这是用户在 2024-11-29 22:38 为 https://app.immersivetranslate.com/word/ 保存的双语快照页面,由 沉浸式翻译 提供双语支持。了解如何保存?

DEHP 暴露诱导生精细胞自噬,损害小鼠睾丸生殖功能

刘志 1,#, 毛坤1, #, 阎浩 1, 尹子 1, 高欣琦1, 佘朗庆 1, 刘浩福1, 苏恒1, 李青1, 王思宇 1, 祁扬燕2,3,4李亚钊 5, 潘戈2,3,4,*2,3,4,*
刘志 1,#, 毛坤1,#, 阎浩 1, 尹子 1, 高欣琦1, 佘朗庆 1, 刘浩福1, 苏恒1, 李青1, 王思宇 1, 祁扬燕2,3,4李亚钊 5, 潘戈2,3,4,*2,3,4,*

1 习安交通大学医学系, 习安, 陕西, 710061;

2习安交通大学医学院基础医学院病理学教研室 ,习安,710061;

3习安交通大学医学院遗传学与发育生物学研究所 , 习安, 710061;

4 习安交通大学医学院生殖医学中心 , 习安, 710061;

5 习安交通大学第一附属医院转化医学中心 ,中国710061习安。

#作者对这项工作做出了同样的贡献。

*通信:

Pan Ge,习安交通大学医学院基础医学院病理学系 ,中国710061省陕西省习安市。电子邮件:gepan@xjtu.edu.cn。 手机: (+86)18149493365.

习安交通大学医学院基础医学院病理学系 周 Dang-xia China , 习安, 710061电子邮件:zdxtougao@163.com。

抽象

背景和目标: 近年来,全球精子质量下降,男性不育症随之增加。此外,随着人们的环保意识越来越强,环境毒性在男性生殖障碍中的作用也受到了广泛关注。虽然DEHP作为一种常见的塑化剂,广泛用于食品包装、化妆品和日用品,但有大量研究报道其致癌性,尤其对生殖系统的损害。 然而,关于 DEHP 暴露导致生殖损伤机制的研究很少。
背景和目标: 近年来,全球精子质量下降,男性不育症随之增加。此外,随着人们的环保意识越来越强,环境毒性在男性生殖障碍中的作用也受到了广泛关注。虽然DEHP作为一种常见的塑化剂,广泛用于食品包装、化妆品和日用品,但有大量研究报道其致癌性,尤其对生殖系统的损害。然而,关于 DEHP 暴露导致生殖损伤机制的研究很少。

方法: 在本研究中,我们创建了 DEHP 强饲的 ICR 小鼠模型 DEHP 暴露的小鼠精原细胞模型。首先,我们通过组织形态学和 Johnson 评分系统评估了生殖损伤的水平。然后,通过使用一系列测试和化验技术检测细胞和组织氧化应激的水平。最后, 通过 western blottingRT-qPCR 和免疫荧光检测自噬和丝裂细胞增多症相关基因表达的变化。
方法:在本研究中,我们创建了 DEHP 强饲的 ICR 小鼠模型和 DEHP 暴露的小鼠精原细胞模型。首先,我们通过组织形态学和 Johnson 评分系统评估了生殖损伤的水平。然后,通过使用一系列测试和化验技术检测细胞和组织氧化应激的水平。最后, 通过 western blottingRT-qPCR 和免疫荧光检测自噬和丝裂细胞增多症相关基因表达的变化。

结论:总之,我们的研究表明,DEHP 可能通过诱导氧化应激导致生殖细胞中自噬程序的启动而造成生殖损伤 。其中,自噬相关基因 P62 的表达降低,LC3B 和 Atg12 升高。此外,氧化应激相关 ROS 、 GSH 和 MDA 的含量也有所增加。而经具有抗氧化作用的 VC 处理后,其含量均降低。同时,有丝分裂细胞增多症的存在也降低了自噬的程度。 这些可能为 DEHP 暴露相关的生殖毒性损伤提供潜在的治疗靶点。
结论:总之,我们的研究表明,DEHP 可能通过诱导氧化应激导致生殖细胞中自噬程序的启动而造成生殖损伤 。其中,自噬相关基因 P62 的表达降低,LC3B 和 Atg12 升高。此外,氧化应激相关 ROS 、 GSH 和 MDA 的含量也有所增加。而经具有抗氧化作用的 VC 处理后,其含量均降低。同时,有丝分裂细胞增多症的存在也降低了自噬的程度。这些可能为 DEHP 暴露相关的生殖毒性损伤提供潜在的治疗靶点。

Keywords: DEHP, reproductive toxicity, cellular autophagy, oxidative stress
关键词:DEHP, 生殖毒性, 细胞自噬, oxidative stress

Introduction
介绍

Several epidemiological studies have shown a significant decrease in the quality of male sperm over the past fifty years. (10.1093/humupd/dmac035). Male reproductive toxicity pertains to the negative impact of foreign substances on the male reproductive system (10.1016/j.tox.2021.152780), among which environmental toxic factors play an important role. Multiple studies have confirmed potential links between exposure to environmental toxins and reduced male reproductive capacity and injury. (10.1016/j.bpobgyn.2022.102298).
几项流行病学研究表明,在过去 50 年中,男性精子的质量显着下降。(10.1093/humupd/dmac035 的 intent 文档)。男性生殖毒性是指外来物质对男性生殖系统的负面影响 (10.1016/j.tox.2021.152780),其中环境毒性因素起重要作用。多项研究证实了暴露于环境毒素与男性生殖能力下降和受伤之间存在潜在联系。(10.1016/j.bpobgyn.2022.102298)。

Di(2-ethylhexyl) phthalate (DEHP) is a commonly used plasticizer in plastics to increase their elasticity and toughness. Because it is widely used in the plastics industry, there is a high risk of human exposure. DEHP can enter the body through the gastrointestinal tract, respiratory tract and skin, and is primarily ingested orally. (10.1007/s00253-014-6183-8). The substance rapidly breaks down into monoester compounds within the human body and is subsequently eliminated through urine
(10.1007/s00253-014-6183-8)。该物质在人体内迅速分解成单酯化合物,随后通过尿液排出
(10.1016/j.envint.2018.07.029). Various studies have demonstrated that DEHP and its metabolites have diverse effects on cardiovascular function, renal function, reproductive function, and more (10.1016/j.scitotenv.2021.147371), with particular concern regarding their impact on the reproductive system. DEHP exposure is an environmental toxicant that is closely associated with male reproductive toxicity. It adversely affects the male reproductive system by causing a decrease in testosterone levels, testicular and epididymal mass, sperm quality, prostate lesions, and metabolic alterations (10.1016/j.repbio.2020.07.005). Nonetheless, the mechanisms of injury induced by DEHP exposure remain unclear, and autophagy is believed to play a significant role in the process.
邻苯二甲酸二(2-乙基己基)酯 (DEHP) 是塑料中常用的增塑剂,用于增加其弹性和韧性。由于它广泛用于塑料工业,因此人体接触的风险很高。DEHP 可通过胃肠道、呼吸道和皮肤进入人体,主要经口摄入。 (10.1007/s00253-014-6183-8)。该物质在人体内迅速分解成单酯化合物,随后通过尿液排出 (10.1016/j.envint.2018.07.029)。各种研究表明,DEHP 及其代谢物对心血管功能、肾功能、生殖功能等有多种影响 (10.1016/j.scitotenv.2021.147371),特别关注它们对生殖系统的影响。DEHP 暴露是一种与男性生殖毒性密切相关的环境毒物。它通过导致睾丸激素水平、睾丸和附睾质量、精子质量、前列腺病变和代谢改变降低,对男性生殖系统产生不利影响 (10.1016/j.repbio.2020.07.005)。尽管如此,DEHP 暴露诱导的损伤机制仍不清楚,自噬被认为在此过程中起着重要作用

