Cadmium attenuates testosterone synthesis by promoting ferroptosis and blocking autophagosome-lysosome fusion

doi:10.1016/j.freeradbiomed.2021.09.028

Free Radical Biology and Medicine
自由基生物学和医学

Volume 176, 20 November 2021, Pages 176-188
第 176 卷，2021 年 11 月 20 日，第 176-188 页

https://doi.org/10.1016/j.freeradbiomed.2021.09.028IF: 7.1 Q1 IF: 7.1 Q1
https://doi.org/10.1016/j.freeradbiomed.2021.09.028IF：7.1 Q1 如果：7.1 Q1 Get rights and content 获取权利和内容

Highlights 亮点

•
Cadmium attenuates testosterone synthesis.
镉会减弱睾酮的合成。
•
Cadmium induces ferroptosis, characterized by iron overload and lipid peroxidation.
镉会诱发铁死亡，其特征是铁过载和脂质过氧化。
•
The overexpression of heme oxygenase 1, which releases Fe²⁺ from heme, participates in ferroptosis.
血红素加氧酶 1 的过度表达（从血红素中释放 Fe ²⁺ ）参与铁死亡。
•
Cadmium leads to autophagic vesicles accumulation with the blockage of autophagosome-lysosome fusion.
镉导致自噬囊泡积聚并阻碍自噬体-溶酶体融合。
•
The autophagy disruption affects the intake of cholesterol and testosterone biosynthesis.
自噬破坏会影响胆固醇的摄入和睾酮的生物合成。

Abstract 抽象的

Ferroptosis is a newly defined programmed cell death pathway characterized by iron overload and lipid peroxidation. Increasing studies show that autophagy regulates testosterone synthesis and promotes ferroptosis. Testosterone is essential for sexual development and the maintenance of male characteristics. The deficiency of testosterone induced by cadmium (Cd) can severely affect male fertility. However, the underlying mechanism of testosterone reduction after Cd exposure remains blurry. In this study, we found that Cd affected iron homeostasis and elicited ferroptosis, ultimately reducing testosterone production. Mechanically, our findings revealed that Cd-induced ferroptosis depended upon the excessive activation of Heme oxygenase 1 (HMOX1) and the release of free iron from heme. Additionally, Cd exposure promoted autophagosome formation but blocked autophagosome-lysosome fusion, which attenuated the absorption of total cholesterol and triglycerides, further aggravating testosterone synthesis disorder. Collectively, Cd induced ferroptosis by iron homeostasis dysregulation, mediated by excessive activation of HMOX-1. The disruption of autophagy flow contributed to Cd-induced testicular dysfunction and attenuated testosterone synthesis.
铁死亡是一种新定义的程序性细胞死亡途径，其特征是铁过载和脂质过氧化。越来越多的研究表明自噬调节睾酮合成并促进铁死亡。睾酮对于性发育和男性特征的维持至关重要。镉（Cd）引起的睾酮缺乏会严重影响男性的生育能力。然而，镉暴露后睾酮减少的潜在机制仍然模糊。在这项研究中，我们发现镉影响铁稳态并引起铁死亡，最终减少睾酮的产生。从机械角度来看，我们的研究结果表明，Cd 诱导的铁死亡依赖于血红素加氧酶 1 (HMOX1) 的过度激活和血红素中游离铁的释放。此外，镉暴露促进自噬体形成，但阻碍自噬体-溶酶体融合，从而减弱总胆固醇和甘油三酯的吸收，进一步加剧睾酮合成障碍。总的来说，Cd 通过 HMOX-1 过度激活介导的铁稳态失调诱导铁死亡。自噬流的破坏导致镉诱导的睾丸功能障碍和睾酮合成减弱。

Graphical abstract 图文摘要

Keywords 关键词

Cadmium

Testosterone synthesis

Ferroptosis

Autophagy

Iron homeostasis

镉

睾酮合成

铁死亡

自噬

铁稳态

Abbreviations 缩写

Cd 光盘

cadmium 镉

LH 黄体激素

Luteinizing hormone 黄体生成素

FSH 卵泡刺激素

Follicle stimulating hormone
卵泡刺激素

ROS 活性氧

reactive oxygen species 活性氧

MtROS 活性氧

mitochondrial reactive oxygen species
线粒体活性氧

Fer-1

ferrostatin-1 铁他汀-1

Dfom 德福姆

desferioxamine; 去铁胺；

MMP 基质金属蛋白酶

mitochondrial membrane potential
线粒体膜电位

GSH 还原型谷胱甘肽

glutathione 谷胱甘肽

MDA 丙二醛

malondialdehyde 丙二醛

LPO

Lipid peroxidation 脂质过氧化

SOD 草皮

Superoxide dismutase 超氧化物歧化酶

ETC

electron transport chain 电子传递链

ATP

Adenosine Triphosphate 三磷酸腺苷

GPXs GPX

glutathione peroxidases 谷胱甘肽过氧化物酶

Gpx4

glutathione peroxidase 4 谷胱甘肽过氧化物酶4

GSH & GSSG 谷胱甘肽和谷胱甘肽

glutathione and oxidized glutathione disulfide;
谷胱甘肽和氧化型谷胱甘肽二硫化物；

T-CHO

total cholesterol 总胆固醇

triglyceride; 甘油三酯；

F–CHO F-CHO

free cholesterol 游离胆固醇

LIP 唇

labile Fe pool 不稳定铁池

NRF2

Nuclear factor-E2-related factor 2
核因子-E2相关因子2

HO-1

Heme oxygenase 1
血红素加氧酶1

SLC7A11

Solute carrier family 7 member 11 FSP1, Ferroptosis suppressor protein 1
溶质载体家族 7 成员 11 FSP1，铁死亡抑制蛋白 1

FTH1 光纤到户1

ferritin heavy chain 1 铁蛋白重链1

FTL 超光速

ferritin light chain 铁蛋白轻链

TfR1 转铁蛋白R1

transferrin receptor 1 转铁蛋白受体1

FPN1

ferroportin 1 铁转运蛋白1

IRP1 and IRP2 IRP1 和 IRP2

iron regulatory protein 1 and iron regulatory protein 2
铁调节蛋白1和铁调节蛋白2

P450scc

cytochrome P450 family 11 subfamily A member 1
细胞色素 P450家族 11 亚家族 A 成员 1

Cty C C城

cytochrome c
细胞色素c

znpp 锌磷

Zinc Protoporphyrin 原卟啉锌

SR-BI

scavenger receptor class B, type I
B 类清道夫受体，I 型

LDLR 低密度脂蛋白受体

low-density lipoprotein receptor
低密度脂蛋白受体

ATG5

Autophagy related 5 自噬相关5

ATG7

Autophagy related 7 自噬相关7

p62

sequestosome 1 隔离体1

LC3

Microtubule-associated protein 1 light chain 3
微管相关蛋白1轻链3

1. Introduction 一、简介

The infertility rate continues to rise in this century and about 15% of couples are infertile [1], amounting to 48.5 million couples [2]. Globally, approximately 40–50% of infertility cases are due to male factors [2,3]. The causes of male infertility are complex and at least 30% of cases remain etiologically unclear [4]. Although genetic factors can explain a certain percentage of the cases, increasing environmental pollution might also contribute to the persistent increase of male infertility [5,6].
本世纪不孕率持续上升，约15%的夫妇不孕[ 1 ]，达到4850万对夫妇[ 2 ]。全球范围内，大约 40-50% 的不孕不育病例是由男性因素造成的 [ 2 , 3 ]。男性不育的原因很复杂，至少有30%的病例病因尚不清楚[ 4 ]。尽管遗传因素可以解释一定比例的病例，但环境污染的加剧也可能导致男性不育症的持续增加[ 5 , 6 ]。

Cadmium (Cd) is a toxic heavy metal and ubiquitous environmental endocrine disruptor, which is derived from agricultural and industrial sources. Cd is widely present in contaminated foods, water, soil and air at low concentrations [[7], [8], [9], [10], [11]]. Notably, cigarette smoking is an increasingly significant route of Cd exposure [12,13]. Continuous low-dose Cd exposure can lead to its chronic accumulation in the human body, due to a long half-life of 25–30 years. Studies have shown that environmental exposure to Cd can affect male fertility via multiple pathways, seriously threatening male reproductive health [[14], [15], [16], [17], [18]]. Previous studies on Cd-induced damage to male fertility focused on semen quality [19], which includes sperm concentration, motility and morphology. Although a growing number of epidemiological studies indicate that environmental exposure to Cd is also linked with changes in serum testosterone level [[20], [21], [22]], no consensus has been reached. For example, Zeng et al. reported that with occupational cadmium exposure, the levels of serum testosterone were significantly increased after adjusting for confounders such as age, alcohol consumption and smoking habits [22]. Jurasović et al. also found that serum Cd level was significantly associated with an elevation in serum testosterone levels [21]. However, Chen et al. found that serum Cd level was negatively correlated with testosterone [20]. Gabrielsen et al. reported that erectile dysfunction was related to serum Cd level [23]. Testosterone is indispensable for sexual development and the maintenance of male characteristics. The deficiency of testosterone can seriously affect male fertility. However, the underlying mechanism of changes in serum testosterone level after Cd exposure has not been fully elucidated.
镉 (Cd) 是一种有毒重金属，是普遍存在的环境内分泌干扰物，来源于农业和工业来源。镉以低浓度广泛存在于受污染的食品、水、土壤和空气中[ [7] 、 [8] 、 [9] 、 [10] 、 [11] ]。值得注意的是，吸烟是镉暴露的一个日益重要的途径[ 12 , 13 ]。由于半衰期长达 25-30 年，持续低剂量镉暴露可导致其在人体内慢性积累。研究表明，环境中镉暴露可通过多种途径影响男性生育能力，严重威胁男性生殖健康[ [14] 、 [15] 、 [16] 、 [17] 、 [18] ]。先前关于镉对男性生育能力损害的研究主要集中在精液质量[ 19 ]，包括精子浓度、活力和形态。尽管越来越多的流行病学研究表明环境中镉暴露也与血清睾酮水平的变化有关[ [20] 、 [21] 、 [22] ]，但尚未达成共识。例如，曾等人。报道称，随着职业镉暴露，在调整年龄、饮酒和吸烟习惯等混杂因素后，血清睾酮水平显着升高[ 22 ]。尤拉索维奇等人。还发现血清镉水平与血清睾酮水平升高显着相关[ 21 ]。然而，陈等人。发现血清Cd水平与睾酮呈负相关[ 20 ]。加布里埃尔森等人。报道勃起功能障碍与血清Cd水平有关[ 23 ]。睾酮对于性发育和男性特征的维持是不可或缺的。睾酮缺乏会严重影响男性的生育能力。然而，镉暴露后血清睾酮水平变化的潜在机制尚未完全阐明。

