Elsevier

Environmental Pollution  环境污染

Volume 292, Part A, 1 January 2022, 118297
第 292 卷 A 部分，2022 年 1 月 1 日，118297
Environmental Pollution

High-fat diet exacerbated decabromodiphenyl ether-induced hepatocyte apoptosis via intensifying the transfer of Ca2+ from endoplasmic reticulum to mitochondria
高脂饮食通过强化Ca 2+从内质网向线粒体的转移，加剧十溴二苯醚诱导的肝细胞凋亡

https://doi.org/10.1016/j.envpol.2021.118297 IF: 7.6 Q1 IF: 7.6 Q1 Get rights and content  获取权利和内容

Highlights  亮点

  • •
    The hepatotoxicity of BDE-209 was enhanced by high fat diet (HFD).
    高脂肪饮食 (HFD) 会增强 BDE-209 的肝毒性。
  • •
    BDE-209 induced hepatocytes apoptosis via ER/Ca2+/mitochondria pathway.
    BDE-209 通过 ER/Ca 2+ /线粒体途径诱导肝细胞凋亡。
  • •
    The enhanced ER-mitochondria interaction by HFD promoted Ca2+ transfer.
    HFD 增强的 ER-线粒体相互作用促进了 Ca 2+转移。

Abstract  抽象的

Polybrominated diphenyl ether (PBDE) as the flame retardant is heavily used in daily necessities, causing adverse health effects on humans. This study aimed to evaluate the hepatotoxicity of decabromodiphenyl ether (BDE-209), the most widely used PBDE, in lean and high-fat diet (HFD)-treated obese mice and elucidate the underlying mechanism. Firstly, the increasing levels of TG and proinflammatory factors in the liver and ALT and AST in serum demonstrated the hepatic damage caused by BDE-209 and further exacerbated by HFD. Tunel image revealed that BDE-209 induced more severe hepatocyte apoptosis with the assistant of HFD. Next, the mechanism analysis showed that the pro-apoptotic action of BDE-209 was in an endoplasmic reticulum (ER)/Ca2+ flux/mitochondria-dependent manner, concluded from the impairment of mitochondrial membrane potential, the enhancive protein expression of p-PERK/PERK, p-IRE1/IRE1, ATF6, CHOP, Bax/Bcl-2, cleaved caspase-3/caspase-3, IP3R1 and Sig-1R, and the over-transfer of Ca2+ from ER to mitochondria. Such proposed mechanism was further confirmed by the IP3R1 siRNA transfection cell experiment, where apoptotic rate was reduced in parallel with the reduced mitochondrial Ca2+ level. Finally, the higher expression of PACS-2 protein and the expanded ER contributed to the enriched ER-mitochondria interaction, reflected by the closer distance between ER and mitochondria visually displayed in the TEM image in HFD groups. This change was conducive to the rapid delivery of apoptosis signals via Ca2+, as proven, mechanically explaining the strengthening effect of HFD on BDE-209 hepatotoxicity. These findings detailedly explained the mechanism of BDE-209 hepatotoxicity and clarified the auxiliary effect of HFD, providing a theoretical basis for further studying other analogs.
多溴二苯醚（PBDE）作为阻燃剂大量用于日常生活用品中，对人类健康造成不良影响。本研究旨在评估十溴二苯醚 (BDE-209)（最广泛使用的 PBDE）对瘦肉和高脂饮食 (HFD) 治疗的肥胖小鼠的肝毒性，并阐明其潜在机制。首先，肝脏中TG和促炎因子以及血清中ALT和AST水平的升高证明了BDE-209引起的肝损伤，并且HFD进一步加剧了肝损伤。 Tunel图像显示，在HFD的辅助下，BDE-209诱导了更严重的肝细胞凋亡。接下来，机制分析表明，BDE-209的促凋亡作用是内质网（ER）/Ca 2+通量/线粒体依赖性方式，从线粒体膜电位受损、增强p蛋白表达得出结论。 -PERK/PERK、p-IRE1/IRE1、 ATF6 、CHOP、Bax/Bcl-2、切割的 caspase-3/caspase-3、 IP3R1 和 Sig-1R，以及 Ca 2+从内质网过度转移到线粒体。 IP3R1 siRNA转染细胞实验进一步证实了这种机制，其中细胞凋亡率随着线粒体Ca 2+水平的降低而降低。最后，PACS-2蛋白的较高表达和扩大的ER有助于丰富的ER-线粒体相互作用，这反映在HFD组的TEM图像中视觉上显示的ER和线粒体之间的距离更近。 事实证明，这种变化有利于通过Ca 2+快速传递细胞凋亡信号，从机械上解释了HFD对BDE-209肝毒性的增强作用。这些研究结果详细解释了BDE-209的肝毒性机制，阐明了HFD的辅助作用，为进一步研究其他类似物提供了理论基础。