Autophagy, as an important eukaryotic intracellular homeostatic regulatory mechanism, is responsible for the removal of cellular components such as accumulated proteins and damaged organelles to maintain intracellular homeostasis. (10.1038/s41419-022-04906-6) Autophagy contributes to cellular adaptation to changing internal and external environments and promoting survival and development (10.1089/ars.2013.5371). However, dysfunctions in autophagy are also involved in the pathogenesis of diverse diseases, playing a crucial role in the development of some (10.15252/embj.2021108863). The autophagy process entails four essential stages, namely initiation, nucleation, maturation, and degradation (10.1016/j.cell.2011.10.026). Autophagy dysfunction can occur due to various abnormal triggers and stressors, which affect these stages. Oxidative stress plays a vital role as a mediator in the development of autophagy dysfunction (10.1038/cdd.2014.150).
自噬作为一种重要的真核生物细胞内稳态调节机制,负责去除积累的蛋白质和受损的细胞器等细胞成分,以维持细胞内稳态。(10.1038/s41419-022-04906-6) 自噬有助于细胞适应不断变化的内部和外部环境并促进生存和发育 (10.1089/ars.2013.5371)。然而,自噬功能障碍也参与多种疾病的发病机制,在一些疾病的发展中起着至关重要的作用 (10.15252/embj.2021108863)。自噬过程包括四个基本阶段,即起始、成核、成熟和降解 (10.1016/j.cell.2011.10.026)。自噬功能障碍可能是由于影响这些阶段的各种异常触发因素和压力源而发生的。氧化应激作为自噬功能障碍发展的介质起着至关重要的作用 (10.1038/cdd.2014.150)。

Cellular homeostasis is a necessary condition for cells to implement optimal functions. (10.1038/sj.cdd.4401765) And mitochondria are the most momentous energy organelles, playing an irreplaceable role in intracellular homeostasis. Therefore, mitochondrial autophagy is an important part of cellular autophagy. In addition, mitochondria are involved in the regulation of second messenger levels including reactive oxygen species. It has been shown that dysfunctional mitochondria increase oxidative stress (10.1016/j.bbadis.2016.11.010). A recent study has demonstrated that damaged mitochondria can be translocated extracellularly by a membrane-bound structure named migrasome, a process known as mitocytosis (10.1016/j.cell.2021.04.027). When mitochondria are slightly damaged, migrasome will transfer them to the cell membrane and then be treated, which is conducive to cell survival. What's more, this process is not positively correlated with the degree of damage, so severely damaged mitochondria cannot prevent cell damage through mitochondrial exocytosis. (10.1021/acs.jafc.2c08601)
细胞稳态是细胞实现最佳功能的必要条件。(10.1038/sj.cdd.4401765)而线粒体是最重要的能量细胞器,在细胞内稳态中起着不可替代的作用。因此,线粒体自噬是细胞自噬的重要组成部分。此外,线粒体参与第二信使水平的调节,包括活性氧。已经表明,功能失调的线粒体会增加氧化应激 (10.1016/j.bbadis.2016.11.010)。最近的一项研究表明,受损的线粒体可以通过一种名为迁移体的膜结合结构在细胞外易位,这一过程称为有丝分裂症 (10.1016/j.cell.2021.04.027)。当线粒体受到轻微损伤时,迁移体会将其转移到细胞膜上,然后进行处理,有利于细胞存活。更重要的是,这个过程与损伤程度没有正相关,因此严重受损的线粒体无法通过线粒体胞吐作用阻止细胞损伤。(10.1021/acs.jafc.2c08601)

Hence, exploring the relationship between mitocytosis and DEHP-induced oxidative stress and cellular autophagy may provide new ideas for DEHP reproductive toxicity. In order to investigate the male reproductive toxicity of DEHP and the mechanisms of autophagy and oxidative stress in its damage, and mitocytosis in this regulation process, we performed a series of bio-histological measurements, molecular biology experiments, and various assay techniques using animal and cellular treatment models. We hope to provide strong evidence for the environmental toxicity of DEHP and its mechanism of injury.
因此,探索有丝分裂细胞增多症与 DEHP 诱导的氧化应激和细胞自噬之间的关系可能为 DEHP 生殖毒性提供新思路。 为了研究 DEHP 的男性生殖毒性以及和氧化应激在其损伤中的机制,以及该调节过程中的有丝分裂细胞增多,我们使用动物和细胞处理模型进行了一系列生物组织学测量、分子生物学实验和各种检测技术。我们希望为 DEHP 的环境毒性及其损伤机制提供强有力的证据。

Materials and methods
材料和方法

Animals and treatment
动物和治疗

The male ICR mice (37 ± 2.87 g) were purchased from the Experimental Animal Center of Xi’an Jiaotong University. These mice were randomly and equally divided into 5 groups (n = 10): water group named blank control group (Con), water group containing 2% Tween-80 named solvent control group (Scon), and three experimental groups. According to the design of our study, the mice of experimental groups were gavaged with different concentrations of DEHP solution for 4 consecutive weeks, which were respectively named 100 mg/kg DEHP (D100), 200 mg/kg DEHP (D200), and 400 mg/kg DEHP (D400). The solution was prepared by dissolving different doses of DEHP (Shanghai Macklin Biochemical Co., Ltd, Shanghai, China) in high-pressure purified water containing 2 % Tween-80 (Xi'an Fuli Chemical Factory, Xi'an, China). All mice were kept with food and water available randomly in a appropriate temperature and humidity room (22 ± 2 ℃, 40-60% humidity), maintained with a cycle of 12 h each of light and darkness. All experiments were performed in accordance with the Medical Ethics Committee of Xi’an Jiaotong University.
雄性 ICR 小鼠 (37 只± 2.87 g) 购自习安交通大学实验动物中心 将这些小鼠随机均匀分为 5 组 (n = 10):水组称为空白对照组 (Con),含有 2% Tween-80 的水组称为溶剂对照组 (Scon),和 3 个实验组。 根据本研究的设计,实验组小鼠 用不同浓度的 DEHP 溶液连续灌胃 4 周,分别命名为 100 mg/kg DEHP (D100)、200 mg/kg DEHP (D200) 和 400 mg/kg DEHP (D400)。 通过将不同剂量的 DEHP(上海麦克林生化有限公司,中国上海)溶解在含有 2% 吐温-80(习安富利化工厂,中国习安)的高压纯化水中来制备溶液。 将所有小鼠与随机提供的食物和水保存在 适当的温度和湿度室 (22 ± 2 °C,40-60% 湿度) 中,保持光明和黑暗各 12 小时的周期。 所有实验均按照习安交通大学医学伦理委员会进行