Earlier research on the mechanism of Cd reproductive toxicity mainly focused on three aspects: oxidative stress imbalance, apoptosis pathway activation and endocrine disruption, whereby oxidative stress imbalance is considered the most common. Cd exposure can cause an imbalance between the production and degradation of cellular reactive oxygen species (ROS), leading to lipid peroxidation, mitochondrial dysfunction and enzymes inactivation [3,[24], [25], [26], [27], [28], [29], [30]]. Nonetheless, no study to date has linked ferroptosis to the etiology of Cd-induced testicular dysfunction. Hence, we hypothesized that ferroptosis might play a crucial role in testicular dysfunction induced by Cd contamination.
早期对Cd生殖毒性机制的研究主要集中在氧化应激失衡、凋亡途径激活和内分泌干扰三个方面，其中氧化应激失衡被认为是最常见的。镉暴露会导致细胞活性氧（ROS）的产生和降解之间的不平衡，导致脂质过氧化、线粒体功能障碍和酶失活[ 3 ， [24] ， [25] ， [26] ， [27] ， [ 28] 、 [29] 、 [30] ]。尽管如此，迄今为止还没有研究将铁死亡与镉引起的睾丸功能障碍的病因联系起来。因此，我们假设铁死亡可能在镉污染引起的睾丸功能障碍中发挥至关重要的作用。

Ferroptosis is a newly defined form of programmed cell death induced by small molecules (e.g., erastin, RSL3) and related factors. Ferroptosis execution is characterized by iron overload and lipid peroxidation [31,32]. The molecular mechanism of ferroptosis primarily involves canonical ferroptosis induction and non-canonical ferroptosis induction. The canonical pathway is to directly or indirectly inhibit the activity of glutathione peroxidases (GPXs), especially glutathione peroxidase 4 (GPX4). The non-canonical pathway is to increase the labile iron pool (LIP), which directly catalyzing free radical formation via the fenton reaction, further propagating lipid peroxidation [32]. It has been reported that ferroptosis is present in numerous disease models, including hemochromatosis [33], neurodegenerative diseases [[34], [35], [36]], ischemia-reperfusion injury [37], heart failure [38] and cancers [[39], [40], [41]].
铁死亡是一种新定义的程序性细胞死亡形式，由小分子（例如erastin、RSL3）和相关因子诱导。铁死亡执行的特点是铁过载和脂质过氧化[ 31 , 32 ]。铁死亡的分子机制主要包括经典铁死亡诱导和非经典铁死亡诱导。典型途径是直接或间接抑制谷胱甘肽过氧化物酶(GPX)，尤其是谷胱甘肽过氧化物酶 4 (GPX4) 的活性。非经典途径是增加不稳定铁池（LIP），其通过芬顿反应直接催化自由基形成，进一步促进脂质过氧化[ 32 ]。据报道，铁死亡存在于许多疾病模型中，包括血色素沉着症[ 33 ]、神经退行性疾病[ [34] 、 [35] 、 [36] ]、缺血再灌注损伤[ 37 ]、心力衰竭[ 38 ]和癌症[ [39] 、 [40] 、 [41] ]。

Several studies support the notion that ferroptosis is an autophagy-dependent cell death process [42,43]. Importantly, ROS and lipid peroxidation can trigger excessive autophagy and lysosome activity [[44], [45], [46]], while autophagy can in turn promote ferroptosis [42] via NCOA4-facilitated ferritinophagy, RAB7A-dependent lipophagy, BECN1-mediated system xc⁻ inhibition, STAT3-induced lysosomal membrane permeabilization, and HSP90-associated chaperone-mediated autophagy [43]. Testosterone is mainly produced in Leydig cells, and it is reported that autophagy regulates testosterone synthesis by facilitating cholesterol uptake in Leydig cells [47]. Herein, we explored the relationship between ferroptosis/autophagy and testosterone reduction after Cd treatment in mice.
多项研究支持铁死亡是一种自噬依赖性细胞死亡过程的观点[ 42 , 43 ]。重要的是，ROS和脂质过氧化可引发过度的自噬和溶酶体活性[ [44] 、 [45] 、 [46] ]，而自噬又可通过NCOA4促进的铁蛋白自噬、RAB7A依赖性自噬、BECN1-促进铁死亡[ 42 ]介导的系统 xc ^-抑制、STAT3 诱导的溶酶体膜透化，以及HSP90 相关分子伴侣介导的自噬 [ 43 ]。睾酮主要在间质细胞中产生，据报道，自噬通过促进间质细胞摄取胆固醇来调节睾酮合成[ 47 ]。在此，我们探讨了小鼠镉治疗后铁死亡/自噬与睾酮减少之间的关系。

2. Materials and methods 2 材料与方法

2.1. Ethics statement 2.1.道德声明

All the animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Tongji Medical College, Huazhong University of Science and Technology (S2457). All experiments with mice were implemented ethically according to the Guide for the Care and Use of Laboratory Animals.
所有动物实验均经华中科技大学同济医学院动物护理和使用机构委员会（IACUC）批准（ S2457 ）。所有小鼠实验均根据《实验动物护理和使用指南》道德地进行。

2.2. Animals and experimental groups
2.2.动物和实验组

Eight-week-old adult male C57BL/6J mice were raised for 7 days before the study in a temperature and humidity controlled animal facility (22–25 °C, 50% relative humidity) with a12-h light/dark cycles. The animals had free access to food and water.
研究前，将八周大的成年雄性 C57BL/6J 小鼠在温度和湿度受控的动物设施（22-25°C，50% 相对湿度）中饲养 7 天，光照/黑暗周期为 12 小时。动物可以自由获取食物和水。

A total of 10 mice were randomly assigned into two groups. As described in our previous study [48,49], the control group was treated with 0.9% NaCl (0 mg CdCl₂), and the treatment group was intraperitoneally injected with CdCl₂ at a dose of 1.0 mg per kg of body weight for 1 week. Cd chloride was purchased from Sigma Chemical Co. (St. Louis, MO, USA).
总共10只小鼠被随机分为两组。如我们之前的研究[ 48 , 49 ]所述，对照组用0.9% NaCl（0 mg CdCl ₂ ）治疗，治疗组腹腔注射CdCl _{2 ，}剂量为1.0 mg/kg体重，持续时间1周。氯化镉购自 Sigma Chemical Co.（美国密苏里州圣路易斯）。

2.3. Cd content in the testis
2.3.睾丸中镉含量

As previously reported [48], Cd content in testis was detected by graphite furnace atomic absorption spectrometry (GFAAS). The samples were baked and weighed. We added 3 ml nitric acid and 0.5 ml perchloric acid to dissolve into a flowing liquid. And then the samples were baked on a 280 °C hotplate for 1 h. After the samples cooled, 5 ml pure water was added to volume. The operating conditions for GFAAS require atomization at 1600 °C for Cd after a heating phase (110 °C and then 1308 °C) and pyrolysis at 500 °C for Cd. The wavelength was 228.80 nm for Cd.
先前报道[ 48 ]，采用石墨炉原子吸收光谱法（GFAAS）检测睾丸中的Cd含量。将样品烘烤并称重。我们加入3毫升硝酸和0.5毫升高氯酸溶解成流动液体。然后将样品在280℃电炉上烘烤1小时。待样品冷却后，加入5ml纯水至刻度。 GFAAS 的操作条件要求在加热阶段（110 °C，然后 1308 °C）后在 1600 °C 下对 Cd 进行雾化，并在 500 °C 下对 Cd 进行热解。 Cd 的波长为 228.80 nm。

2.4. Cell culture 2.4.细胞培养

Mouse Testis Leydig Cell line TM3, was cultured in Dulbecco's modified Eagle's medium: F-12 (DMEM/F12) (Gibco, USA) supplemented with 2.5% fetal bovine serum (FBS) (Gibco, USA), 5% horse serum (Solarbio, China), and 1% penicillin-streptomycin antibiotics (Sigma-Aldrich, USA) in an incubator with 5% CO₂ at 37 °C.
小鼠睾丸Leydig 细胞系 TM3，在 Dulbecco 改良 Eagle 培养基中培养：F-12 (DMEM/F12)（Gibco，美国），补充有 2.5% 胎牛血清 (FBS)（Gibco，美国）、5% 马血清 (Solarbio) ，中国）和 1% 青霉素-链霉素抗生素（Sigma-Aldrich，美国），置于培养箱中37 °C 时5% CO ₂ 。

2.5. Determination of sex hormones (T, LH and FSH)
2.5.性激素（T、LH 和 FSH）的测定

Serum samples were obtained from orbital blood samples by clotting overnight at 4 °C before centrifugation for 10 min at 1000×g and storage in aliquots at −80 °C for later use. Similarly, cell culture supernatants of TM3 cells were collected and stored in aliquots at −80 °C for later use.
血清样品是通过在 4°C 下凝固过夜，然后以 1000× g离心10 分钟，并在 -80°C 下等分储存以备后用而从眼眶血样中获得的。类似地，收集TM3细胞的细胞培养上清液并等分储存于-80°C以备后用。

Reagent kits for the T assay were purchased from ELK Biotechnology (Wuhan, China), and Reagent kits for the LH and FSH assays were purchased from Cloud-Clone Corp. (Houston, USA). The assays were conducted according to the manufacturer's instructions.
T测定的试剂盒购自ELK Biotechnology（中国武汉），LH和FSH测定的试剂盒购自Cloud-Clone Corp.（休斯顿，美国）。根据制造商的说明进行测定。

2.6. Histopathological analyses of the testis
2.6。睾丸的组织病理学分析

For Hematoxylin and eosin staining (HE), testis sections were paraffin-embedded, de-waxed, rehydrated, and stained with HE. The images were captured using an inverted microscope.
对于苏木精和伊红染色 (HE)，将睾丸切片石蜡包埋、脱蜡、再水化并用 HE 染色。使用倒置显微镜捕获图像。

2.7. Transmission electron microscopy
2.7.透射电子显微镜

Testis samples were fixed with 2.5% glutaraldehyde for 2 h at 4 °C, post-fixed with 1% osmium tetroxide, and embedded in Epon 812. Blocks were cut into semi-thin sections and stained with methanolic uranyl acetate and lead citrate. The testicular ultrastructure was investigated by transmission electron microscopy (TEM) (JEM 1200-EX; Hitachi, Ltd, Tokyo, Japan) at 80 kV.
睾丸样本用2.5%戊二醛在4℃下固定2小时，后用1%四氧化锇固定，并包埋于Epon 812中。将块切成半薄切片并用甲醇乙酸双氧铀和柠檬酸铅染色。通过透射电子显微镜 (TEM)（JEM 1200-EX；日立有限公司，东京，日本）在 80 kV 下研究睾丸超微结构。

2.8. Cell viability 2.8.细胞活力

Cell viability was assessed using a CCK-8 assay Kit (A311-01/02, Vazyme, China) according to the manufacturer's instructions. In experimental groups, cells in 96-well plates were treated with Cd at the dosage of 1, 2.5, 5, 10 and 20 μg/ml, while the control group was mock-treated with sodium chloride. At the end of exposure, the culture medium was replaced with 100 μl CCK-8 solution (containing 90 μl cell culture medium and 10 μl CCK-8 reagent), and incubated for 1 h. Then, the absorbance at 450 nm was measured using a conventional microplate reader.
根据制造商的说明，使用 CCK-8 测定试剂盒（A311-01/02，Vazyme，中国）评估细胞活力。实验组中，96孔板中的细胞用1、2.5、5、10和20μg/ml剂量的Cd处理，而对照组用氯化钠模拟处理。暴露结束后，更换培养基为100 μl CCK-8溶液（含90 μl细胞培养基和10 μl CCK-8试剂），孵育1 h。然后，使用常规酶标仪测量 450 nm 处的吸光度。