Keywords  关键词

Decabromodiphenyl ether
Hepatotoxicity
Intrinsic apoptotic pathway
Ca2+ flux
IP3R1
PACS-2

十溴二苯醚
肝毒性
内在凋亡途径
Ca 2+熔剂
IP3R1
PACS-2

1. Introduction  一、简介

Decabromodiphenyl ether (BDE-209) is a member of the polybrominated diphenyl ethers (PBDEs), containing ten bromine atoms. Benefiting from the excellent flame-retardant capacity, BDE-209 has been widely used in electronic products, plastics, and building materials (Ni et al., 2013), accounting for >70% of total usage of PBDEs (Shaoyong et al., 2021). However, the concern about population health risk is growing due to the reality that BDE-209 is persistent in the environment and finally appears in the human body through the food and air. Several studies have testified that BDE-209 disturbed thyroid hormones level, impaired fertility, and caused damage to the neuro (Li et al., 2019; Sun et al., 2017; Wang et al., 2019). In particular, the potential hepatotoxicity of BDE-209 attracts increasing attention because the liver is the critical metabolic and detoxification organ. An experiment carried out in a human embryonic stem cell-based liver differentiation model found that BDE-209 might exert developmental hepatotoxicity in humans (Liang et al., 2019). As a result, BDE-209 has been banned in many countries, including European Union, the United States, and Canada, but not in China so far (Akortia et al., 2016).
十溴二苯醚(BDE-209) 是多溴二苯醚(PBDE) 的一员，含有十个溴原子。得益于优异的阻燃能力，BDE-209已广泛应用于电子产品、塑料、建筑材料等领域( Ni et al., 2013 )，占PBDEs总用量的70%以上( Shaoyong et al., 2013 )。 2021 ）。然而，由于 BDE-209 在环境中持久存在并最终通过食物和空气进入人体，人们对人口健康风险的担忧日益增加。多项研究证明，BDE-209 会扰乱甲状腺激素水平，损害生育能力，并对神经造成损害（ Li et al., 2019 ； Sun et al., 2017 ； Wang et al., 2019 ）。特别是，BDE-209的潜在肝毒性引起了越来越多的关注，因为肝脏是关键的代谢和解毒器官。在基于人胚胎干细胞的肝脏分化模型中进行的实验发现，BDE-209可能对人类产生发育性肝毒性（ Liang et al., 2019 ）。因此，BDE-209已在许多国家被禁止，包括欧盟、美国和加拿大，但迄今为止在中国尚未被禁止（ Akortia等，2016 ）。
The worse is that the hepatotoxicity of BDE-209 to the obese might be underestimated. Owing to the lipophilicity of the hydrocarbon construction, BDE-209 tends to accumulate in adipose tissue (Klinčić et al., 2020). Therefore, the aggravating hepatic lipid deposition in the overweight people would theoretically increase the content of BDE-209, resulting in the enhanced toxicity. Actually, some flame retardants with similar physiological and biochemical properties to BDE-209 have been found to exert stronger hepatotoxicity when assisted with a high-fat diet (HFD). In HFD-induced obese mice, BDE-47 and bisphenol-A triggered more severe hepatic steatosis and fibrosis than that in normal diet (ND)-treated mice (Figueiredo et al., 2020; Yang et al., 2019). Besides, metabolism analysis revealed that administered with HFD, polychlorinated biphenyl-153 produced a significant metabolic difference from HFD alone or ND + polychlorinated biphenyl-153, consistent with the worsened obesity/non-alcoholic fatty liver disease pathology (Shi et al., 2012). Consequently, considering the prevalence of obesity worldwide, it is necessary to reassess the liver impairment caused by BDE-209.
更糟糕的是，BDE-209 对肥胖者的肝毒性可能被低估。由于碳氢化合物结构的亲脂性，BDE-209 倾向于在脂肪组织中积聚（ Klinčić 等人，2020 ）。因此，超重人群肝脏脂质沉积加剧，理论上会增加BDE-209的含量，导致毒性增强。事实上，一些与BDE-209具有相似生理生化特性的阻燃剂被发现在高脂饮食（HFD）的辅助下会产生更强的肝毒性。在 HFD 诱导的肥胖小鼠中，BDE-47 和双酚 A 引发比正常饮食 (ND) 治疗的小鼠更严重的肝脏脂肪变性和纤维化（ Figueiredo 等，2020 ； Yang 等，2019 ）。此外，代谢分析显示，与单独使用 HFD 或 ND + 多氯联苯 153 联合使用 HFD 时，多氯联苯 153 产生显着的代谢差异，这与肥胖/非酒精性脂肪肝病病理恶化一致（ Shi et al., 2012） ）。因此，考虑到全球肥胖的流行，有必要重新评估 BDE-209 引起的肝脏损害。
The endoplasmic reticulum (ER) is a crucial organelle responsible for modifying most secreted proteins, controlling Ca2+ homeostasis, synthesizing lipids in cells, and so on (Schwarz & Blower, 2016). Suffering from physiological or pathological damage, the ER would be in stress so that cannot fold proteins correctly, which could be relieved by the initiated unfold protein response (UPR) (Almanza et al., 2019). However, the persistent ER stress will cause cell apoptosis, one of which is ignited by the prolonged transfer of Ca2+ from the ER to the mitochondria, named mitochondria-dependent apoptosis (Danese et al., 2021; Pinton et al., 2008; Rimessi et al., 2020). This mode of apoptosis has been superficially studied in bisphenol A and BDE-47, where no altered Ca2+ homeostasis was explored (Huang et al., 2021; Zhang et al., 2015). Although the impairment of BDE-209 to mitochondrial function has been discovered by Pereira et al. (2017), other related studies are almost empty. Besides, Zhang et al. (2017) found that the BDE-47-inhibited testosterone production was related to serum insulin which could be increased by HFD, explaining the aggravated reproduction toxicity of BDE-47 in the obese rats. This phenomenon implied that in addition to increasing the content, HFD might enhance the toxicity via participating in the pathway through which toxicants play a role. Hence, a comprehensive study must be conducted to illuminate the hepatotoxicity mechanism of BDE-209 and the impact of HFD on it.
内质网（ER）是负责修饰大多数分泌蛋白、控制Ca 2+稳态、合成细胞内脂质等的重要细胞器（ Schwarz & Blower, 2016 ）。受到生理或病理损伤，内质网会处于应激状态，无法正确折叠蛋白质，这可以通过启动未折叠蛋白反应（UPR）来缓解（ Almanza et al., 2019 ）。然而，持续的内质网应激会导致细胞凋亡，其中一种是由Ca 2+从内质网长时间转移到线粒体而引发的，称为线粒体依赖性细胞凋亡（ Danese et al., 2021 ; Pinton et al., 2008）里梅西等人，2020 ）。这种细胞凋亡模式已在双酚 A和 BDE-47 中进行了初步研究，但未发现 Ca 2+稳态发生改变（ Huang 等，2021 ； Zhang 等，2015 ）。尽管Pereira 等人已经发现 BDE-209 对线粒体功能的损害。 (2017) ，其他相关研究几乎空白。此外，张等人。 (2017)发现BDE-47抑制的睾酮产生与高脂饮食可增加血清胰岛素有关，这解释了BDE-47在肥胖大鼠中加重的生殖毒性。这一现象表明，HFD除了增加含量外，还可能通过参与毒物发挥作用的途径来增强毒性。因此，必须进行全面的研究来阐明BDE-209的肝毒性机制以及HFD对其的影响。
In the present study, the liver function was evaluated in the lean and HFD-treated obese mice to elucidate and compare the hepatotoxicity of BDE-209. Next, the mechanism of BDE-209-induced hepatocyte apoptosis in an ER/Ca2+/mitochondria dependent manner has been explored. Finally, cell assay, determination of proteins and gene expression, and transmission electron microscope (TEM) experiment were performed to answer how HFD exacerbating the apoptosis through the Ca2+ flux.
在本研究中，评估了瘦小鼠和 HFD 治疗的肥胖小鼠的肝功能，以阐明和比较 BDE-209 的肝毒性。接下来，探讨了BDE-209以ER/Ca 2+ /线粒体依赖性方式诱导肝细胞凋亡的机制。最后，通过细胞分析、蛋白质和基因表达测定以及透射电子显微镜（TEM）实验来回答HFD如何通过Ca 2+通量加剧细胞凋亡。