Hematoxylin and eosin (H&E) staining
苏木精和伊红 (H&E) 染色

Male mouse testis tissues were fixed in 4% paraformaldehyde (PFA) and embedded in paraffin. Testicular tissues were cut into 4 µm sections, deparaffinized, and rehydrated. After the slide has been immersed in hematoxylin for 3 min, it is rinsed with running water for 3 min. Then, the slides were stained with a 1% eosin solution for 30 s. The sections were orderly dehydrated using 85%, 90%, 95% alcohol and three changes of 100% alcohol, each for 1 min. Finally, alcohol was extracted three times by xylene washing. The glass slides were sealed with tree glue, and images were observed on the microscope (Nikon, Tokyo, Japan).
将雄性小鼠睾丸组织固定在 4% 多聚甲醛 (PFA) 中并包埋在石蜡中。将睾丸组织切成 4 μm 切片,脱蜡并再水化。玻片浸入苏木精3分钟用流水冲洗 3分钟。然后,用 1% 伊红溶液对载玻片染色 30 秒。使用 85%、90%、95% 酒精和 3 次 100% 酒精有序脱水切片,每次 1 分钟。最后,经二甲苯洗涤萃取醇 3 次。用树胶密封载玻片,并在显微镜上观察图像(尼康,东京,日本)。

Pathological morphometric indices and Johnson’s score
病理形态测量指标和 Johnson 评分

The diameter and height of the seminiferous tubules were measured separately under a 200× microscope. Additionally, the occurrence of spermatozoa in the seminiferous tubules and the degree of spermatogenic disorders were assessed by 2 independent specialties using the Johnson Score, i.e., the lower the score, the more severe the spermatogenic disorder.
在 200× 显微镜下分别测量生精小管的直径和高度。此外,生精小管中精子的发生和生精疾病的程度由 2 个独立的专业使用 Johnson 评分进行评估,即分数越低,生精障碍越严重。

Sperm count and sperm morphology
精子数量和精子形态

After the male mice were given intragastric (i.g.) doses of the specified concentrations of DEHP (0, 100, 200, and 400 mg/kg), sperm from epididymal tissue was collected into centrifuge tubes. The sperm suspension was then evenly spread across the center of a slide in a rectangular shape, with a moderate range. After air-drying, the sperm smear was fixed using ice-cold methanol. Following further air-drying, the smears were stained with eosin at room temperature for one hour and then the excess dye was washed with PBS for three times. Eventually, the smear was observed and counted under a microscope. At the same time, abnormalities in sperm morphology were observed and sperm deformity rates were calculated in multiple fields of view.
雄性小鼠胃内 () 剂量的指定浓度的 DEHP (0、100、200 和 400 mg/kg) 后,将附睾组织的精子收集到离心管中。然后将精子悬液以矩形均匀分布在载玻片的中心,范围适中。风干后,用冰冷的甲醇固定精子涂片。进一步风干后,将弥散条带在室温下用伊红染色 1 小时,然后用 PBS 洗涤过量的染料 3 次。最终,在显微镜下观察和计数涂片。同时,观察到精子形态异常,并在多个视野中计算精子畸形率。

Cell culture and treatment
细胞培养和处理

Mouse spermatocyte-derived GC-2 spd (ts) cells (GC-2 cells) (iCell Bioscience Inc, Shanghai, China) were cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 1% antibiotics in a 37 ℃ incubator with 5% CO2. The adherent growth of cells was observed with a microscope, and cells in the logarithmic growth phase were used in the experiment. The cells were randomly divided into four groups, blank control group (Con) and three experimental groups treated with different doses (50, 100, and 200μmmol/L) of DEHP (D50, D100, and D200). The experimental cells were treated with DEHP for 48 h.
小鼠精原细胞来源的 GC-2 spdts) 细胞(GC-2 细胞)(iCell Bioscience Inc,中国上海)在含有 10% 胎牛血清 (FBS) 和 1% 抗生素的 DMEM 培养基中,在 37 °C 和 5% CO 2 的培养箱中培养。用显微镜观察细胞的贴壁生长,实验中使用对数生长期细胞。将细胞随机分为 4 组,空白对照组 (Con) 和 3 个实验组 (D50、100 和 200μmmol/L) 处理 DEHP (D50、D100 和 D200)。实验细胞用 DEHP 处理 48 h。

Cell viability assay
细胞活力检测

Cell viability was analyzed by Cell Counting Kit‐8 (CCK-8). CCK-8 assay was conducted to measure GC-2 cell viability treated with different concentrations of DEHP. The cell suspension was seeded with 100 μL / well into 96 well plates. The culture plates were precultured in an incubator at 37℃ with 5% CO2. Then, ten microliters of the CCK-8 reagent (Beyotime Biotechnology, Shanghai, China) was added into each well. Notably, the bubbles in the pores should avoid being generated, which will affect the reading of the absorbance. The 96-well plates were incubated for 1 hours at 37°C. The absorbance was detected at 450 nm by a microplate reader (Thermo Fisher Scientific, Waltham, Massachusetts, USA) using a well without cells as blank.
通过细胞计数试剂盒-8 (CCK-8) 分析细胞活力。进行 CCK-8 测定以测量用不同浓度的 DEHP 处理的 GC-2 细胞活力。将细胞悬液以 100 μL/孔接种到 96 孔板中。将培养板在 37°C 和 5% CO2 的培养箱中预培养。然后,每个孔中加入 10 微升 CCK-8 试剂(Beyotime Biotechnology,中国上海)。 值得注意的是,应避免在孔中产生气泡,这会影响吸光度的读数。将 96 孔板在 37°C 下孵育 1 小时。 使用无细胞的孔作为空白,通过酶标仪(Thermo Fisher Scientific,Waltham,Massachusetts,USA)在 450 nm 处检测吸光度。

Trypan blue staining
台盼蓝染色

The GC-2 cells from different experimental groups were digested by trypsinization and resuspended into cell suspension. Next, they were stained with 0.4% trypan blue staining solution (Carnoss Technology Co., Ltd, Wuhan, China) at room temperature for 5 min. Ultimately, cell suspension was counted and calculated the ratio of living cells.
通过胰蛋白酶消化来自不同实验组的 GC-2 细胞,并重悬于细胞悬液中。接下来,在室温下用 0.4% 台盼蓝染色液 (Carnoss Technology Co., Ltd, Wuhan, China) 染色 5 分钟。最后,对细胞悬液进行计数并计算活细胞的比例。

GSH and MDA measurement
GSH 和 MDA 测量

Testis samples and GC-2 cells were homogenized with a homogenizer and then centrifuged at 12000g for 15 min at 4 ℃. According to the commercial kits following the manufacturer's instructions, the resultant supernatants were retained for measuring the levels of reduced glutathione (GSH) and Malondialdehyde (MDA).
用匀浆器匀浆睾丸样品和 GC-2 细胞,然后在 4 °C 下以 12000g 离心 15 分钟。根据遵循制造商说明的商业试剂盒,保留所得上清液用于测量还原型谷胱甘肽 (GSH) 和丙二醛 (MDA) 的水平