2.9. ROS and mtROS measurement
2.9. ROS 和 mtROS 测量

TM3 cells were cultured in confocal dishes and then exposed to Cd at a concentration of 2.5 μg/ml for 12 h. Then, DCFH-DA (Beyotime, China) was added at a final concentration of 10 μM. After incubation for 20 min at 37 °C, the cells were washed with warm serum-free DMEM/F12 and immediately observed under a confocal microscope (ZEISS LSM 780). As for mtROS, TM3 cells were incubated with 5 μM MitoSOX™ Red Mitochondrial Superoxide Indicator (M36008, Thermo, USA) in HBSS medium at 37 °C for 10 min, protected from the light. Then, the cells were gently washed three times with warm serum-free DMEM/F12 and immediately observed under a confocal microscope (ZEISS LSM 780). The fluorescence intensity was analyzed using ZEN 3.2 (blue edition). The fluorescence intensity of single cells was analyzed using CellProfiler 4.0.7.
TM3细胞在共聚焦培养皿中培养，然后暴露于浓度为2.5μg/ml的Cd中12小时。然后添加DCFH-DA（Beyotime，中国），终浓度为10 μM。 37°C 孵育 20 分钟后，用温热的无血清 DMEM/F12 洗涤细胞，并立即在共聚焦显微镜(ZEISS LSM 780) 下观察。对于 mtROS，TM3 细胞与 5 μM MitoSOX™ 红色线粒体超氧化物指示剂（M36008，Thermo，美国）在 HBSS 培养基中于 37 °C 避光孵育 10 分钟。然后，用温热的无血清DMEM/F12轻轻洗涤细胞3次，并立即在共聚焦显微镜（ZEISS LSM 780）下观察。使用ZEN 3.2（蓝色版）分析荧光强度。使用CellProfiler 4.0.7分析单细胞的荧光强度。

2.10. Iron assay 2.10.铁测定

The intracellular iron concentration was measured using FerroOrange (F374, Dojindo, Japan). TM3 cells were cultured in confocal dishes and then exposed to Cd at the concentration of 2.5 μg/ml for 12 h. Then, the supernatant was discarded and the cells were washed with HBSS twice. Subsequently, the 1 μM FerroOrange working solution was added to the cells. After incubation for 30 min at 37 °C, the cells were observed under a confocal microscope (ZEISS LSM 780). The fluorescence intensity of single cells was analyzed using CellProfiler 4.0.7.
使用 FerroOrange (F374, Dojindo, Japan) 测量细胞内铁浓度。 TM3细胞在共聚焦培养皿中培养，然后暴露于浓度为2.5μg/ml的Cd中12小时。然后，弃去上清液并用HBSS洗涤细胞两次。随后，将 1 μM FerroOrange 工作溶液添加到细胞中。 37°C 孵育 30 分钟后，在共聚焦显微镜（ZEISS LSM 780）下观察细胞。使用CellProfiler 4.0.7分析单细胞的荧光强度。

2.11. Biochemical assays 2.11.生化检测

For the measurement of the Lipid peroxidation (LPO), malondialdehyde (MDA), Superoxide dismutase (SOD), GPx (glutathione peroxidase), GSH & GSSG (glutathione and oxidized glutathione disulfide), Adenosine Triphosphate (ATP), triglyceride (TG), total cholesterol (T-CHO), and free cholesterol (F–CHO) levels in TM3 cells, cell homogenates were prepared and the assays were preformed according to the manufacturer's instructions of the respective kits, listed as follows: LPO assay kit (A106-1-2; Nanjing Jiancheng Bioengineering Institute), MDA assay kit (A003-1-2; Nanjing Jiancheng Bioengineering Institute), SOD assay kit (S0101S, Beyotime, China), GSH-Px assay kit (S0058,Beyotime, China), GSH & GSSG assay kit (S0053, Beyotime, China), ATP assay kit (S0026, Beyotime, China), TG assay kit (A110-1-1; Nanjing Jiancheng Bioengineering Institute), TC assay kit (A111-1-1; Nanjing Jiancheng Bioengineering Institute), FC assay kit (BC1890, Solarbio, China) and NEFA assay kit (A042-1-1; Nanjing Jiancheng Bioengineering Institute).
用于测量脂质过氧化(LPO)、丙二醛(MDA)、超氧化物歧化酶(SOD)、GPx（谷胱甘肽过氧化物酶）、GSH 和 GSSG（谷胱甘肽和氧化型谷胱甘肽二硫化物）、三磷酸腺苷 (ATP)、甘油三酯(TG)、 TM3 细胞、细胞中的总胆固醇 (T-CHO) 和游离胆固醇 (F–CHO) 水平根据各试剂盒制造商的说明制备匀浆并进行测定，如下所示：LPO测定试剂盒（A106-1-2；南京建成生物工程研究所）、MDA测定试剂盒（A003-1-2；南京建成生物工程研究所）生物工程研究所），SOD测定试剂盒（S0101S，碧悠天，中国），GSH-Px测定试剂盒（S0058，碧悠天，中国）， GSH & GSSG 检测试剂盒（S0053，Beyotime，中国）、ATP 检测试剂盒（S0026，Beyotime，中国）、TG 检测试剂盒（A110-1-1；南京建成生物工程研究所）、TC 检测试剂盒（A111-1-1；南京建成生物工程研究所），FC检测试剂盒（BC1890，Solarbio，中国）和NEFA检测试剂盒（A042-1-1；南京建成生物工程研究所）。

2.12. Measurement of mitochondrial membrane potential (MMP)
2.12.线粒体膜电位（MMP）的测量

MMP was measured using the JC-1 staining assay kit (C2006, Beyotime, China). TM3 cells were treated with 2.5 μg/ml Cd for 12 h and stained with 1 × JC-1 for 20 min at 37 °C in the dark, washed twice with JC-1 staining buffer and observed by confocal microscopy (ZEISS LSM 780). The fluorescence intensity was analyzed using ZEN 3.2 (blue edition).
使用JC-1染色试剂盒（C2006，Beyotime，中国）测量MMP。 TM3细胞用2.5 μg/ml Cd处理12小时，并在37°C避光下用1×JC-1染色20分钟，用JC-1染色缓冲液洗涤两次，并通过共聚焦显微镜（ZEISS LSM 780）观察。使用ZEN 3.2（蓝色版）分析荧光强度。

2.13. Quantitative real-time polymerase chain reaction (qRT-PCR)
2.13.实时定量聚合酶链式反应 (qRT-PCR)

The RNA-easy Isolation Reagent (R701-01, Vazyme, China) was used for total RNA extraction. The quantity and quality of the RNA was assessed using a NanoDrop ND-2000 spectrophotometer (Thermo Scientific) at A260/A280 nm. The HiScript III RT SuperMix for qPCR (+gDNA wiper) (R323-01, Vazyme, China) was used for the reverse transcription. RT-qPCR was performed using a commercial SYBR Green RT-qPCR master mix (Q111-C1, Vazyme, China) in accordance with the manufacturer's instructions. The temperature program consisted of an initial single cycle of 5 min at 95 °C, followed by 40 cycles of 10 s at 95 °C, 30 s at 60 °C and 30 s at 72 °C. PCR reactions were implemented using a StepOnePlus™ realtime PCR system (Version 2.2.2, Applied Biosystems, USA). The cycle threshold (Ct) values were recorded, and relative expression of the target genes was calculated using the 2^–ΔΔCT method. GAPDH was used as the internal control to normalize the data. PCR primers were synthesized by Sangon Biotech (China). The primer sequences are listed in Supplementary Table 1. All experiments were repeated three times.
总RNA提取采用RNA-easy Isolation Reagent（R701-01，Vazyme，中国）。使用 NanoDrop ND-2000 分光光度计 (Thermo Scientific) 在 A260/A280 nm 下评估 RNA 的数量和质量。 HiScript III RT SuperMix for qPCR（+gDNA擦拭器）（R323-01，Vazyme，中国）用于逆转录。 RT-qPCR 使用商业 SYBR Green RT-qPCR 主混合物（Q111-C1，Vazyme，中国）按照制造商的说明进行。温度程序包括 95 °C 下 5 分钟的初始单循环，然后是 95 °C 下 10 秒、60 °C 下 30 秒和 72 °C 下 30 秒的 40 个循环。 PCR 反应使用 StepOnePlus™ 实时 PCR 系统（2.2.2 版，Applied Biosystems，美国）进行。记录循环阈值（Ct），并使用2 ^-ΔΔCT方法计算目标基因的相对表达量。 GAPDH用作内部对照以标准化数据。 PCR引物由生工生物工程公司（中国）合成。引物序列列于补充表1中。所有实验重复三次。

2.14. Western blot analysis
2.14.蛋白质印迹分析

Total proteins from testicular tissues were extracted immediately after the mice were sacrificed. The cultured TM3 cells were digested with trypsin and proteins were extracted using Cell lysis buffer for Western and IP (P0013, Beyotime, China). The protein concentration in the extracts was determined using a BCA protein assay kit (BL521A, Biosharp, China). Proteins were denatured, separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to a polyvinylidene fluoride (PVDF) membrane, which was then blocked in 5% non-fat milk at room temperature for 2 h. The membranes were then washed three times using Tris buffered saline tween (TBST) for 10 min each. Subsequently, the membranes were incubated with primary antibodies according to the manufacturer's instructions, including SLC7A11 (26864-1-AP, 1:1000, Proteintech, China), GPX4 (14432-1-AP, 1:1000, Proteintech, China), FSP1 (16105-1-AP, 1:1000, Proteintech, China), NRF2 (A1244, 1:1000, ABclonal, China), HO-1 (27282-1-AP, 1:1000, Proteintech, China), FTH1 (A19544, 1:1000, ABclonal, China), LDLR (ab52818, 1:1000, Abcam, USA), SR-BI (ab217318, 1:2000, Abcam, USA), ATG7 (8558, 1:2000, Cell Signaling Technology, USA), ATG5 (12994, 1:2000, Cell Signaling Technology, USA), p62 (18420-1-AP, 1:1000, Proteintech, China), LC3A/B (12741, 1:2000, Cell Signaling Technology, USA), P450 scc (13363-1-AP, 1:1000, Proteintech, China), Cytochrome C (ab133504, 1:4000, Abcam, USA), TfR1 (ab269513, 1:1000, Abcam, USA), IRP1/2 (ab181153, 1:1000, Abcam, USA), FTL (ab109373, 1:4000, Abcam, USA), TFR 1 (ab269513, 1:5000, Abcam, USA), SLC40A1 (26601-1-AP, 1:800, Proteintech, China), GAPDH (60004-1-Ig, 1:1000, Proteintech, China), and β-Actin (66009-1-Ig, 1:1000, Proteintech, China). Next, the membranes were rinsed and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies. The specific immunoreactive protein bands were developed using ECL reagent (G2014-50 ML, Servicebio, China).
处死小鼠后立即从睾丸组织中提取总蛋白。培养的TM3细胞用胰蛋白酶消化，并使用用于Western和IP的细胞裂解缓冲液（P0013，Beyotime，中国）提取蛋白质。使用BCA蛋白测定试剂盒（BL521A，Biosharp，中国）测定提取物中的蛋白浓度。蛋白变性，十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分离，转移至聚偏氟乙烯（PVDF）膜上，5%脱脂牛奶室温封闭2 h。然后使用 Tris 缓冲盐水吐温 (TBST) 将膜洗涤 3 次，每次 10 分钟。随后，根据制造商的说明将膜与一抗一起孵育，包括SLC7A11（26864-1-AP，1：1000，Proteintech，中国），GPX4（14432-1-AP，1：1000，Proteintech，中国）， FSP1 (16105-1-AP, 1:1000, Proteintech, 中国), NRF2 (A1244, 1:1000, ABclonal, 中国), HO-1 (27282-1-AP, 1:1000, Proteintech, 中国), FTH1 (A19544, 1:1000, ABclonal, 中国), LDLR (ab52818, 1:1000, Abcam ，美国），SR-BI（ab217318，1：2000，Abcam， USA)、ATG7 (8558, 1:2000, Cell Signaling Technology, USA)、ATG5 (12994, 1:2000, Cell Signaling Technology, USA)、p62 (18420-1-AP, 1:1000, Proteintech, China), LC3A/B（12741，1:2000，Cell Signaling Technology，美国），P450 scc (13363-1-AP, 1:1000, Proteintech, China), 细胞色素 C (ab133504, 1:4000, Abcam, USA), TfR1 (ab269513, 1:1000, Abcam, USA), IRP1/2 (ab181153, 1 ：1000，Abcam，美国），FTL（ab109373， 1:4000, Abcam, 美国), TFR 1 (ab269513, 1:5000, Abcam, 美国), SLC40A1 (26601-1-AP, 1:800, Proteintech, 中国), GAPDH (60004-1-Ig, 1: 1000，Proteintech，中国）和β-肌动蛋白（66009-1-Ig， 1:1000，Proteintech，中国）。接下来，冲洗膜并用辣根过氧化物酶 (HRP) 偶联的二抗孵育。使用ECL试剂（G2014-50 ML，Servicebio，中国）显色特异性免疫反应蛋白条带。