2. Materials and methods  2 材料与方法

2.1. Chemical materials  2.1.化工原料

The BED-209 was purchased from Sigma-Aldrich Co. Ltd. (Shanghai, China) and corn oil from Rhawn Reagent Co., Ltd. (Shanghai, China). Triacylglycerol (TG), total cholesterol (TC), aspartate transaminase (AST), and alanine transaminase (ALT) kits were brought from Biobase Holding Group Co., Ltd. (Jinan, China), and tumor necrosis factor-α (TNF-α), interleukin 1 (IL-1), interleukin 4 (IL-4), interleukin 6 (IL-6) and interleukin 10 (IL-10) kits from Meimian industrial Co., Ltd. (Jiangsu, China). HepG2 cells (SWEXB10107) were gifted from Shanghai Chinese Academy of Sciences, and all cell culture reagents were purchased from Hyclone Technologies, Inc. (Logan, Utah, USA) unless otherwise stated. Sodium palmitate and sodium oleate were purchased from Kunchuang Technology Development Co., Ltd. (Xian, China).
BED-209 购自 Sigma-Aldrich Co. Ltd.（中国上海），玉米油购自 Rhawn 试剂有限公司（中国上海）。三酰甘油（TG）、总胆固醇（TC）、天冬氨酸转氨酶（AST）、丙氨酸转氨酶（ALT）试剂盒购自Biobase控股集团有限公司（中国济南），肿瘤坏死因子-α（TNF- α)、白细胞介素 1 (IL-1)、白细胞介素 4 (IL-4)、白细胞介素 6 (IL-6) 和白细胞介素 10 (IL-10)试剂盒来自美棉实业有限公司(中国江苏)。 HepG2细胞(SWEXB10107)由上海中国科学院赠送，所有细胞培养试剂均购自Hyclone Technologies, Inc.(Logan, Utah, USA)，除非另有说明。棕榈酸钠和油酸钠购自昆创科技发展有限公司（中国西安）。