Reactive Oxygen Species (ROS) detection
活性氧 (ROS) 检测

The ROS Assay Kit (Labgic Technology Co., Ltd, Beijing, China) was used to detect the ROS level in GC-2 cells treated with different gradient concentrations of DEHP. Firstly, untreated or treated GC-2 cells were plated in 6-well plates. 2’, 7’-dichlorofluorescein-diacetate (DCFH-DA), an intra-cellular ROS indicator that can be oxidized into fluorescent 2’,7’-dichlorofluorescein (DCF), was diluted into DCFH-DA solution with serum-free medium (1:1000). Then cells were incubated with 10 μmol/L DCFH-DA solution for 30 min. Finally, cells were washed in a serum-free medium three times to fully remove DCFH-DA. The ROS level of cells was observed by an inverted fluorescence microscope respectively.
ROS 检测试剂盒(Labgic Technology Co., Ltd,中国北京)用于检测用不同梯度浓度的 DEHP 处理的 GC-2 细胞中的 ROS 水平。首先,将未处理或处理的 GC-2 细胞接种在 6 孔板中。将 2',7'-二氯荧光素-二乙酸酯 (DCFH-DA) 稀释到含有无血清培养基 (1:1000) 的 DCFH-DA 溶液中,这是一种细胞内 ROS 指示剂,可氧化成荧光 2',7'-二氯荧光素 (DCF)。然后将细胞与 10 μmol/L DCFH-DA 溶液孵育 30 分钟。最后,将细胞在无血清培养基中洗涤 3 次以完全去除 DCFH-DA。分别通过倒置荧光显微镜观察细胞的 ROS 水平。

Real-time quantitative polymerase chain reaction (RT-qPCR)
实时定量聚合酶链反应 (RT-qPCR)

Total RNA was extracted from tissues and cells by TRIzol reagent (GenStar, Beijing, China). Ultraviolet spectrophotometer (Denovix, Wilmington, Delaware, USA) was used for purity detection and content measurement. RNA samples with an A260/A280 ratio of 1.8-2.0 were selected to reverse transcription to synthesize cDNA, and cDNA was used as the template for fluorescence quantitative PCR reaction. The reaction condition was set as 95˚C for 2 min, 95˚C for 15 s, and 60˚C for 30 s for 40 cycles. GAPDH is an internal reference gene. The experimental data were calculated using the 2-ΔΔCt method. All experiments were repeated at least 3 times. All primer sequences are listed in Table 1.
通过 TRIzol 试剂 (GenStar, Beijing, China) 从组织和细胞中提取总 RNA。紫外分光光度计 (Denovix, Wilmington, Delaware, USA) 用于纯度检测和含量测量。选取 A260/A280 比值为 1.8-2.0 的 RNA 样品逆转录合成 cDNA,以 cDNA 为模板进行荧光定量 PCR 反应。反应条件设置为 95°C 2 min、95°C 15 s 和 60°C 30 s,循环 40 次。GAPDH 是一个内部参考基因。实验数据采用 2-ΔΔCt 方法计算。所有实验至少重复 3 次。所有引物序列均列于表 1 中。

Table 1. Primer sequences.
表 1.引物序列。

Gene
基因

F/R

Primer sequence (5’- 3’)
引物序列 (5'- 3')

LC3B

Forward
向前

GTCCTGGACAAGACCAAGTTCC

Reverse
反向

CCATTCACCAGGAGGAAGAAGG

P62

Forward
向前

GCTCTTCGGAAGTCAGCAAACC

Reverse
反向

GCAGTTTCCCGACTCCATCTGT

Atg12

Forward
向前

GAAGGCTGTAGGAGACACTCCT

Reverse
反向

GGAAGGGGCAAAGGACTGATTC

BECN1

Forward
向前

CAGCCTCTGAAACTGGACACGA

Reverse
反向

CTCTCCTGAGTTAGCCTCTTCC

GAPDH

Forward
向前

CATCACTGCCACCCAGAAGACTG

Reverse
反向

ATGCCAGTGAGCTTCCCGTTCAG

Western blotting
Western 印迹

The total protein was extracted from tissues and cells by RIPA lysate. After denaturation, the protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Then it was transferred to polyvinylidene fluoride (PVDF) membrane constant current and blocked with 5 % skim milk for 2 h. The primary antibody (P62, LC3B, Atg12 and BECN1) was diluted and then added to the membranes, placed at 4 °C overnight incubation. The next day, the membranes were washed with PBST 3 times, and then the secondary antibody (Wanleibio, Shenyang, China) was added to the membranes and incubated at 37 ℃ for 1 h. After the second antibody incubation, the membranes were also washed 3 times. Finally, the bands were displayed on a chemiluminescence imager (Syngene, Cambridge, Cambridgeshire, UK) with a luminescent solution, and the gray values of the proteins were measured.
通过 RIPA 裂解物从组织和细胞中提取总蛋白。变性后,通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳 (SDS-PAGE) 分离蛋白质样品。然后将其转移到聚偏二氟乙烯 (PVDF) 膜恒流中,并用 5% 脱脂牛奶封闭 2 h。稀释一抗(P62、LC3B、Atg12 和 BECN1),然后加入到膜中,在 4 °C 下孵育过夜。第二天,用 PBST 洗涤膜 3 次,然后将二抗 (Wanleibio, Shenyang, China) 加入膜中并在 37 °C 下孵育 1 小时。第二次抗体孵育后,膜也洗涤 3 次。最后,用发光溶液在化学发光成像仪 (Syngene, Cambridge, Cambridgeshire, UK) 上显示条带,并测量蛋白质的灰度值。

Immunofluorescence analysis
免疫荧光分析

After being dewaxed and dehydrated, the paraffin sections were incubated overnight with the primary antibody at 4 ℃. The primary antibodies were LC3B (1:400; 18725-1-AP, Proteintech), P62 (1:500; 18420-1-AP, Proteintech) and BECN1 (1:50; sc-48341, SANTA). The fluorescent secondary antibodies CoraLite594-conjugated Goat Anti-Mouse IgG (1:400; SA00013-3, proteintech) and CoraLite488-conjugated Goat Anti-Rabbit IgG (1:400; SA00013-2, proteintech) were darkly incubated at 37 ℃ and then the nucleus was stained with DAPI (C0065, Beijing Solarbio Science & Technology Co.,Ltd., Beijing, China) in 37 ℃. Finally, the sections were observed under a fluorescence microscope (Nikon, Tokyo, Japan).
脱蜡和脱水后,将石蜡切片与一抗在 4 °C 下孵育过夜。一抗为 LC3B (1:400; 18725-1-AP,Proteintech)、P62(1:500;18420-1-AP,Proteintech)和 BECN1(1:50;sc-48341,SANTA)。荧光二抗 CoraLite594 偶联的山羊抗小鼠 IgG (1:400;SA000 13-3,proteintechCoraLite488 偶联的山羊抗兔 IgG (1:400;SA000 13-2,proteintech在 37 °C 下暗温育,然后在 37 °C 下用 DAPI(C0065,北京阳光生物科技有限公司,中国北京)对细胞核进行染色。最后,在荧光显微镜下观察切片 (Nikon, Tokyo, Japan)。