2.15. Immunofluorescence staining and observation by confocal microscope
2.15。免疫荧光染色及共聚焦显微镜观察

The testes were paraffin-embedded and cut into 4-μm sections using a microtome (Leica, Germany). Each section was de-waxed, followed by antigen retrieval by boiling in 10 mM sodium-citrate buffer pH 6.0 for 10 min. After blocking with 5% goat serum (GS), the primary antibody was added to the sections and incubated at 4 °C overnight, followed by washing with 5% GS 3 times for 10 min each, and incubation with the secondary antibody (SA00013-2, 1:200 or SA00013-3, 1:200, both from Proteintech, China). The nuclei were counter-stained with DAPI in the dark. For cellular immunofluorescence, the cells were cultured on a cover glass for 24 h, fixed with 4% paraformaldehyde (PFA) for 30 min, and stained as detailed above. For BODIPY493/503 staining (D3922, Invitrogen, USA), the cover glasses were fixed in 4% PFA, rinsed three times with PBS, and incubated with 1 μg/ml BODIPY–PBS solution at room temperature for 10 min, followed by immunofluorescence staining as detailed above. The images were acquired using a confocal microscope (ZEISS LSM 780). The fluorescence intensity was analyzed using ZEN 3.2 (blue edition).
将睾丸石蜡包埋并使用切片机（Leica，德国）切成 4 μm 的切片。将每个切片脱蜡，然后在 10 mM 柠檬酸钠缓冲液 pH 6.0 中煮沸 10 分钟进行抗原修复。用5%山羊血清（GS）封闭后，在切片中加入一抗，4℃孵育过夜，然后用5% GS洗涤3次，每次10分钟，与二抗（SA00013- 2, 1:200 或 SA00013-3, 1:200，均来自 Proteintech，中国）。在黑暗中用DAPI对细胞核进行复染色。对于细胞免疫荧光，将细胞在盖玻片上培养 24 小时，用 4% 多聚甲醛 (PFA) 固定 30 分钟，并按照上文详述进行染色。对于 BODIPY493/503 染色（D3922，Invitrogen，美国），将盖玻片固定在 4% PFA 中，用 PBS 冲洗 3 次，并与 1 μg/ml BODIPY-PBS 溶液在室温下孵育 10 分钟，然后进行免疫荧光染色如上所述。使用共焦显微镜 (ZEISS LSM 780) 获取图像。使用ZEN 3.2（蓝色版）分析荧光强度。

2.16. mCherry-GFP-LC3B transfection and confocal microscopy
2.16。 mCherry-GFP-LC3B 转染和共聚焦显微镜

mCherry-GFP-LC3B adenoviral particles were purchased from Hanbio (AP20101508, China). Cells were infected with adenoviral particles at 20 multiplicity of infection (MOI) for 24 h. Following Cd treatment, autophagy was observed using the confocal microscopy (ZEISS LSM 780). Autophagy flux was determined by calculating the number of yellow and red puncta.
mCherry-GFP-LC3B 腺病毒颗粒购自汉恒生物（AP20101508，中国）。用腺病毒颗粒以20感染复数(MOI)感染细胞24小时。 Cd 处理后，使用共聚焦显微镜 (ZEISS LSM 780) 观察自噬。通过计算黄色和红色斑点的数量来确定自噬通量。

2.17. Lyso-Tracker Red and confocal microscopy
2.17。 Lyso-Tracker 红色和共聚焦显微镜

TM3 cells were cultured into confocal dishes and then exposed to Cd at the concentrations of 2.5 μg/ml for 12 h. After that, cells were treated with Lyso-Tracker Red (50 nM, C1046, Beyotime, China) and Hoechst 33342 at 37 °C for 30 min. After incubation, cells were washed with warm serum-free DMEM/F12 and immediately observed under confocal microscopy (ZEISS LSM 780). The fluorescence intensity was analyzed by Image - Pro Plus 6.0 software.
将TM3细胞培养到共聚焦培养皿中，然后暴露于浓度为2.5μg/ml的Cd中12小时。之后，用Lyso-Tracker Red（50 nM，C1046，Beyotime，中国）和Hoechst 33342在37℃下处理细胞30分钟。孵育后，用温热的无血清 DMEM/F12 洗涤细胞，并立即在共聚焦显微镜 (ZEISS LSM 780) 下观察。通过Image-Pro Plus 6.0软件分析荧光强度。

2.18. Statistical analysis
2.18。统计分析

GraphPad Prism 8 (GraphPad Inc., USA) was used for the visualization of graphs and data analysis. All data are presented as the means ± standard deviations (SD) from at least 4 biological replicates. Differences between groups were analyzed using Student's t-test or single-factor analysis of variance (one-way ANOVA). Differences with P-values of less than 0.05 were considered statistically significant.
GraphPad Prism 8（GraphPad Inc.，美国）用于图形可视化和数据分析。所有数据均以至少 4 次生物重复的平均值±标准差 (SD) 形式呈现。使用学生t检验或单因素方差分析（单向方差分析）分析组间差异。 P 值小于 0.05 的差异被认为具有统计显着性。

3. Results 3. 结果

3.1. Cd impaired testosterone production and induced ferroptosis in mice
3.1.镉损害小鼠睾酮生成并诱导铁死亡

Adult male C57BL/6 J mice were treated with Cd at a dose of 1 mg/kg for 1 week. After Cd exposure, the Cd content in testis was remarkably increased (p < 0.01; Fig. 1A), whereas the serum testosterone level was significantly reduced (p < 0.05; Fig. 1B). Meanwhile, Cd induced the elevation of serum LH level (p < 0.05; Fig. 1B) with no significant difference in serum FSH level (Fig. 1B). Testicular morphology was damaged by Cd. As shown in Fig. 1C, the structure of the seminiferous tubules was disrupted with loose arrangement, even severe vacuolization; the cytoplasm of Leydig cells was stained lighter and exhibited edema in Cd-treated group. The ultrastructure of the testes was further observed by transmission electron microscopy (Fig. 1D). In Leydig cells, the mitochondria were shrunken with an increase of mitochondrial membrane density and decrease of mitochondrial cristae. These data indicated that Cd impaired the morphology of Leydig cells and testosterone production in mice testes.
成年雄性 C57BL/6 J 小鼠以 1 mg/kg 的剂量接受 Cd 治疗 1 周。镉暴露后，睾丸中的镉含量显着增加（p％3C 0.01；图1A ），而血清睾酮水平显着降低（p％3C 0.05；图1B ）。同时，Cd 诱导血清 LH 水平升高（p < 0.05；图 1 B），而血清 FSH 水平没有显着差异（图 1 B）。 Cd 损害睾丸形态。如图1C所示，生精小管结构被破坏，排列疏松，甚至严重空泡化； Cd处理组Leydig细胞胞浆染色较浅，水肿。通过透射电子显微镜进一步观察睾丸的超微结构（图1D ）。在间质细胞中，线粒体收缩，线粒体膜密度增加，线粒体嵴减少。这些数据表明，镉损害了小鼠睾丸中间质细胞的形态和睾酮的产生。

To explore whether Cd induced ferroptosis in vivo, the expression of corresponding markers in testicular tissues was assessed via western blotting and/or immunofluorescence staining. Glutathione peroxidase 4 (GPX4) possesses a unique ability to detoxify hydroperoxides and protect biological membranes from peroxidation damage. GPX4 is a widely-reported biomarker for identifying the occurrence of ferroptosis, since its direct or indirect inhibition can induce ferroptosis [32]. Solute carrier family 7 member 11 (SLC7A11), a component of system Xc⁻, imports extracellular cystine and exports intracellular glutamate [50]. Cystine is indispensable for glutathione (GSH) biosynthesis. Ferroptosis suppressor protein 1 (FSP1) is a glutathione-independent ferroptosis suppressor. Depletion of GSH indirectly deactivates Gpx4, resulting in the accumulation of lipid hydroperoxides [32]. At the same time, the protein levels of ferritin heavy chain (FTH1) were determined to assess intercellular iron storage.
为了探讨镉是否在体内诱导铁死亡，通过蛋白质印迹和/或免疫荧光染色评估睾丸组织中相应标记物的表达。谷胱甘肽过氧化物酶 4 (GPX4) 具有独特的解毒氢过氧化物和保护生物膜免受过氧化损伤的能力。 GPX4是一种广泛报道的用于识别铁死亡发生的生物标志物，因为它的直接或间接抑制可以诱导铁死亡[ 32 ]。溶质载体家族 7 成员 11 (SLC7A11) 是系统 Xc ⁻的一个组成部分，输入细胞外胱氨酸并输出细胞内谷氨酸[ 50 ]。胱氨酸是谷胱甘肽（GSH）生物合成所不可缺少的。铁死亡抑制蛋白 1 (FSP1) 是一种不依赖于谷胱甘肽的铁死亡抑制剂。 GSH 的消耗间接使 Gpx4 失活，导致脂质氢过氧化物的积累 [ 32 ]。同时，测定铁蛋白重链（FTH1）的蛋白质水平以评估细胞间铁储存。

Compared with the control group, the protein expression of GPX4 was obviously down-regulated in Cd-treated group (Fig. 1E). Conversely, the protein expression of both SLC7A11 and FSP1 were significantly upregulated in the Cd-exposed group, which may be indicative of a protective mechanism against ferroptosis in response to Cd. To further validate these results, we used immunofluorescence staining to determine the average protein expression in the Leydig cells of testes. As shown in Fig. 1F, the average fluorescence intensity of GPX4 was remarkably decreased, while Heme oxygenase 1 (HMOX-1, HO-1), SLC7A11 and FTH1 were noticeably increased in Cd-treated group. Taken together, the protein expression of ferroptosis markers through western blot and immunofluorescence staining supported that Cd induced ferroptosis in the Leydig cells of testes.
与对照组相比，Cd处理组GPX4蛋白表达明显下调（图1E ）。相反，在 Cd 暴露组中，SLC7A11 和 FSP1 的蛋白表达均显着上调，这可能表明针对 Cd 响应的铁死亡的保护机制。为了进一步验证这些结果，我们使用免疫荧光染色来确定睾丸 Leydig 细胞中的平均蛋白表达。如图1F所示，Cd处理组中GPX4的平均荧光强度显着降低，而血红素加氧酶1（HMOX-1，HO-1）、SLC7A11和FTH1显着增加。综上所述，通过蛋白质印迹和免疫荧光染色得到的铁死亡标志物的蛋白表达支持镉诱导睾丸间质细胞铁死亡。