2.2. Animals and experimental protocol
2.2.动物和实验方案

This experiment was approved by the Law of Animal Experiment, University of Nanchang (approval number: SYXK (GAN) 2015–0001; expiry date: December 29, 2020). Forty male C57BL/6 mice (aged 4 weeks) were purchased from Hunan SJA Laboratory Animal Co., Ltd. (Hunan, China) and housed in a specific pathogen-free facility at 23 °C with a 12/12 h light−dark cycle. After 1 week of acclimatization, randomly selected half mice were provided with ND, and the rest ate HFD. Besides, half of the mice (n = 10) in both ND and HFD groups were administered intragastrically with 100 mg/kg body weight (BW) of BDE-209 dissolved in 2 ml/kg BW corn oil, abbreviated as ND209 and HDF209, respectively. As a control, 2 ml/kg BW corn oil was fed to the other 10 mice, named NDC and HDFC, respectively. Both the ND and HFD were produced based on the laboratory animal feed production standard (GB/T 34,240–2017) by Nanjing Shengmin Scientific Research Animal Farm (Jiangsu, China), and the compositions were given in Table S1. The reasons for using a dose of 100 mg/kg bw were explained in detail in the discussion section.
本实验经南昌大学动物实验法批准（批准文号：SYXK（GAN）2015-0001；有效期：2020年12月29日）。 40只雄性C57BL/6小鼠（4周龄）购自湖南三甲实验动物有限公司（中国湖南），饲养在23℃、12/12小时明暗条件下的特定无病原体设施中。循环。适应1周后，随机选取一半小鼠喂食ND，其余小鼠喂食HFD。此外，ND组和HFD组的一半小鼠（n = 10）均以100 mg/kg体重（BW）的BDE-209溶解在2 ml/kg BW玉米油中灌胃，缩写为ND209和HDF209，分别。作为对照，将 2 ml/kg BW 玉米油喂给另外 10 只小鼠，分别命名为 NDC 和 HDFC。 ND和HFD均由南京生民科研动物场（中国江苏）按照实验动物饲料生产标准（GB/T 34,240-2017）生产，成分见表S1 。使用 100 mg/kg bw 剂量的原因已在讨论部分详细解释。
The experiment lasted for 8 weeks. During the entire experimental period, the food consumption and BW of each mouse were monitored twice a week. At the end of the experiment, after overnight fasting for 12 h, mice were anesthetized with ether to collect blood by removing the eyeballs, and then they were sacrificed by breaking the spine. Livers were removed, washed with phosphate buffered saline (PBS), and weighed. Part of the liver tissue was fixed for morphological and immunohistochemical analysis while the rest were frozen in liquid nitrogen and then stored at −80 °C until use.
实验持续了8周。在整个实验期间，每周两次监测每只小鼠的食物消耗和体重。实验结束时，隔夜禁食12小时后，用乙醚麻醉小鼠，摘除眼球采血，然后折断脊柱处死。取出肝脏，用磷酸盐缓冲盐水(PBS)洗涤，并称重。部分肝组织固定用于形态学和免疫组织化学分析，其余部分冷冻在液氮中，然后保存在-80°C直至使用。

2.3. Serum biochemistry and determination of liver lipid
2.3.血清生化及肝脂测定

These parameters were measured under the guidance of instructions in kits. Briefly, after incubation at room temperature for 2 h, the serum was separated by centrifugation under 3000 rpm at 4 °C for 10 min, and used to determine the AST and ALT contents by a Chemray 240 Automatic Biochemical Analyzer (Rayto Life and Analytical Sciences Co., Ltd., Shenzhen, China). TC and TG levels in the liver were also detected by the Automatic Biochemical Analyzer, but the results were calibrated by total protein content measured using a BCA Protein Assay Kit (Beyotime, Peking, China).
这些参数是在试剂盒说明书的指导下测量的。简而言之，室温孵育2小时后，4℃、3000rpm离心10分钟分离血清，并使用Chemray 240自动生化分析仪（Rayto Life and Analytical Sciences）测定AST和ALT含量。有限公司，深圳，中国）。肝脏中的TC和TG水平也通过自动生化分析仪检测，但结果是通过使用BCA蛋白测定试剂盒（Beyotime，北京，中国）测量的总蛋白含量来校准的。