Statistical analysis
统计分析

SPSS 18.0 software (SPSS, Chicago, IL, USA) was used to analyze the data. The experimental data were expressed as mean ± standard deviation. And all the data were repeated in at least three independent trials. In addition, one-way analysis of variance (ANOVA) was used for component comparison between multiple groups. The least significant difference (LSD) was used to determine the significance of differences between groups. Significance is indicated by asterisks: *P < 0.05, **P < 0.01.
使用 SPSS 18.0 软件 (SPSS, Chicago, IL, USA) 对数据进行分析。实验数据表示为平均值±标准差。所有数据都在至少三项独立试验中重复进行。此外,单因素方差分析 (ANOVA) 用于多组之间的成分比较。使用最不显著的差异 (LSD) 来确定组间差异的显着性。显著性用星号表示:*P < 0.05,**P < 0.01。

Results
结果

3.1 DEHP decreases body weight and organ coefficient of mice, and destroys seminiferous tubules and affects spermatogenesis.
3.1 DEHP 降低小鼠的体重和器官系数,破坏生精小管并影响精子发生。

Compared with the control group, DEHP-treated mice showed a significant decrease in body weight (Figure 1a), and testicular organ coefficient (Figure 1b), and the extent of the decrease was related to the exposure dose of DEHP. Futhermore, compared with the control group, 100 mg/kg DEHP-exposed mice showed no significant damage to the seminiferous tubules, while 200 mg/kg and 400 mg/kg DEHP-exposed mice showed shedding of spermatogonial cells and reduction in the layers of the seminiferous epithelium (Figure 1c). Meanwhile, as shown in Figure 1d, the diameter of the seminiferous tubules also decreased and the height of the seminiferous epithelium was reduced after DEHP exposure compared to the control group. The Johnson's score results also showed that Johnson's score decreased after an increased dose of DEHP exposure (Figure 1e). Further observations and analyses of semen quality showed that DEHP exposure affected sperm count, viability and morphology (Figure 1f&g).
与对照组相比,DEHP 处理的小鼠显示体重(图 1a)和睾丸器官系数(图 1b)显着降低,降低的程度与 DEHP 的暴露剂量有关。 此外与对照组相比,100 mg/kg DEHP 暴露的小鼠对生精管没有显着损伤,而 200 mg/kg 和 400 mg/kg DEHP 暴露的小鼠显示精原细胞脱落和生精上皮层减少(图 1c)。同时,如图 1d 所示,与对照组相比,DEHP 暴露后生精小管的直径也减小,生精上皮的高度降低。Johnson 评分结果还显示,在 DEHP 暴露剂量增加后,Johnson 评分下降(图 1e)。对精液质量的进一步观察和分析表明,DEHP 暴露会影响精子数量、活力和形态(图 1f&g)。

Figure 1. Effects of DEHP on the reproductive system of mice.
图 1.DEHP 对小鼠生殖系统的影响。

(a) Changes in incremental body weight of mice. (b) Change in testicular organ coefficient in mice. (c) Histopathological observation of testicular (H&E staining, ×400). (d) The height of spermatogenic epithelium. (e)Johnson’s score. (f) Microscopic observation of sperm smears. (g) Sperm abnormality rate. All results are expressed as the mean ±SD. *P < 0.05, **P < 0.01, compared with the control group.
(a) 小鼠体重增量的变化。(b) 小鼠睾丸器官系数的变化。(c) 睾丸的组织病理学观察(H&E 染色,×400)。(d) 生精上皮的高度。(五)约翰逊的分数。(f) 精子涂片的显微镜观察。(g) 精子异常率。所有结果均表示为平均值 ±SD。*P < 0.05,**P < 0.01,与对照组相比。

3.2 DEHP activates oxidative stress in testicular tissue and GC-2 cells, and affects the viability and survival of GC-2 cells.
3.2 DEHP 激活睾丸组织和 GC-2 细胞中的氧化应激,并影响 GC-2 细胞的活力和存活。

In vivo experiments on mice were conducted to detect the level of ROS and GSH content in mouse testicular tissue. It was found that the ROS and MDA levels in the DEHP treatment group increased with increasing concentration, while the GSH content showed a decreasing trend (Figure 2a). This showed that DEHP induced a highly oxidative environment around the tissues.
对小鼠进行体内实验,检测小鼠睾丸组织中 ROS 和 GSH 含量的水平。结果发现,DEHP 处理组的 ROS 和 MDA 水平随着浓度的增加而增加,而 GSH 含量呈下降趋势(图 2a)。这表明 DEHP 诱导了组织周围的高度氧化环境。

Similarly, in vitro cell experiments on GC-2 cells, the control group, 50mM DEHP treatment group, 100mM DEHP treatment group and 200mM DEHP treatment group showed increasing ROS and MDA levels and decreasing GSH content with increasing DEHP concentration, consistent with the in vivo experimental results (Figure 2b). At the same time, we co-treated cells with vitamin C and 100 μmmol/L DEHP, performing the same detection. Compared to the 100μmmol/L DEHP treatment group, the ROS and MDA levels of the co-treated group significantly decreased (Figure 2c).
同样,GC-2 细胞的体外细胞实验,对照组、50mM DEHP 处理组、100mM DEHP 处理组和 200mM DEHP 处理组显示,随着 DEHP 浓度的增加,ROS 和 MDA 水平增加,GSH 含量降低,与体内实验结果一致(图 2b)。同时,我们将细胞与维生素 C 和 100 μmmol/L DEHP 共处理,进行相同的检测。与 100μmmol/L DEHP 处理组相比,共处理组的 ROS 和 MDA 水平显着降低(图 2c)。

We further used the CCK-8 kit to respectively detect the cell viability of each group. The results showed that the measured OD values of the DEHP treatment groups were much lower than the control group, with significantly reduced cell vitality. Compared with the 100 μmmol/L DEHP treatment group, the cell viability and survival rate increased with the addition of vitamin C (Figure 2d&e).
我们进一步使用 CCK-8 试剂盒分别检测每组的细胞活力。结果表明,DEHP 处理组测得的 OD 值远低于对照组,细胞活力显著降低。与 100 μmmol/L DEHP 处理组相比,添加维生素 C 后细胞活力和存活率增加(图 2d&e)。

Figure 2. Oxidative stress related levels in testicular tissues of mice and GC-2 cells.
图 2.小鼠和 GC-2 细胞睾丸组织中的氧化应激相关水平。

(a) Contents of GSH(降), MDA(升) and ROS(升,不知道放拍摄原图还是柱状图) in testicular tissues of mice. (b) Contents of GSH, MDA and ROS in GC-2 cells. (c) Contents of GSH, MDA and ROS in GC-2 cells alleviated by Vitamin C. (d) CCK-8 assay of cell viability. (e) Trypan blue staining of cell counting. All results are expressed as the mean ±SD. *P < 0.05, **P < 0.01, compared with the control group.
(a) 小鼠睾丸组织中 GSH(降)、MDA(升)和 ROS(升,不知道放拍摄原图还是柱状图)的含量。(b) GC-2 细胞中 GSH 、 MDA 和 ROS 的含量。(c) 维生素 C 减轻的 GC-2 细胞中 GSH、MDA 和 ROS 的含量。(d) CCK-8 细胞活力测定。(e) 细胞计数的台盼蓝染色。所有结果均表示为平均值 ±SD。*P < 0.05,**P < 0.01,与对照组相比。