Combining with the results of testosterone reduction and ultrastructure disruption in Leydig cells, we therefore speculated that Cd-induced testosterone reduction in mice might be associated with ferroptosis in Leydig cells.
结合间质细胞睾酮减少和超微结构破坏的结果，我们推测镉诱导的小鼠睾酮减少可能与间质细胞铁死亡有关。

3.2. Cd induced ferroptosis in cultured TM3 cells
3.2. Cd 诱导培养的 TM3 细胞铁死亡

To determine the effect of Cd on testosterone synthesis in mouse testis Leydig cells, a Cd-treated TM3 cell model was established in vitro. The appropriate concentration of Cd was determined using a CCK-8 assay (to evaluate cell viability). As shown in Fig. 2A, 2.5 μg/ml of Cd was selected as the optimal treatment concentration for further experiments. Subsequently, we measured testosterone levels in the TM3 cells suspension after incubation with 2.5 μg/ml Cd for 2, 6, and 24 h. Consistent with the animal model, testosterone levels in the supernatant of TM3 cells were significantly reduced after Cd-exposure for 2 h (p < 0.0001) and continued to decline with prolonged exposure time (Fig. 2B).
为了确定Cd对小鼠睾丸间质细胞睾酮合成的影响，体外建立了Cd处理的TM3细胞模型。使用 CCK-8 测定法确定 Cd 的适当浓度（以评估细胞活力）。如图2A所示，选择2.5μg/ml的Cd作为进一步实验的最佳处理浓度。随后，我们测量了 TM3 细胞悬浮液与 2.5 μg/ml Cd 孵育 2、6 和 24 小时后的睾酮水平。与动物模型一致，镉暴露2小时后，TM3细胞上清液中的睾酮水平显着降低（p％3C 0.0001），并且随着暴露时间的延长而继续下降（图2B ）。

To further explore whether testosterone reduction was related to ferroptosis, we assessed the levels of ROS in Cd-treated TM3 cells using the fluorescent probe DCFH-DA. Compared with the control group, ROS were significantly increased in the Cd-treated group (P < 0.0001; Fig. 2C). Since ferroptosis is mainly characterized by intracellular iron overload and lipid peroxidation, we also measured the content of intracellular ferrous iron using the FerroOrange probe and the levels of lipid peroxidation using LPO and MDA assay kits. Similar to typical ferroptosis inducers, Cd triggered an excessive increase of iron content in TM3 cells (Fig. 2E). Consistently, there was also a significant increase in lipid peroxidation as measured via MDA and LPO (Fig. 2D). The levels of the antioxidant enzymes SOD, and GSH-Px in TM3 cells were also assessed. The levels of SOD and GSH-Px were slightly increased in the Cd-exposed group, which was in compile with the leves of GSH and GSSG. This might be a compensatory mechanism for maintaining the oxidative balance in the cells (Fig. 2F).
为了进一步探讨睾酮减少是否与铁死亡相关，我们使用荧光探针DCFH-DA 评估了 Cd 处理的 TM3 细胞中 ROS 的水平。与对照组相比，Cd处理组的ROS显着增加（P< 0.0001；图2C ）。由于铁死亡的主要特征是细胞内铁超载和脂质过氧化，因此我们还使用FerroOrange探针测量了细胞内二价铁的含量，并使用LPO和MDA检测试剂盒测量了脂质过氧化水平。与典型的铁死亡诱导剂类似，Cd 引发 TM3 细胞中铁含量的过度增加（图 2 E）。一致的是，通过 MDA 和 LPO 测量，脂质过氧化也显着增加（图 2D ）。还评估了 TM3 细胞中抗氧化酶SOD和 GSH-Px的水平。镉暴露组SOD和GSH-Px水平略有升高，与GSH和GSSG水平一致。这可能是维持细胞氧化平衡的一种补偿机制（图2F ）。

We next investigated the mRNA and protein levels of ferroptosis markers. As shown in Fig. 2G and H, the mRNA levels of Nrf2, Slc7a11 and HO-1 genes and the protein levels of NRF2 and HO-1 were dramatically elevated in the Cd-exposed group. Conversely, the mRNA level of Fsp1 gene was reduced by Cd. Notably, although there was no significant difference in the mRNA level of Gpx4 gene compared to the control group, the protein level of GPX4 changed over time. When exposed to Cd for 6 h, the protein level of GPX4 increased to a certain extent, but when the exposure time was extended to 24 h, the protein level of GPX4 decreased significantly (Fig. 2I).
接下来我们研究了铁死亡标记物的 mRNA 和蛋白质水平。如图2G和H所示，在Cd暴露组中， Nrf2 、 Slc7a11和HO-1基因的mRNA水平以及NRF2和HO-1的蛋白质水平显着升高。相反，Cd 降低了Fsp1基因的 mRNA 水平。值得注意的是，虽然与对照组相比， Gpx4基因的mRNA水平没有显着差异，但GPX4的蛋白水平随着时间的推移而发生变化。当Cd暴露6 h时，GPX4的蛋白水平有一定程度的升高，但当暴露时间延长至24 h时，GPX4的蛋白水平显着下降（图2 I）。

Cellular iron homeostasis consists of iron uptake, trafficking and export [51,52]. Iron uptake in cells occurs by transferrin receptor 1 (TfR1) which binds with circulating transferrin. Followed by endosomal acidification and dissociation of iron from transferrin, ferrous iron may then be used, stored into ferritin (consisiting FTH1 and FTL) or effluxed across the cell membrane by ferroportin 1 (FPN1) [51]. Within the cells, iron homeostasis is under exquisite regulation [53]. Iron regulatory proteins (IRP1 and IRP2) could sense cellular iron concentration and post-transcriptionally regulate the expression of iron metabolism genes to maitain cellular iron homeostasis. Therefore, we further investigated the protein expression of iron metabolism markers. As shown in Fig. 2H, the protein expression of IRP1/2, FPN1, FTH1 and FTL were significantly upregulated, whereas that of TfR1 was significantly down-regulated. These data indicated that celluar free iron were in excess after Cd exposure and iron from endogenous origins might be the main cause of cellular iron overload, leading to the feeroptotic cell death.
细胞铁稳态由铁的吸收、运输和输出组成[ 51 , 52 ]。细胞中的铁摄取是通过与循环转铁蛋白结合的转铁蛋白受体 1 (TfR1) 进行的。随后内体酸化和铁从转铁蛋白解离，然后可以使用二价铁，将其储存到铁蛋白（由 FTH1 和 FTL 组成）中或通过铁转运蛋白1 (FPN1)跨细胞膜流出[ 51 ]。在细胞内，铁稳态受到精细调节[ 53 ]。铁调节蛋白（IRP1和IRP2）可以感知细胞铁浓度并在转录后调节铁代谢基因的表达以维持细胞铁稳态。因此，我们进一步研究了铁代谢标志物的蛋白表达。如图2H所示，IRP1/2、FPN1、FTH1和FTL的蛋白表达显着上调，而TfR1的蛋白表达显着下调。这些数据表明，镉暴露后细胞游离铁过量，内源性铁可能是细胞铁超载的主要原因，导致细胞死亡。

In brief, Cd exposure elicited characteristic changes of ferroptosis in TM3 cells, including cytosolic iron overload and lipid peroxidation.
简而言之，镉暴露引起了 TM3 细胞铁死亡的特征性变化，包括胞质铁超载和脂质过氧化。

3.3. Dfom and Fer-1 inhibited cellular ferroptosis and ameliorated testosterone reduction due to Cd exposure
3.3. Dfom 和 Fer-1 抑制细胞铁死亡并改善镉暴露导致的睾酮减少

To further investigate the role of ferroptosis in Cd-induced testicular cell dysfunction, the ferroptosis inhibitors including lipid peroxidation inhibitors (ferrostatin-1, Fer-1) and iron chelators (desferioxamine, Dfom) were utilized. As shown in Fig. 3A, the cell viability of Cd-exposed TM3 cells was partly alleviated by Fer-1 and Dfom. This was similar to erastin, a canonical ferroptosis inducer. Compared with the untreated Cd-exposed group, pretreatment with Dfom conspicuously increased the testosterone levels in the TM3 cell suspension after Cd exposure for 2 h, 6 h, and 24 h (Fig. 3B). Additionally, the levels of ROS and Fe²⁺ were obviously reduced by pretreatment with Dfom and/or Fer-1 (Fig. 3C and D), which can be explained by the fact that the iron overload could immediately trigger ROS production via the Fenton reaction. Taken together, these data suggest that Dfom and Fer-1 inhibited Cd-induced ferroptosis and ameliorated the resulting dysfunction in TM3 cells.
为了进一步研究铁死亡在镉诱导的睾丸细胞功能障碍中的作用，使用了铁死亡抑制剂，包括脂质过氧化抑制剂（ferrostatin-1，Fer-1）和铁螯合剂（去铁胺，Dfom）。如图3A所示，Fer-1和Dfom部分降低了暴露于Cd的TM3细胞的细胞活力。这与erastin（一种典型的铁死亡诱导剂）类似。与未处理的Cd暴露组相比，Dfom预处理显着增加了Cd暴露2小时、6小时和24小时后TM3细胞悬液中的睾酮水平（图3B ）。此外，通过Dfom和/或Fer-1预处理，ROS和Fe ²⁺的水平明显降低（图3 C和D ），这可以通过以下事实来解释：铁过载可以通过以下方式立即触发ROS产生：芬顿反应。总而言之，这些数据表明 Dfom 和 Fer-1 抑制 Cd 诱导的铁死亡并改善 TM3 细胞中由此产生的功能障碍。

3.4. Cd suppressed testosterone production through hmox-1-mediated ferroptosis and mitochondrial damage
3.4. Cd 通过 hmox-1 介导的铁死亡和线粒体损伤抑制睾酮产生

HO-1, as an inducible enzyme, catalyzes the degradation of heme to carbon monoxide (CO), biliverdin, and ferrous iron. The expression of HO-1 has been shown to be enhanced in ferroptosis, but the role it plays in ferroptosis has not yet been illuminated. Since the inducible HO-1 rapidly responds to a variety of stimuli, such as oxidative stress, hypoxia, and inflammation [54], it functions as an antioxidant and cytoprotective molecule [55,56]. However, overexpressed or sustained HO-1 production has been shown to have pro-oxidant effects and enhance ferroptosis via increasing the labile Fe pool (LIP) [[57], [58], [59]]. In this study, we found that Cd induced HO-1 expression in TM3 cells, and excessive activation of HO-1 accelerated the Cd-triggered ferroptotic cell death.
HO-1 作为一种诱导酶，催化血红素降解为一氧化碳(CO)、胆绿素和二价铁。 HO-1 的表达已被证明在铁死亡中增强，但其在铁死亡中所起的作用尚未阐明。由于诱导型 HO-1 能快速响应多种刺激，如氧化应激、缺氧和炎症 [ 54 ]，因此它可作为抗氧化剂和细胞保护分子 [ 55 , 56 ]。然而，过表达或持续产生 HO-1 已被证明具有促氧化作用，并通过增加不稳定铁池 (LIP) 来增强铁死亡 [ [57] 、 [58] 、 [59] ]。在本研究中，我们发现Cd诱导TM3细胞中HO-1的表达，HO-1的过度激活加速了Cd触发的铁死亡细胞死亡。