2.4. Glucose tolerance test
2.4.葡萄糖耐量试验

With reference to the method of Nagy and Einwallner (2018), the fasting glucose tolerance test was performed at week 6. After fasting for 16 h, the glucose in the blood from the tail of mice was measured with a blood glucose meter (Bayer, Germany). 30 min later, 0.2 mL of glucose solution was intragastrically administered at a dose of 2 g/kg BW, and the values of blood glucose at 0, 30, 60, 90, and 120 min were recorded.
参照Nagy和Einwallner(2018)的方法，第6周进行空腹糖耐量试验。禁食16h后，用血糖仪(Bayer，Bayer，德国）。 30分钟后，以2g/kg体重的剂量灌胃0.2mL葡萄糖溶液，记录0、30、60、90和120分钟的血糖值。

2.5. Evaluation of hepatic mitochondrial function
2.5.肝线粒体功能评估

2.5.1. Mitochondrial membrane potential
2.5.1.线粒体膜电位

After removed from mice, the hepatic mitochondria were rapidly isolated by standard differential centrifugation, under the guidance of the Tissue Mitochondria Isolation Kit instruction (Beyotime, Peking, China). The measurement of mitochondrial membrane potential (MMP) was finished using JC-1 kit (Beyotime, Peking, China) within 2 h. The red fluorescence intensity of JC-1 aggregates, and the green fluorescence intensity of JC-1 monomer were measured by a multimode reader (PerkinElmer, Massachusetts, UK).
从小鼠体内取出肝线粒体后，在组织线粒体分离试剂盒说明书（Beyotime，北京，中国）的指导下，通过标准差速离心快速分离肝线粒体。使用JC-1试剂盒（Beyotime，北京，中国）在2小时内完成线粒体膜电位（MMP）的测量。 JC-1 聚集体的红色荧光强度和 JC-1单体的绿色荧光强度通过多模式读数器（PerkinElmer，马萨诸塞州，英国）测量。

2.5.2. Mitochondrial DNA copy number
2.5.2.线粒体 DNA 拷贝数

The total DNA was extracted using the Rapid Animal Genomic DNA Isolation Kit (Sangon Biotech, Shanghai, China) according to the manufacturer's instruction. The B6 gene used forward primer 5′-AACCTGGCACTGAGTCACCA-3′, reverse primer 5′- GGGTCTGAGTGTATATATCATGA-3’; The 18s RNA gene used forward primer 5′- CGCGGTTCTATTTTGTTGGT′, reverse primer 5′-AGTCGGCATCGTTTATGGTC-3′ were taken from the literature (Xu et al., 2021) and provided by Sangon Biotech Co., Ltd. (Shanghai, China). Mitochondrial DNA (MtDNA) copy number in the liver was evaluated by qRT-PCR utilizing TB Green™ Premix Ex Taq™ (Tli RNaseH Plus, RR820A) from Takara Bio Inc. (Shiga, Japan) following the manufacturer's protocol. The data were obtained by the CFX Connect Real-Time System (Bio-Rad Laboratories, California, USA), and 2−ΔΔCt (MtDNA to nuclear DNA) was equivalent to the MtDNA copy number (Song et al., 2018).
根据制造商的说明，使用快速动物基因组DNA分离试剂盒（生工生物科技，上海，中国）提取总DNA。 B6基因所用正向引物5'-AACCTGGCACTGAGTCACCA-3'，反向引物5'-GGGTCTGAGTGTATATATCATGA-3'；所用18s RNA基因正向引物5'-CGCGGTTCTATTTTGTTGGT'，反向引物5'-AGTCGGCATCGTTTATGGTC-3'取自文献（ Xu et al., 2021 ），由生工生物科技有限公司（中国上海）提供。使用来自 Takara Bio Inc.（日本滋贺）的 TB Green™ Premix Ex Taq™（Tli RNaseH Plus，RR820A）按照制造商的方案，通过 qRT-PCR 评估肝脏中的线粒体 DNA (MtDNA) 拷贝数。数据通过CFX Connect实时系统（Bio-Rad Laboratories，加利福尼亚州，美国）获得，2 −ΔΔCt （MtDNA到核DNA）相当于MtDNA拷贝数（ Song等，2018 ）。

2.5.3. Hepatic ATP content
2.5.3.肝ATP含量

Hepatic ATP levels were detected by ATP Assay Kit according to the manufacturer's instruction and quantitated by a luminometer loaded on a multimode reader (PerkinElmer, Massachusetts, USA).
根据制造商的说明，通过 ATP 检测试剂盒检测肝脏 ATP 水平，并通过装载在多模式读数器（PerkinElmer，马萨诸塞州，美国）上的光度计进行定量。

2.6. ELISA validation  2.6。 ELISA 验证

The contents of hepatic TNF-α, IL-1, IL-4, and IL-10 were quantified by ELISA kits following the manufacturers’ instructions.
按照制造商的说明，通过ELISA试剂盒对肝脏 TNF-α、IL-1、IL-4 和 IL-10 的含量进行定量。

