	< 0.001	0.093	0.719	< 0.001
注：^a为t值，^b为χ²值，^c为U值。

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