Excessive activation of HO-1 in TM3 cells induced by Cd led to the release of ferrous iron due to heme degradation, triggering a non-canonical ferroptosis pathway by increasing LIP. As shown in Fig. 4A, the protein expression of HO-1 was obviously upregulated in the Cd-exposed group. To further validate the protein expression of HO-1, we used immunofluorescence staining to determine the relative protein expression in TM3 cells (Fig. 4B). The increased cellular free iron concentration induced the protein expression of Ferritin Heavy Chain 1 (FTH1), which was confirmed by the upregulation of Fth1 mRNA level (Fig. 2G). Notably, these changes were alleviated by the ferroptosis inhibitors Fer-1 and Dfom to some extent (Fig. 4A).
Cd 诱导的 TM3 细胞中 HO-1 过度激活，导致血红素降解而释放亚铁，通过增加 LIP 触发非典型铁死亡途径。如图4A所示，Cd暴露组HO-1蛋白表达明显上调。为了进一步验证HO-1的蛋白表达，我们使用免疫荧光染色来确定TM3细胞中的相对蛋白表达（图4B ）。细胞游离铁浓度的增加诱导了铁蛋白重链1（FTH1）的蛋白表达，这通过Fth1 mRNA水平的上调得到证实（图2G ）。值得注意的是，铁死亡抑制剂 Fer-1 和 Dfom 在一定程度上减轻了这些变化（图 4 A）。

Heme is an iron-containing porphyrin molecule that serves as a prosthetic group in proteins that function in diverse metabolic pathways [60]. Overexpression of HO-1 induced by Cd not only triggered ferroptosis but also led to the degradation of important heme-containing proteins, such as cytochrome P450 family 11 subfamily A member 1 (P450scc) and components of the mitochondrial respiratory chain complex. P450scc is a rate-limiting enzyme in the synthesis of testosterone. Within the mammalian cells, the electron transport chain (ETC) of mitochondria is an important source of ROS, potentially contributing to ferroptosis [32]. Furthermore, most of the mitochondrial respiratory chain complexes, such as cytochrome c (Cyt C), contain different forms of heme [60]. To confirm the effect of overactive HO-1 induced by Cd on P450scc and Cyt C, we examined the protein expression of P450scc and Cyt C. As shown in Fig. 4C, the protein levels of P450scc and Cyt C were slightly increased after Cd treatment for 6 h, whereas this change was alleviated for 12 h. Elevated P450scc and Cyt C might be degraded as a substrate of HO-1, which in turn can promote ferroptosis by releasing iron. When we used a competitive HO-1 inhibitor, Zinc Protoporphyrin (znpp), to inhibit the activity of HO-1, the decline in Cyt C could be partially reversed (Fig. 4D). Unfortunately, znpp alone significantly reduced the protein expression of P450scc within the cells. In other words, when we used znpp as an inhibitor of HO-1 to preserve P450scc, the effectiveness was not very good (Fig. 4D).
血红素是一种含铁卟啉分子，作为蛋白质的辅基，在多种代谢途径中发挥作用[ 60 ]。 Cd 诱导的 HO-1 过度表达不仅引发铁死亡，而且导致重要的含血红素蛋白的降解，例如细胞色素 P450家族 11 亚家族 A 成员 1 (P450scc) 和线粒体呼吸链复合物的成分。 P450scc是睾酮合成中的限速酶。在哺乳动物细胞内，线粒体的电子传递链（ETC）是ROS的重要来源，可能导致铁死亡[ 32 ]。此外，大多数线粒体呼吸链复合物，例如细胞色素c （Cyt C），都含有不同形式的血红素[ 60 ]。为了证实Cd诱导的过度活跃的HO-1对P450scc和Cyt C的影响，我们检测了P450scc和Cyt C的蛋白表达。如图4C所示，Cd后P450scc和Cyt C的蛋白水平略有增加。治疗6小时后，这种变化减轻12小时。升高的 P450scc 和 Cyt C 可能作为 HO-1 的底物被降解，HO-1 反过来又可以通过释放铁来促进铁死亡。当我们使用竞争性HO-1抑制剂锌原卟啉（znpp）来抑制HO-1的活性时，Cyt C的下降可以部分逆转（图4D ）。不幸的是，单独使用 znpp 会显着降低细胞内 P450scc 的蛋白表达。换句话说，当我们使用znpp作为HO-1的抑制剂来保存P450scc时，效果不是很好（图4D ）。

Studies also suggest that some exogenous stimulation could result in the overexpression and mitochondrial translocation of HO-1, ultimately leading to mitochondrial dysfunction [58,61]. To further confirm the effect of overactive HO-1 induced by Cd on the mitochondrial function, we examined the mitochondrial membrane potentials (MMP), ATP levels and mtROS levels. As shown in Fig. 4E, the intensity of red fluorescence and the red/green fluorescence ratio were significantly reduced after Cd treatment, indicating that the MMP of cells was conspicuously reduced. However, the intensity of green fluorescence was not significantly increased as expected. These phenomena were partially alleviated by the ferroptosis inhibitors Dfom and Fer-1 (Fig. 4E). Accordingly, the ATP levels were remarkably reduced after Cd treatment (Fig. 4F). What’ more, Cd obviously augmented the levels of mtROS, which was partially reversed by znpp (Fig. 4G).
研究还表明，一些外源刺激可能导致 HO-1 过度表达和线粒体易位，最终导致线粒体功能障碍 [ 58 , 61 ]。为了进一步证实Cd诱导的过度活跃的HO-1对线粒体功能的影响，我们检测了线粒体膜电位（MMP）、ATP水平和mtROS水平。如图4E所示，Cd处理后红色荧光强度和红绿荧光比值均显着降低，表明细胞的MMP显着降低。然而，绿色荧光的强度并未如预期那样显着增加。铁死亡抑制剂 Dfom 和 Fer-1 部分缓解了这些现象（图 4 E）。因此，Cd处理后ATP水平显着降低（图4F ）。更重要的是，Cd 明显增加了 mtROS 的水平，而 znpp 部分逆转了这种水平（图 4 G）。

Taken together, these data indicated that Cd induced mitochondrial dysfunction and ferroptosis through the excessive activation of HO-1, ultimately affecting testosterone synthesis in mitochondria.
综上所述，这些数据表明，Cd 通过过度激活 HO-1 诱导线粒体功能障碍和铁死亡，最终影响线粒体中睾酮的合成。

3.5. Cd suppressed testosterone production and cholesterol uptake by blocking autophagosome-lysosome fusion
3.5.镉通过阻断自噬体-溶酶体融合来抑制睾酮产生和胆固醇摄取

The decline of testosterone levels following Cd exposure might be attributed to a decrease in the activities of enzymes engaged in testosterone biosynthesis or cholesterol reduction in the testes [62]. To further explore the reasons for the decrease in testosterone levels after Cd exposure, we determined the levels of total cholesterol (T-CHO), triglycerides (TG) and free cholesterol (F–CHO) in Cd-exposed TM3 cells.
镉暴露后睾酮水平的下降可能是由于睾丸中参与睾酮生物合成的酶活性下降或胆固醇减少所致[ 62 ]。为了进一步探讨镉暴露后睾酮水平下降的原因，我们测定了镉暴露TM3细胞中总胆固醇（T-CHO）、甘油三酯（TG）和游离胆固醇（F-CHO）的水平。

As shown in Fig. 5A, T-CHO and TG were significantly decreased in TM3 cells after Cd treatment with 2.5 μg/ml for 6 and 24 h. Immunofluorescence staining showed that microtubule-associated protein 1 light chain 3 (LC3) was remarkably increased, while bodipy staining was significantly reduced after Cd treatment for 24 h (Fig. 5B). Consequently, we speculated that lipophagy was enhanced, promoting the degradation of lipid droplets. However, the levels of free cholesterol did not increase significantly (Fig. 5C), which was inconsistent with the expected results.
如图5A所示，用2.5μg/ml的Cd处理6和24小时后，TM3细胞中的T-CHO和TG显着降低。免疫荧光染色显示，Cd处理24小时后，微管相关蛋白1轻链3（LC3）显着增加，而bodipy染色显着减少（图5B ）。因此，我们推测自噬作用增强，促进了脂滴的降解。然而，游离胆固醇水平并没有显着增加（图5C ），这与预期结果不一致。

To further investigate this discrepancy, we determined the mRNA and protein levels of the scavenger receptor class B, type I (SR-BI) and the low-density lipoprotein receptor (LDLR), two major lipoprotein receptors that mediated the selective uptake of cholesterol esters from high-density lipoprotein, low-density lipoprotein, or very-low-density lipoprotein [47,63]. Interestingly, we found that the mRNA and protein levels of LDLR and SR-BI were markedly reduced following Cd treatment (Fig. 5D and E). Gao et al. [47] found that autophagy defect in Leydig cells resulted in the downregulation of SR-BI, leading to defective cholesterol uptake and a deficiency of testosterone synthesis. We therefore speculated that Cd treatment might affect the autophagy levels in TM3 cells and thus affect the intake of cholesterol.
为了进一步研究这种差异，我们测定了 B 类清道夫受体I 型 (SR-BI) 和低密度脂蛋白受体 (LDLR) 的 mRNA 和蛋白质水平，这两种主要的脂蛋白受体介导选择性摄取胆固醇酯来自高密度脂蛋白、低密度脂蛋白或极低密度脂蛋白[ 47 , 63 ]。有趣的是，我们发现Cd处理后LDLR和SR-BI的mRNA和蛋白水平显着降低（图5D和E）。高等人。 [ 47 ]发现Leydig细胞的自噬缺陷导致SR-BI下调，导致胆固醇摄取缺陷和睾酮合成缺陷。因此我们推测Cd治疗可能会影响TM3细胞的自噬水平，从而影响胆固醇的摄入。