2.7. Quantitative real-time polymerase chain reaction
2.7.实时定量聚合酶链反应

Using Trizol assay and PrimeScript RT Reagent Kit with gDNA Eraser (Takara, Shiga, Japan), the cDNA from hepatic tissues were prepared. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed with TB Green™ Premix Ex Taq™ (Tli RNaseH Plus) (Takara, Shiga, Japan). The corresponding primers were provided by Sangon Biotech Co., Ltd. (Shanghai, China), and detailed primer sequences were shown in Table S2. The changes of gene expression were detected in the CFX Connect Real-Time System (Bio-Rad Laboratories, California, USA) and calculated as 2−ΔΔCt, where glyceraldehyde 3 phosphoric acid dehydrogenase (GAPDH) was used as the internal control gene.
使用Trizol测定和带有 gDNA Eraser 的 PrimeScript RT Reagent Kit（Takara，滋贺，日本），制备来自肝组织的 cDNA。使用 TB Green™ Premix Ex Taq™ (Tli RNaseH Plus)（日本滋贺县宝）进行定量实时聚合酶链反应 (qRT-PCR)。相应引物由中国上海生工生物科技有限公司提供，详细引物序列见表S2 。在CFX Connect实时系统（Bio-Rad Laboratories，加利福尼亚州，美国）中检测基因表达的变化并计算为2 −ΔΔCt ，其中甘油醛3磷酸脱氢酶（GAPDH）用作内参基因。

2.8. Western blot  2.8.蛋白质印迹

The used method was as Wu et al. (2020) with some modifications. The total protein in hepatic tissues was extracted using RIPA Lysis Buffer (Beyotime, Peking, China). After separated by SDS-PAGE, transferred into a poly(vinylidene difluoride) membrane, blocked by 5% skimmed milk, and orderly incubated with the primary and secondary antibodies, the expression of targeted proteins were detected using an ECL Kit (Solarbio, Peking, China) and recorded by the ChemiDoc Imaging Systems/Image Lab software (Bio-Rad). GAPDH or β-actin was the internal reference for equality of sample loading. The information on the antibodies used was listed in Table S3.
所用方法如Wu等人。 （2020）进行了一些修改。使用RIPA Lysis Buffer（Beyotime，北京，中国）提取肝组织中的总蛋白。 SDS-PAGE分离后，转入聚偏二氟乙烯膜，5%脱脂牛奶封闭，依次与一抗和二抗孵育，使用ECL试剂盒（Solarbio，北京，中国）并由 ChemiDoc 成像系统/图像实验室软件 (Bio-Rad) 记录。 GAPDH 或 β-肌动蛋白是样品加载相等的内参。表S3列出了所用抗体的信息。

2.9. Hepatic tissue HE staining
2.9.肝组织HE染色

Hepatic tissues were fixed overnight with 4% paraformaldehyde, followed by embedded in paraffin, sliced into 4 μm, and stained with hematoxylin and eosin (HE) (Baiqiandu, Wuhan, China). The stained areas were observed using an upright optical microscope (Nikon Eclipse CI, Tokyo, Japan) under a magnification of 200 × .
肝组织用4％多聚甲醛固定过夜，然后石蜡包埋，切成4μm，并用苏木精和伊红（HE）（中国武汉百千渡）染色。使用正置光学显微镜（Nikon Eclipse CI，东京，日本）在 200 × 的放大倍数下观察染色区域。

2.10. Tunel staining  2.10.隧道染色

Fixed hepatic tissues with 4% paraformaldehyde were embedded in paraffin, sliced into 4 μm, and stained by the TUNEL technique using an in-situ apoptosis detection kit (Roche, Basle, Switzerland) according to the instruction provided by the manufacturer. The pictures were taken using an inverted fluorescence microscope (Nikon Eclipse Ti-SR, Tokyo, Japan), and the apoptosis rate was calculated as the ratio of the number of TUNEL positive signal cells to the total number of cells via Image-Pro Plus 6.
将用4%多聚甲醛固定的肝组织包埋在石蜡中，切成4μm的切片，并使用原位细胞凋亡检测试剂盒（Roche，Basle，Switzerland）按照制造商提供的说明书通过TUNEL技术染色。使用倒置荧光显微镜（Nikon Eclipse Ti-SR，Tokyo，Japan）拍摄照片，通过Image-Pro Plus 6计算凋亡率，即TUNEL阳性信号细胞数与细胞总数的比值。 。