3.3 DEHP induces autophagy leading to reproductive injury in mice
3.3 DEHP 诱导自噬导致小鼠生殖损伤

We detected the expression levels of the autophagy key proteins P62, LC3B and Atg12 in the testicular tissues of mice in each group. The results showed that compared to the control group, the protein contents of P62 decreased, LC3B and Atg12 in the DEHP exposure group increased in a dose-dependent manner (Figure 3a). The trends of their mRNA relative expression levels were also the same as above (Figure 3b-d). Additionally, proteins P62, LC3B and BECN1 were also immunofluorescence stained. And their fluorescence intensity increased in a dose-dependent manner (Figure 3e). These results suggested that DEHP can induce autophagy in mouse testicular tissues.
我们检测了各组小鼠睾丸组织中自噬关键蛋白 P62 、 LC3B 和 Atg12 的表达水平。结果表明,与对照组相比,DEHP 暴露组 P62 的蛋白质含量降低,LC3B 和 Atg12 呈剂量依赖性增加(图 3a)。它们的 mRNA 相对表达水平的趋势也与上述相同 (图 3b-d)。此外,蛋白 P62 、 LC3B 和 BECN1 也进行免疫荧光染色。它们的荧光强度以剂量依赖性方式增加(图 3e)。这些结果表明 DEHP 可以诱导小鼠睾丸组织中的自噬。

Figure 3. Autophagy-related changes induced by DEHP in testicular tissues of mice.
图 3.DEHP 在小鼠睾丸组织中诱导的自噬相关变化

(a) The protein expression of P62(降), LC3B(升), and Atg12 (升)in testicular tissues of mice. (b-c) The relative mRNA experssion of P62(不一定), LC3B(升) and Atg12(升) in testicular tissues of mice. (e) Immunofluorescence staining of P62, LC3B, and BECN1 in testicular tissues of mice. All results are expressed as the mean ±SD. *P < 0.05, **P < 0.01, compared with the control group.
(a) 小鼠睾丸组织中 P62 (降) 、 LC3B (升) 和 Atg12 (升) 的蛋白表达。(b-c)小鼠睾丸组织中 P62(不一定)、LC3B(升)和 Atg12(升)的相对 mRNA 表达。(e) 小鼠睾丸组织中 P62、LC3B 和 BECN1 的免疫荧光染色。所有结果均表示为平均值 ±SD。*P < 0.05,**P < 0.01,与对照组相比。

3.4 The level of autophagy in DEHP-treated GC-2 cells increased in vitro and could be inhibited by autophagy inhibitors.
3.4 DEHP 处理的 GC-2 细胞中的自噬水平在体外增加,并且可以被自噬抑制剂抑制。

The protein of GC-2 cells cultured by the above method was extracted. Western Blotting showed that the autophagy-related protein P62 decreased, whilst the contents of LC3B and Atg12 increased in DEHP-treated mouse spermatocytes (Figure 4a). The relative expression of mRNA also showed the same trend by performing RT-qPCR (Figure 4b-d). On this basis, we co-treated cells with the autophagy inhibitor 3-MA and 100μM DEHP. Compared with the 100μM DEHP treatment group, the contents of P62, LC3B and Atg12 in cells added with the autophagy inhibitor decreased significantly (Figure 4e), as well as their relative expression of mRNA (Figure 4f-h). Detection using the CCK-8 kit also showed higher cell viability when the inhibitor was added (Figure 4i). A series of results indicated that autophagy participates in DEHP-induced reproductive system injury and may play a major role.
提取上述方法培养的 GC-2 细胞的蛋白质。Western Blotting 显示,在 DEHP 处理的小鼠精母细胞中,自噬相关蛋白 P62 降低,而 LC3B 和 Atg12 的含量增加(图 4a)。通过进行 RT-qPCR,mRNA 的相对表达也显示出相同的趋势(图 4b-d)。在此基础上,我们用自噬抑制剂 3-MA 和 100μM DEHP 共同处理细胞。与 100μM DEHP 处理组相比,添加自噬抑制剂的细胞中 P62、LC3B 和 Atg12 的含量显着降低(图 4e),以及它们的 mRNA 相对表达(图 4f-h)。使用 CCK-8 试剂盒的检测也显示,当添加抑制剂时,细胞活力更高(图 4i)。一系列结果表明,自噬参与 DEHP 诱导的生殖系统损伤,并可能发挥主要作用。

Figure 4. Autophagy-related changes induced by DEHP in GC-2 cells.
图 4.DEHP 在 GC-2 细胞中诱导的自噬相关变化。

(a) The protein expression of P62, LC3B and Atg12 in GC-2 cells. (b-d) The relative mRNA experssion of P62, LC3B and Atg12 in GC-2 cells. (b) CCK-8 assay of cell viability and Trypan blue staining of cell counting. (e) The protein expression of P62, LC3B, and Atg12 in GC-2 cells inhibited by 3-MA. (f-h) The relative mRNA experssion of P62, LC3B and Atg12 in GC-2 cells inhibited by 3-MA. (i) CCK-8 assay of cell viability and Trypan blue staining of cell counting. All results are expressed as the mean ±SD. *P < 0.05, **P < 0.01, compared with the control group.
(a) GC-2 细胞中 P62 、 LC3B 和 Atg12 的蛋白表达。(b-d) GC-2 细胞中 P62 、 LC3B 和 Atg12 的相对 mRNA 表达。(b) 细胞活力的 CCK-8 测定和细胞计数的台盼蓝染色。(e) 受 3-MA 抑制的 GC-2 细胞中 P62 、 LC3B 和 Atg12 的蛋白表达。(f-h) 3-MA 抑制 GC-2 细胞中 P62 、 LC3B 和 Atg12 的相对 mRNA 表达。(i) 细胞活力的 CCK-8 测定和细胞计数的台盼蓝染色。所有结果均表示为平均值 ±SD。*P < 0.05,**P < 0.01,与对照组相比。

3.5 The key role of autophagy in DEHP-induced reproductive injury in mice
3.5 自噬在 DEHP 诱导的小鼠生殖损伤中的关键作用

The experimental results show that DEHP may induce autophagy by causing oxidative stress (Figure 5). Therefore, in the process of DEHP-induced reproductive damage in mice, we seized the two key points of oxidative stress and autophagy. And reverse verification was performed by adding corresponding inhibitors.
实验结果表明,DEHP 可能通过引起氧化应激来诱导自噬(图 5)。因此,在 DEHP 诱导的小鼠生殖损伤过程中,我们抓住了氧化应激和自噬两个关键点。并通过添加相应的抑制剂进行反向验证。