To test the hypothesis, we measured the protein expression of autophagy-related genes in TM3 cells. Compared with the control group, the expression of autophagy related 7 (ATG7), and autophagy related 5 (ATG5) was obviously down-regulated in Cd-exposed TM3 cells, while sequestosome 1 (p62) was significantly upregulated (Fig. 5F). These results indicated that Cd exposure downregulated autophagy in TM3 cells. However, LC3 expression was significantly upregulated in Cd-exposed TM3 cells according to both western blotting and immunofluorescence (Fig. 5B and F), which may be caused by the blocking of autophagy flow. In order to further verify the situation of autophagy flow, we evaluated the extent of autophagosome and autolysosome formation by using mCherry-EGFP-LC3. Consistent with our previous study [48], Cd significantly increased red and green puncta (Fig. 5G). Crucially, the abundance of yellow dots in the merged images was also dramatically increased, indicating that Cd induced and increased autophagosome formation, accompanied by the blockage of autophagic flow. Interestingly, we used Lyso-Tracker RED to detect the content of lysosomes and found that the lysosomes were significantly reduced after Cd treatment (Fig. 5H). The reduction of lysosome might block the fusion of autophagosome and lysosome to some extent.
为了验证这一假设，我们测量了 TM3 细胞中自噬相关基因的蛋白表达。与对照组相比，Cd暴露的TM3细胞中自噬相关7（ATG7）和自噬相关5（ATG5）的表达明显下调，而sequestosome 1 （p62）的表达显着上调（图5F ）。这些结果表明镉暴露下调了 TM3 细胞的自噬。然而，根据蛋白质印迹和免疫荧光，在Cd暴露的TM3细胞中LC3表达显着上调（图5B和F），这可能是由于自噬流受阻所致。为了进一步验证自噬流的情况，我们利用mCherry-EGFP-LC3评估了自噬体和自溶酶体形成的程度。与我们之前的研究一致[ 48 ]，Cd显着增加了红色和绿色斑点（图5G ）。至关重要的是，合并图像中黄点的丰度也显着增加，表明镉诱导并增加了自噬体的形成，并伴随着自噬流的阻塞。有趣的是，我们使用Lyso-Tracker RED检测溶酶体的含量，发现Cd处理后溶酶体明显减少（图5H ）。溶酶体的减少可能在一定程度上阻碍了自噬体与溶酶体的融合。

Collectively, these results indicated that Cd exposure promoted autophagosome formation but blocked autophagosome-lysosome fusion, which attenuated the absorption of T-CHO and TG, further aggravating testosterone synthesis disruption.
总的来说，这些结果表明，镉暴露促进了自噬体的形成，但阻碍了自噬体-溶酶体的融合，从而减弱了 T-CHO 和 TG 的吸收，进一步加剧了睾酮合成的破坏。

4. Discussion 4. 讨论

The present study demonstrated that Cd treatment disturbed iron homeostasis, ultimately leading to ferroptosis and testosterone reduction. Mechanically, we found that Cd-induced ferroptotic cell death is mediated by the overactivation of HO-1, which releases excessive free iron from heme. Fortunately, ferroptosis caused by Cd as a result of iron-dependent lipid peroxidation and testosterone production can be pharmacologically rescued by the ferroptosis inhibitors Fer-1 and Dfom. Additionally, Cd exposure led to autophagy disruption by blocking autophagosome-lysosome fusion, which attenuating the absorption of TG and TC, further aggravating the testosterone synthesis disorder (see Fig. 6).
目前的研究表明，镉治疗扰乱了铁稳态，最终导致铁死亡和睾酮减少。从机械角度来看，我们发现 Cd 诱导的铁死亡细胞死亡是由 HO-1 过度激活介导的，HO-1 从血红素中释放过量的游离铁。幸运的是，由于铁依赖性脂质过氧化和睾酮产生而由 Cd 引起的铁死亡可以通过铁死亡抑制剂 Fer-1 和 Dfom 在药理学上得到挽救。此外，Cd暴露通过阻断自噬体-溶酶体融合导致自噬破坏，从而减弱TG和TC的吸收，进一步加剧睾酮合成障碍（见图6 ）。

HO-1 catalyzes the degradation of heme to CO, biliverdin, and ferrous iron, and its critical role in heme metabolism makes it an important factor with both protective and detrimental effects, depending on the context [64]. The antioxidative activity of HO-1 relies on the synthesis of biliverdin, which can scavenge or neutralize ROS. Nevertheless, the concomitantly released ferrous iron is a major pro-oxidant that boosts the fenton-mediated decomposition of peroxides [32]. The expression of HO-1 has been shown to be induced in ferroptosis [[55], [56], [57], [58], [59]], but its exact role has not yet been illuminated. Knockout of HO-1 promoted ferroptosis in proximal tubular cells and hepatocellular carcinoma cells [55,56]. However, pharmacological inhibition or silencing of HO-1 confers resistance to ferroptosis induced by withaferin A, erastin, and BAY [32,[57], [58], [59]]. In our study, we found that overactivition of HO-1 increased LIP, ROS and lipid peroxidation, which could be pharmacologically rescued by the ferroptosis inhibitors Fer-1 and DFOM. Thus, excessive upregulation of HO-1 could be a trigger for ferroptosis, while moderate upregulation could be protective against ferroptosis.
HO-1 催化血红素降解为CO2 、胆绿素和二价铁，其在血红素代谢中的关键作用使其成为具有保护作用和有害作用的重要因素，具体取决于具体情况[ 64 ]。 HO-1的抗氧化活性依赖于胆绿素的合成，胆绿素可以清除或中和ROS。然而，同时释放的二价铁是一种主要的促氧化剂，可促进芬顿介导的过氧化物分解[ 32 ]。 HO-1 的表达已被证明在铁死亡中被诱导[ [55] 、 [56] 、 [57] 、 [58] 、 [59] ]，但其确切作用尚未阐明。 HO-1 的敲除促进近端肾小管细胞和肝细胞癌细胞的铁死亡[ 55 , 56 ]。然而，HO-1 的药理抑制或沉默可抵抗由醉茄素 A 、erastin 和 BAY 诱导的铁死亡[ 32 ]、 [57] 、 [58] 、 [59] ]。在我们的研究中，我们发现 HO-1 的过度激活会增加 LIP、ROS 和脂质过氧化，这可以通过铁死亡抑制剂 Fer-1 和 DFOM 在药理学上得到挽救。因此，HO-1 的过度上调可能会引发铁死亡，而适度上调可能会预防铁死亡。

In addition, we further investigated the concrete mechanism of HO-1 in ferroptosis. Overexpression of HO-1 can interfere with P450scc and Cyt C-mediated ETC, triggering iron overload and further inducing a surge of mtROS and mitochondrial dysfunction. All of these colud be partially pharmacologically rescued by the competitive HO-1 inhibitor, znpp. These findings provide a valuable reference for further verification of the molecular regulatory network of ferroptosis. However, previous studies suggested that znpp were associated with a variety of effects apart from inhibition of HO-1 and such nonspecific effects of znpp are more pronounced at higher concentrations (≥0.5 mM) [65]. Although we tried different inhibitor concentrations, even at a concentration as low as 0.625 μM, we could not avoid the influence of znpp itself on P450scc. As a result, znpp could not protect the protein content of P450scc within the cells after Cd treatment. In order to better verify the mechanism of HO-1 in ferroptosis, we will use genetic inhibition of HO-1 in the future studies to avoid nonselective effects of znpp.
此外，我们还进一步研究了HO-1在铁死亡中的具体机制。 HO-1 的过度表达会干扰 P450scc 和 Cyt C 介导的 ETC，引发铁过载，并进一步诱导 mtROS 激增和线粒体功能障碍。所有这些都可以通过竞争性 HO-1 抑制剂 znpp 在药理学上得到部分挽救。这些发现为进一步验证铁死亡的分子调控网络提供了有价值的参考。然而，先前的研究表明，除了抑制 HO-1 之外，znpp 还与多种作用相关，并且 znpp 的这种非特异性作用在较高浓度（≥0.5 mM）下更为明显[ 65 ]。尽管我们尝试了不同的抑制剂浓度，即使是低至0.625 μM的浓度，我们也无法避免znpp本身对P450scc的影响。结果，在Cd处理后，znpp不能保护细胞内P450scc的蛋白质含量。为了更好地验证HO-1在铁死亡中的作用机制，我们将在未来的研究中利用HO-1的基因抑制来避免znpp的非选择性效应。

Previous studies [42,43] indicated that ferroptosis might be a type of autophagy-dependent programmed cell death. However, ROS and lipid peroxidation due to Cd exposure not only triggered ferroptosis and activated the formation of autophagic vesicles, but also blocked autophagic flow. It was reported that autophagy could promote ferroptosis via NCOA4-facilitated ferritinophagy, RAB7A-dependent lipophagy, BECN1-mediated system xc⁻ inhibition, STAT3-induced lysosomal membrane permeabilization, and HSP90-associated chaperone-mediated autophagy [42,43]. The blockage of autophagic flow can restrain ferroptosis to some extent, but whether this is beneficial or harmful to cell survival is not clear. Moreover, a previous study [66] has shown that Cd could affect the lysosomal membrane permeabilization through divalent ion channels, which in turn affected lysosomal function. Similarly, whether the Cd-induced blockage of autophagic flow confirmed in this study was due to impaired lysosomal function remains unclear, and this question certainly merits further research.
先前的研究[ 42 , 43 ]表明铁死亡可能是一种自噬依赖性程序性细胞死亡。然而，Cd暴露引起的ROS和脂质过氧化不仅引发铁死亡并激活自噬囊泡的形成，而且还阻断自噬流。据报道，自噬可以通过NCOA4促进的铁蛋白自噬、RAB7A依赖性脂肪自噬、BECN1介导的系统xc-抑制、STAT3诱导的溶酶体膜透化和HSP90相关分子伴侣介导的自噬促进^铁死亡[ 42 , 43 ]。自噬流的阻断可以在一定程度上抑制铁死亡，但这对于细胞生存是有利还是有害尚不清楚。此外，先前的研究[ 66 ]表明，Cd可以通过二价离子通道影响溶酶体膜的通透性，进而影响溶酶体功能。同样，本研究证实的镉诱导的自噬流阻断是否是由于溶酶体功能受损仍不清楚，这个问题当然值得进一步研究。

What is more, although a growing number of epidemiological studies indicate that environmental exposure to Cd is linked with changes in serum testosterone level [[20], [21], [22]], no consensus has been reached. In addition, the current datas are only limited to epidemiology, and the mechanism of Cd exposure on serum testosterone level needs to be further explored. In our study, we used a mouse model to further explore the effects of cadmium exposure on serum testosterone and its possible mechanisms. As a result, we found that cadmium reduced testosterone production by inducing ferroptosis and autophagy disruption.
此外，尽管越来越多的流行病学研究表明环境中镉暴露与血清睾酮水平的变化有关[ [20] 、 [21] 、 [22] ]，但尚未达成共识。此外，目前的数据仅限于流行病学，Cd暴露对血清睾酮水平的影响机制有待进一步探讨。在我们的研究中，我们使用小鼠模型来进一步探讨镉暴露对血清睾酮的影响及其可能的机制。结果，我们发现镉通过诱导铁死亡和自噬破坏来减少睾酮的产生。

Overall, the most striking finding of this study is that overactivation of HO-1 after Cd exposure not only triggered ferroptosis, but also blocked autophagosome-lysosome fusion, affecting testosterone synthesis in general. Although Cd-induced male reproductive injury involves multiple mechanisms, including structural damage to testis vasculature and blood-testis barrier, inflammation, oxidative stress, apoptosis [67] and autophagic cell death [48], this is the first evidence of a role of ferroptosis in Cd-induced male reproductive injury, especially testosterone synthesis dysfunction. Our findings provide a new target for clinical or public health prevention and treatment of reproductive toxicity due to Cd exposure.
总的来说，这项研究最引人注目的发现是，Cd 暴露后 HO-1 的过度激活不仅引发铁死亡，而且还阻断自噬体-溶酶体融合，从而总体上影响睾酮合成。尽管镉引起的男性生殖损伤涉及多种机制，包括睾丸脉管系统和血睾屏障的结构损伤、炎症、氧化应激、细胞凋亡[ 67 ]和自噬性细胞死亡[ 48 ]，但这是铁死亡作用的第一个证据。镉引起的男性生殖损伤，特别是睾酮合成功能障碍。我们的研究结果为临床或公共卫生预防和治疗镉暴露引起的生殖毒性提供了新的目标。

Consent for publication 同意发表

All authors consent for publication.
所有作者均同意发表。

Data availability 数据可用性

All data in this manuscript are available from the authors.
本手稿中的所有数据均可从作者处获得。

Funding 资金

This work is supported by the National Key R&D Program of China (2018YFC1004502).