2.11. Transmission electron microscope
2.11.透射电子显微镜

A small piece of liver (1*1*1 mm3) was rapidly fixed in 2.5% glutaraldehyde for 2–4 h at 4 °C and post-fixed in 1% osmic acid in 0.1 M PBS for 2 h at room temperature. After dehydration, infiltration, embedding, sectioning, and staining, the samples were examined and photographed with a Tecnai G2 20 S-TWIN TEM (FEI Company, Oregon, USA) at 100 kV.
将一小块肝脏 (1*1*1 mm 3 ) 在 2.5%戊二醛中于 4 °C 快速固定 2-4 小时，并在室温下于 1%锇酸的 0.1 M PBS 中后固定 2 小时。经过脱水、渗透、包埋、切片和染色后，使用 Tecnai G2 20 S-TWIN TEM（FEI 公司，俄勒冈州，美国）在 100 kV 下对样品进行检查和拍照。

2.12. Cell experiment  2.12.细胞实验

2.12.1. Cell culture and treatment
2.12.1.细胞培养和治疗

HepG2 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 1% (v/v) penicillin/streptomycin and 10% (v/v) fetal bovine serum (FBS) in a humidified atmosphere of 5% CO2 at 37 °C. After overnight culture, BDE-209 dissolved in dimethyl sulfoxide (DMSO) (Solarbio, Peking, China) or/and the steatosis-induced reagent (SIR) (C sodium oleate/C sodium palmitate = 500/250 μM) was added into the medium for 48 h (ND209 and HFD209). The final concentration of DMSO did not exceed 1%. Cells in the NDC group were treated with equal DMSO, and in the HFDC group were incubated with equal DMSO and SIR.
HepG2细胞在含有1%（v/v）青霉素/链霉素和10%（v/v）胎牛血清（FBS）的Dulbecco改良Eagle培养基（DMEM）中，在5% CO 2的潮湿气氛中于37°C保存。过夜培养后，将 BDE-209 溶解在二甲基亚砜(DMSO)（Solarbio，北京，中国）或/和脂肪变性诱导试剂 (SIR)（C油酸钠/C棕榈酸钠= 500/250 μM）中加入到培养皿中。介质 48 小时（ND209 和 HFD209）。 DMSO的终浓度不超过1%。 NDC组中的细胞用等量的DMSO处理，HFDC组中的细胞用等量的DMSO和SIR孵育。
The siRNA transfection treatment was conducted following the steps of Ding et al. (2020). In brief, DMEM/F-12 was used to dilute Lipofectamine™ 2000 (Invitrogen-Life Technologies, California, USA) and inositol 1,4,5-trisphosphate receptor 1 (IP3R1) siRNA (purchased from RiboBio, Guangzhou, China; IP3R1 siRNA-1: 5′-GGAAGAATGCCTGGAGTTTCA-3’; IP3R1 siRNA-2: 5′-GCAGAAATGATCAAGGAAAGA-3’; IP3R1 siRNA-3: 5′-GGATGACTTTATCTTGGAAGT-3’; Negative control (NC) siRNA: 5′-TTCTCCGAACGTGTCACGT-3′). After incubated for 5 min at room temperature, the two dilutions were mixed and incubated for 20 min at room temperature. Subsequently, HepG2 cells were treated with the mixture containing 50 nM IP3R1 siRNA for 6 h. Discarding the mixture, HepG2 cells were incubated with DMEM/F-12 supplemented with 10% FBS for 48 h and then collected for the following experiments.
siRNA转染处理按照Ding等人的步骤进行。 （2020） 。简而言之，使用 DMEM/F-12 稀释 Lipofectamine™ 2000（Invitrogen-Life Technologies，美国加利福尼亚州）和肌醇 1,4,5-三磷酸受体 1 (IP3R1) siRNA（购自中国广州锐博生物科技有限公司；IP3R1） siRNA-1: 5'-GGAAGAATGCCTGGAGTTTCA-3'; IP3R1 siRNA-2: 5'-GCAGAAATGATCAAGGAAAGA-3'；IP3R1 siRNA-3'：5'-GGATGACTTTATCTTGGAAGT-3'；阴性对照（NC）siRNA：5'-TTCTCCGAACGTGTCACGT-3'）。在室温下孵育5分钟后，将两种稀释液混合并在室温下孵育20分钟。随后，用含有50 nM IP3R1 siRNA的混合物处理HepG2细胞6小时。弃去混合物，将HepG2细胞与补充有10％FBS的DMEM/F-12一起孵育48小时，然后收集用于以下实验。

2.12.2. Effects of SIR and BDE-209 on cell cytotoxicity
2.12.2. SIR 和 BDE-209 对细胞毒性的影响

CCK-8 assay was used to detect the effects of SIR or/and BDE-209 on the viability of HepG2 cells. Briefly, 10 μL CCK-8 reagent (Solarbio, Peking, China) was added to the prepared cells in each well, and the plates were incubated at 37 °C for 1 h. The absorbance was measured at 450 nm using a microplate reader (Thermo Labsystems, Pennsylvania, USA). The viability of HepG2 cells in each group was expressed as the absorbance proportion compared to the NDC group.
采用CCK-8法检测SIR或/和BDE-209对HepG2细胞活力的影响。简而言之，将10μL CCK-8试剂（Solarbio，北京，中国）加入到每孔中准备好的细胞中，并将板在37℃下孵育1小时。使用酶标仪（Thermo Labsystems，宾夕法尼亚州，美国）在 450 nm 处测量吸光度。各组HepG2细胞的存活率表示为与NDC组相比的吸光度比例。