These results suggest that oxidative stress plays a key role in DEHP-induced autophagy. Alleviating oxidative stress with vitamin C can significantly reduce autophagy induced by DEHP and protect cells. Vitamin C may have potential in treating reproductive injuries caused by DEHP exposure through reducing oxidative stress and autophagy.These results suggest that oxidative stress plays a key role in DEHP-induced autophagy. Alleviating oxidative stress with vitamin C can significantly reduce autophagy induced by DEHP and protect cells. Vitamin C may have potential in treating reproductive injuries caused by DEHP exposure through reducing oxidative stress and autophagy.
这些结果表明,氧化应激在 DEHP 诱导的自噬中起关键作用。用维生素 C 缓解氧化应激可以显着减少 DEHP 诱导的自噬并保护细胞。维生素 C 可能通过减少氧化应激和自噬来治疗因 DEHP 暴露引起的生殖损伤。 这些结果表明,氧化应激在 DEHP 诱导的自噬中起关键作用。用维生素 C 缓解氧化应激可以显着减少 DEHP 诱导的自噬并保护细胞。维生素 C 可能通过减少氧化应激和自噬来治疗因 DEHP 暴露引起的生殖损伤。

We found that autophagy is the key mechanism of DEHP-induced reproductive damage. Autophagy inhibitors can significantly inhibit the occurrence of autophagy, improve the viability and survival rate of mouse spermatocytes, thereby blocking the reproductive toxicity of DEHP. This may provide new ideas and potential targets for the prevention and treatment of male infertility caused by DEHP exposure.
我们发现自噬是 DEHP 诱导的生殖损伤的关键机制。自噬抑制剂可显著抑制自噬的发生,提高小鼠精母细胞的活力和存活率,从而阻断DEHP的生殖毒性。这可能为预防和治疗由 DEHP 暴露引起的男性不育症提供新思路和潜在靶点。

Figure 5. Diagram of DEHP-induced reproductive damage in mice
图 5.DEHP 诱导的小鼠生殖损伤图

DEHP may increase the level of autophagy by inducing oxidative stress, thus causing damage to mouse testicular tissue and spermatocytes.
DEHP 可能通过诱导氧化应激来增加自噬水平,从而对小鼠睾丸组织和精母细胞造成损害。

Discussion
讨论

In epidemiological studies, a significant association has been observed between di(2-ethylhexyl) phthalate (DEHP) exposure and disorders of the male reproductive system (10.1016/j.envint.2018.07.029). This correlation has been substantiated by a plethora of prior experimental investigations. Alterations in body weight and the weight of reproductive organs in experimental animals are key parameters for assessing drug toxicity and the impacts of exposure (10.1186/s12935-019-0805-2). Our study elucidates that DEHP exposure precipitated a marked reduction in body weight among ICR mice compared to the control group. Furthermore, the organ coefficients of both the testes and epididymides in male ICR mice exhibited a decline, signifying that DEHP exerts a significant impact on the murine reproductive system. Histological examination of HE-stained sections under the microscope revealed morphological changes in the seminiferous tubules, further corroborating this observation. Through sperm smear analysis, our study further revealed the harmful effects of DEHP on male reproductive function, including sperm quality and quantity. This is consistent with the existing literature ( doi : 10.1016 / j.scitotenv.2022.160432 ). DEHP treatment in ICR mice resulted in a notable increase in the rate of sperm malformation, including defects in the head, neck, and tail regions, as well as a reduction in sperm concentration and motility.
在流行病学研究中,已观察到邻苯二甲酸二(2-乙基己基)酯 (DEHP) 暴露与男性生殖系统疾病之间存在显着关联 (10.1016/j.envint.2018.07.029)。这种相关性已经被先前的大量实验研究所证实。实验动物体重和生殖器官重量的变化是评估药物毒性和暴露影响的关键参数 (10.1186/s12935-019-0805-2)。我们的研究阐明,与对照组相比,DEHP 暴露导致 ICR 小鼠的体重显着降低。此外,雄性 ICR 小鼠睾丸和附睾的器官系数均表现出下降,表明 DEHP 对小鼠生殖系统产生显着影响。在显微镜下对 HE 染色切片的组织学检查显示生精小管的形态学变化,进一步证实了这一观察结果。通过精子涂片分析,我们的研究进一步揭示了 DEHP 对男性生殖功能的有害影响,包括精子的质量和数量。这与现有文献 ( doi : 10.1016 / j.scitotenv.2022.160432 ) 一致。ICR 小鼠的 DEHP 处理导致精子畸形率显着增加,包括头部、颈部和尾部区域的缺陷,以及精子浓度和活力的降低。

DEHP has been shown to increase oxidative stress levels within cells (doi:10.1039/c8tx00263k). A plethora of studies have elucidated that oxidative stress responses, particularly those induced by such compounds, play a pivotal role in the onset of reproductive disorders (doi:10.3109/15376516.2014.989349). The observed decline in sperm count, survival ability, and the escalation in abnormal sperm morphology may be indicative of oxidative stress. Correspondingly, in both tissue and cellular assays, we detected evidence of oxidative stress, as evidenced by changes in the levels of GSH, MDA, and ROS.These findings suggest that oxidative stress damage is a crucial mechanism of DEHP toxicity, consistent with previous studies.(10.1016/j.envpol.2022.120173).
DEHP 已被证明会增加细胞内的氧化应激水平 (doi:10.1039/c8tx00263k)。大量研究阐明,氧化应激反应,尤其是由此类化合物诱导的氧化应激反应,在生殖障碍的发作中起着关键作用 (doi:10.3109/15376516.2014.989349)。观察到的精子数量、存活能力下降和精子形态异常的升级可能表明氧化应激。 相应地,在组织和细胞测定中,我们检测到氧化应激的证据,如 GSH 、 MDA 和 ROS 水平的变化所证明的那样。这些发现表明,氧化应激损伤是 DEHP 毒性的关键机制,与以前的研究一致。10.1016/j.envpol.2022.120173)。

The process of autophagy within cells encompasses a series of alterations in proteins and pathways. Among these, LC3B, a component of the LC3/GABARAP protein family, is instrumental in the generation and maturation of autophagosomes, and is widely recognized as a marker of autophagosomes in mammalian cells (doi:10.1096/fj.201600698R). Another critical protein in autophagy is Atg12, which collaborates with other members of the ATG family to form ubiquitin-like conjugation systems. Atg12 is integral to the expansion of the autophagosomal membrane (doi:10.1186/s12943-020-1138-4). In contrast, P62 functions as an autophagic adapter protein, binding to ubiquitinated proteins and facilitating their transportation to autophagosomes (doi:10.1016/j.freeradbiomed.2015.06.014).
细胞内的自噬过程包括蛋白质和通路的一系列改变。其中,LC3B 是 LC3/GABARAP 蛋白家族的一个组成部分,有助于自噬体的产生和成熟,并被广泛认为是哺乳动物细胞中自噬体的标志物 (doi:10.1096/fj.201600698R)。自噬中的另一种关键蛋白是 Atg12,它与 ATG 家族的其他成员合作形成泛素样偶联系统。Atg12 是自噬体膜扩增不可或缺的一部分 (doi:10.1186/s12943-020-1138-4)。相比之下,P62 作为自噬接头蛋白发挥作用,与泛素化蛋白结合并促进其转运到自噬体 (doi:10.1016/j.freeradbiomed.2015.06.014)。