Authors' contributions

Designed the study and edited the final text: Ling Zeng, Ping Su. Performed the experiments: Ling Zeng, Jinzhao Zhou, Xiaofei Wang, Yanwei Zhang. Wrote the manuscript and prepared the figures: Ling Zeng, Mei Wang. Contributed to manuscript revision and critical discussion: Ling Zeng, Mei Wang, Ping Su.

Declaration of competing interest

The authors report no conflict of interest.

Acknowledgments

The authors thank the editors and reviewers for their significant contributions during the revision period.

Appendix A. Supplementary data

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Multimedia component 1.

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Cited by (59)

Molybdenum and cadmium cause blood–testis barrier dysfunction through ROS-mediated NLRP3 inflammasome activation in sheep

2024, Science of the Total Environment
In this study, 24 healthy male sheep were divided into four groups: the control group, Mo group (45 mg Mo·kg-1·BW), Cd group (1 mg Cd·kg-1·BW), and Mo + Cd group (45 mg Mo·kg-1·BW + 1 mg Cd·kg-1·BW). The experiment was last for 50 d. The results showed that Mo and Cd co-exposure induced histopathological changes and ultrastructural damage, decreased the mRNA and protein expression levels of BTB (blood-testis barrier)-related factors (CX-43, ZO-1, OCLN) (P < 0.05) and the T-SOD and CAT activity (P < 0.05), increased the MDA content (P < 0.05) and the proinflammatory factors levels (P < 0.05) in sheep testes. Moreover, the results showed that a sharp decline in BTB-related factors and antioxidase activity, and a significant increase in reactive oxygen species (ROS) levels (P < 0.05) and the expression levels of NLRP3 inflammasome-related factors (P < 0.05) in primary Sertoli cells (SCs) under Mo and Cd co-exposure. However, treatment with a ROS scavenger or NLRP3 inflammasome inhibitors could relieve BTB damage and oxidative injury, reduce the production of ROS (P < 0.05) and decrease the level of inflammatory factors (P < 0.05). Overall, these results indicated that Mo and Cd co-exposure reduced BTB-related protein levels and promoted ROS production and inflammatory reactions by activating the ROS/NLRP3 inflammasome pathway in sheep testes, which eventually induced reproductive toxicity.
High Fischer ratio oligopeptides from hard-shelled mussel: Preparation and hepatoprotective effect against acetaminophen-induced liver injury in mice

2023, Food Bioscience
High Fischer ratio oligopeptides (HFOPs) have a variety of physiological activities, but there are few literatures reporting the hepatoprotective effects of HFOPs against acetaminophen (APAP)-induced liver injury. The objectives of this study were to establish the preparation process of HFOPs from hard-shelled mussel (Mytilus coruscus) (HFOPs-HM) and investigate the hepatoprotective effects and potential mechanisms of HFOPs-HM against APAP-induced liver injury in mice. Therefore, trypsin and papain were selected as the endo and exogenous proteases for preparing HFOPs-HM according to the degree of hydrolysis (DH) of hard-shelled mussel hydrolysates, and activated carbon of XHJ-200 displayed the best effect to remove aromatic amino acids according to the Fischer ratio (OD220/OD280) and its parameters were optimized as adsorption time of 3.0 h and solid-liquid ratio of 1:10. The HFOPs-HM's Fischer ratio and average molecular weight were 26.03 (>20) and 878.54 Da, respectively. Furthermore, a mice model of liver injury induced by APAP was established. HFOPs-HM could significantly improve liver index and histopathological features, decrease the serum alanine aminotransferase (ALT) and aspartate transaminase (AST) activities, increase the glutathione content and superoxide dismutase (SOD) and catalase (CAT) activities. Further research demonstrated that HFOPs-HM could alleviate liver oxidative stress in liver tissue via activating the Nrf2 pathway to initiate the intracellular antioxidant enzyme system. In addition, HFOPs-HM also suppressed liver inflammation by downregulating the secretion of TNF-α, IL-1β, and IL-6. Thus, HFOPs-HM could serve as an auxiliary functional dietary molecule applied in function products to alleviate drug-induced liver injury.
Melatonin alleviates lead-induced fatty liver in the common carps (Cyprinus carpio) via gut-liver axis

2023, Environmental Pollution
As a widespread aquatic environmental contaminant, Lead (Pb) can provoke hepatic injury in various animals. Melatonin (MT) plays a crucial role in the regulation of inflammatory response. Accumulating evidence elucidates exogenous toxins can elicit hepatic lipid metabolic disorders by influencing the gut microbiome. Nevertheless, the effects of Pb on gut microbiota and hepatic lipid metabolism of the common carps, and whether MT can prevent and cure Pb-induced toxicity via regulating microbiome remains unknown. Here, metagenomic and transcriptomic analysis were subsequently implemented to identify the Pb exposure-triggered prominent alternation of gut-liver signal. In the present study the severe intestinal injury and fatty liver formation caused by Pb in common carp were preliminarily determined. Metagenomic analysis confirmed that the gut microbiome dominant phyla, family and genus of the common carps were Fusobacteria, Fusobacteriaceae and Cetobacterium. Meanwhile, lipopolysaccharide (LPS) biosynthesis pathway was regarded as one of the main responsible for Pb exposure. Subsequently, LPS was demonstrated as the Pb-triggered microbial-derived signal of the common carps by ELISA analysis, and involves in the hepatic metabolic disorders via deteriorating the intestinal barrier. Additionally, it confirmed that hepatocytes ferroptosis associated with Pb-evoked fatty liver of the common carps, and the aggravation of lysosomal dyshomeostasis as well as inhibition of AMPK phosphorylation were referred to lipid metabolic disorders. The results of the present study demonstrated microbial-derived signal induced by aquatic Pb contaminant cause fatty liver formation in the common carps, and the protective effects of MT on Pb toxicity were performed by receding LPS over-synthesis, restraining microbiota-sourced LPS transport, along with attenuation of hepatocytes ferroptosis.
Cadmium exposure induces pyroptosis in testicular tissue by increasing oxidative stress and activating the AIM2 inflammasome pathway

2022, Science of the Total Environment
High doses of cadmium (Cd) cause irreversible injury to the reproductive system, especially testicular tissue. Studies have shown that pyroptosis is involved in Cd-induced tissue damage, but whether pyroptosis is involved in damage to testicular tissue following Cd exposure remains unclear. To investigate the mechanism of pyroptosis in testicular injury induced by Cd exposure, we used 8-week-old male C57BL/6J mice subjected to consecutive 7 days of intraperitoneal injection of cadmium chloride (CdCl₂) at concentrations of 0, 1.0 and 3.0 mg/kg. The results indicated that 3.0 mg/kg CdCl₂ significantly decreased serum testosterone levels, sperm concentration and sperm motility, while increased LDH and IL-1β levels. Testicular HE staining indicated that Cd exposure damaged the interstitial cells and increased the atypical residual bodies. Fluorescence results indicated that 3.0 mg/kg CdCl₂ increased ROS levels, DNA damage, and the number of TUNEL-positive seminiferous tubule cells in testicular tissue. Transcriptome analysis showed that Cd exposure mainly induced inflammatory and chemokine signaling pathways in testicular tissue, with upregulated mRNA levels of Aim2, and reduced mRNA levels of Nlrp3. Further analysis showed that 3.0 mg/kg CdCl₂ increased the expression of testicular HO-1, SOD2, γH2AX and PARP-1, as well as the pyroptosis-related factors GSDMD, GSDME, Caspase-1, ASC and IL-1β. In conclusion, our results provide a possible mechanism by which Cd exposure activates the AIM2 pathway by increasing oxidative stress injury to induce pyroptosis in testicular tissue. This provides a new perspective on testicular damage caused by Cd exposure.
Cadmium induces liver dysfunction and ferroptosis through the endoplasmic stress-ferritinophagy axis

2022, Ecotoxicology and Environmental Safety
Cadmium (Cd) is a type of high-risk heavy metal that can damage organs such as the liver, but its mechanism is not yet clear. Ferroptosis is a newly discovered mode of regulatory cell death. We explored whether ferroptosis is involved in Cd-induced liver damage and the underlying mechanism. Our research showed that Cd induced liver damage by inducing ferroptosis, and the use of ferroptosis inhibitors reduced the degree of liver damage. Moreover, the occurrence of ferroptosis was accompanied by the activation of the PERK-eIF2α-ATF4-CHOP signaling pathway, and inhibiting endoplasmic reticulum (ER) stress reduced ferroptosis demonstrating that ferroptosis induced by Cd is dependent on ER stress. In addition, chloroquine, a common autophagy inhibitor, mitigated ferroptosis caused by Cd exposure. Then, the iron chelator deferoxamine reduced Cd-induced lipid peroxidation and cell death, demonstrating that the iron regulation disorder caused by ferritin phagocytosis contributes to the Cd-induced ferroptosis. In conclusion, our results show that Cd-induced liver toxicity is accompanied by ferroptosis, which contributes to Cd inducing oxidative stress to trigger autophagy and ER stress to promote the process of ferroptosis.
Ferroptosis as a mechanism of non-ferrous metal toxicity

2022, Archives of Toxicology

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Cadmium attenuates testosterone synthesis by promoting ferroptosis and blocking autophagosome-lysosome fusion镉通过促进铁死亡和阻止自噬体-溶酶体融合来减弱睾酮合成

Highlights 亮点

Abstract 抽象的

Graphical abstract 图文摘要

Keywords 关键词

Abbreviations 缩写

1. Introduction 一、简介

2. Materials and methods 2 材料与方法

2.1. Ethics statement 2.1.道德声明

2.2. Animals and experimental groups2.2.动物和实验组

2.3. Cd content in the testis2.3.睾丸中镉含量

2.4. Cell culture 2.4.细胞培养

2.5. Determination of sex hormones (T, LH and FSH)2.5.性激素（T、LH 和 FSH）的测定

2.6. Histopathological analyses of the testis2.6。睾丸的组织病理学分析

2.7. Transmission electron microscopy2.7.透射电子显微镜

2.8. Cell viability 2.8.细胞活力

2.9. ROS and mtROS measurement2.9. ROS 和 mtROS 测量

2.10. Iron assay 2.10.铁测定

2.11. Biochemical assays 2.11.生化检测

2.12. Measurement of mitochondrial membrane potential (MMP)2.12.线粒体膜电位（MMP）的测量

2.13. Quantitative real-time polymerase chain reaction (qRT-PCR)2.13.实时定量聚合酶链式反应 (qRT-PCR)

2.14. Western blot analysis2.14.蛋白质印迹分析

2.15. Immunofluorescence staining and observation by confocal microscope2.15。免疫荧光染色及共聚焦显微镜观察

2.16. mCherry-GFP-LC3B transfection and confocal microscopy2.16。 mCherry-GFP-LC3B 转染和共聚焦显微镜

2.17. Lyso-Tracker Red and confocal microscopy2.17。 Lyso-Tracker 红色和共聚焦显微镜

2.18. Statistical analysis2.18。统计分析