Our analysis of protein levels and mRNA expression of these three key autophagic markers indicated that DEHP exposure results in the downregulation of P62 and upregulation of Atg12 and LC3B expressions. Based on these findings, we posit that DEHP can induce autophagy in the testicular cells of male mice. Autophagy plays a critical role in cell survival under both physiological and certain pathological conditions. However, impaired autophagic function can lead to cell death, a phenomenon known as cell death by autophagy (doi:10.1038/cdd.2011.146). Notably, the introduction of the autophagy inhibitor 3-MA to DEHP-treated cells led to a marked increase in cell viability, implying that DEHP-induced autophagy in male mice's testicular cells may exert cytotoxic effects. Consequently, inhibiting cellular autophagy might provide a therapeutic avenue to mitigate reproductive dysfunction induced by DEHP exposure.
我们对这三个关键自噬标志物的蛋白质水平和 mRNA 表达的分析表明,DEHP 暴露导致 P62 下调以及 Atg12 和 LC3B 表达上调。基于这些发现,我们假设 DEHP 可以在雄性小鼠的睾丸细胞中诱导自噬。自噬在生理和某些病理条件下的细胞存活中起着关键作用。然而,自噬功能受损会导致细胞死亡,这种现象称为自噬导致的细胞死亡 (doi:10.1038/cdd.2011.146)。值得注意的是,将自噬抑制剂 3-MA 引入 DEHP 处理的细胞导致细胞活力显着增加,这意味着 DEHP 诱导的雄性小鼠睾丸细胞自噬可能发挥细胞毒性作用。因此,抑制细胞自噬可能为减轻 DEHP 暴露引起的生殖功能障碍提供一种治疗途径。

Furthermore, treatment of cells exposed to DEHP with vitamin C led to a reduction in oxidative stress levels and a significant increase in cell viability, with levels of autophagy-related proteins trending towards normalization. Vitamin C, an essential micronutrient known for its antioxidant properties, plays a pivotal role in counteracting the onset and progression of various diseases (doi:10.1002/JLB.1MR1219-245R). Our findings highlight the critical role of oxidative stress in inducing autophagy and suggest that vitamin C may play a significant role in combatting reproductive dysfunction caused by DEHP.
此外,用维生素 C 处理暴露于 DEHP 的细胞导致氧化应激水平降低,细胞活力显着增加,自噬相关蛋白水平趋于正常化。维生素 C 是一种必需的微量营养素,以其抗氧化特性而闻名,在对抗各种疾病的发生和发展中起着关键作用 (doi:10.1002/JLB.1MR1219-245R)。我们的研究结果强调了氧化应激在诱导自噬中的关键作用,并表明维生素 C 可能在对抗 DEHP 引起的生殖功能障碍方面发挥重要作用。

Additionally, our study found that mitocytosis occurred in DEHP-treated cells, and the expression of related factors increased. Mitocytosis reduces the degree of cellular autophagy by clearing damaged mitochondria and avoiding their intracellular lysis. Therefore, it is speculated that DEHP induces oxidative stress, activates autophagy mechanisms, and leads to cell death, while downstream mitocytosis serves as a protective factor against reproductive damage caused by DEHP. In other words, cellular autophagy and mitocytosis co-regulate the impact of oxidative stress on testicular cells.
此外,我们的研究发现 DEHP 处理的细胞发生有丝分裂症,并且相关因子的表达增加。 丝粒细胞增多症通过清除受损的线粒体并避免其细胞内裂解来降低细胞自噬的程度。因此,推测 DEHP 诱导氧化应激,激活自噬机制,导致细胞死亡,而下游线粒细胞增多则作为防止 DEHP 引起的生殖损伤的保护因子。换句话说,细胞自噬和有丝分裂细胞作用共同调节氧化应激对睾丸细胞的影响。

In conclusion, the experimental data from our study indicate that DEHP inflicts significant harm on the male reproductive system. The reproductive toxicity of DEHP is mediated through key mechanisms, such as oxidative stress and cellular autophagy. Among them, mitocytosis plays an important role in protecting germ cells from DEHP damage. This study contributes to the broader understanding of the mechanisms underlying DEHP-induced reproductive dysfunction and offers novel insights for its treatment and prevention. Strategies aimed at inhibiting oxidative stress and autophagy may hold promise in alleviating the reproductive toxicity associated with DEHP exposure. Furthermore, vitamin C, as a potent in vivo antioxidant, emerges as a promising candidate for reducing DEHP-induced oxidative damage and safeguarding male reproductive health. Overall, this study enriches our understanding of DEHP toxicity and presents new perspectives for managing its detrimental effects on male fertility.
总之,我们研究的实验数据表明,DEHP 对男性生殖系统造成重大伤害。DEHP 的生殖毒性是通过关键机制介导的,例如氧化应激和细胞自噬。其中,有丝分裂细胞增多在保护生殖细胞免受 DEHP 损伤方面起着重要作用。 本研究有助于更广泛地了解 DEHP 诱导的生殖功能障碍的潜在机制,并为其治疗和预防提供新的见解。旨在抑制氧化应激和自噬的策略可能在减轻与 DEHP 暴露相关的生殖毒性方面具有希望。此外,维生素 C 作为一种有效的体内抗氧化剂,成为减少 DEHP 诱导的氧化损伤和保护男性生殖健康的有前途的候选者。总的来说,这项研究丰富了我们对 DEHP 毒性的理解,并为管理其对男性生育能力的不利影响提供了新的视角。

Acknowledgements and funding
鸣谢和资助

This study was supported by grants 81673224 and 81273018 from the National Natural Science Foundation of China, grants 2024JC-YBQN-0771 and 2024JC-YBQN-0897 from the Natural Science Foundation of Shaanxi Province, grant SJ202210698248 and SJ202210698250 from the National Training Program of lnnovation and Entrepreneurship for Undergraduates, and grant 2022ZDLSF03-10 from the Key Research Plan of Shaanxi Province.
本研究得到了国家自然科学基金81673224和81273018、陕西省自然科学基金 2024JC-YBQN-0771 和 2024JC-YBQN-0897、国家本科生创新创业训练计划 SJ202210698248和SJ202210698250资助,以及陕西省重点研究计划资助 2022ZDLSF03-10。

Authors’ contributions
作者的贡献

Zhi-hao Liu: Formal analysis, Methodology, Writing–original draft, Data curation. Kun Mao: Formal analysis, Writing–original draft, Data curation. Si-yu Wang: Formal analysis, Methodology. Hao-zhe Yan: Data curation. Lang-qing She: Formal analysis. Xin-qi Gao: Software. Zi-xin Yin: Formal analysis. Hao-fu Liu: Software. Pan Ge: Project administration, Formal analysis, Methodology, Supervision.
Zhi-hao Liu:形式分析、方法论、写作-原稿、数据管理。毛坤:形式分析,写作-原稿,数据管理。Si-yu Wang: 形式分析,方法论。 Hao-zhe Yan:数据管理。 Lang-qing She: 形式分析.Xin-qi Gao:软件。Zi-xin Yin:形式分析。Hao-fu Liu:软件。攀戈:项目管理、形式分析、方法论、监督。

Competing interests
利益争夺

The authors declare that they have no competing interests.
作者声明他们没有利益争夺。

References
引用