2.12.3. Oil Red O staining
2.12.3.油红O染色

To detect accumulation of intracellular neutral lipids, HepG2 cells were stained according to the instruction of Modified Oil Red O Staining Kit (Beyotime, Peking, China). After fixed with 10% formalin for 10 min, cells were stained with Oil Red O working solution for 40 min at room temperature. The cells were washed twice with PBS and observed using an Olympus CKX53 inverted microscope (Olympus, Tokyo, Japan) under a magnification of 100 × .
为了检测细胞内中性脂质的积累，按照改良油红O染色试剂盒（Beyotime，北京，中国）的说明书对HepG2细胞进行染色。细胞用10%福尔马林固定10分钟后，用油红O工作液室温染色40分钟。用PBS洗涤细胞两次，并使用Olympus CKX53倒置显微镜（Olympus，Tokyo，Japan）在100×放大倍数下观察。

2.12.4. Ca2+ content in organelle
2.12.4.细胞器中Ca 2+含量

Treated cells were loaded with Hoechst33342 (Beyotime, Peking, China), Mag-Fluo-AM and Rhod-2AM (Maokang Biotech, Shanghai, China) probes. Hoechst33342 nuclear stain can label the cell DNA, allowing measurement of cell count, Mag-Fluo-AM monitors ER Ca2+ concentration, and Rhod-2AM indicated mitochondrial Ca2+ level. Cellular images were acquired by using the Operetta CLSTM High Content Analysis System (PerkinElmer, Massachusetts, USA) or an inverted fluorescence microscope (Nikon Eclipse Ti-SR, Tokyo, Japan).
处理后的细胞装载Hoechst33342（Beyotime，北京，中国）、Mag-Fluo-AM和Rhod-2AM（茂康生物科技，上海，中国）探针。 Hoechst33342 核染色剂可以标记细胞 DNA，从而测量细胞计数，Mag-Fluo-AM 监测 ER Ca 2+浓度，Rhod-2AM 指示线粒体 Ca 2+水平。使用 Operetta CLSTM高内涵分析系统（PerkinElmer，马萨诸塞州，美国）或倒置荧光显微镜（Nikon Eclipse Ti-SR，东京，日本）获取细胞图像。

2.12.5. Flow cytometric analysis of apoptosis
2.12.5。流式细胞术分析细胞凋亡

Annexin V-FITC/Propidium Iodide (PI) double staining cell apoptosis detection kit was purchased from Biological Technology Co., Ltd. (Shanghai, China). Briefly, treated HepG2 cells were digested with 0.25% trypsin, centrifuged at 1500 rpm for 5 min and then washed twice with PBS. Next, the 500 μL cell suspension was incubated with 10 μL Propidium Iodide for 15 min at room temperature in the dark, followed by the load of 5 μL Annexin V-FITC. Finally, cell apoptosis was assessed using the FACSCalibur flow cytometer (BD, New Jersey, USA) within 1 h.
Annexin V-FITC/PI双染细胞凋亡检测试剂盒购自上海生物科技有限公司。简而言之，用0.25%胰蛋白酶消化处理的HepG2细胞，以1500rpm离心5分钟，然后用PBS洗涤两次。接下来，将 500 μL 细胞悬液与 10 μL 碘化丙啶在室温下避光孵育 15 分钟，然后上样 5 μL Annexin V-FITC。最后，使用 FACSCalibur 流式细胞仪（BD，新泽西州，美国）在 1 小时内评估细胞凋亡。

2.13. Statistical analysis
2.13.统计分析

The data were analyzed by the 2 * 2 factorial experimental design using the general linear model procedure of SPSS statistical software (ver. 23.0, SPSS Inc., USA) in the following model: yijk = μ + ai + bj + (ab)ij + eijk (i = 1, 2, j = 1, 2, k = 1, 2, …, nij), where yijk represents the dependent variable, μ is the mean, ai is the effect of BDE-209 (corn oil, BDE-209 dissolved in corn oil), bj is the effect of diet (ND, HFD), (ab)ij is the interaction between BDE-209 and diet, and eijk is the error term. Thereafter, one-way ANOVA followed by the Duncan test was performed to determine the significance of difference at P < 0.05 among all four groups.
使用 SPSS 统计软件（版本 23.0，SPSS Inc.，美国）的一般线性模型程序，通过 2 * 2 析因实验设计对数据进行分析，模型如下： y ijk = μ + a + b + (ab) ij + e ijk ( i = 1, 2, j = 1, 2, k = 1, 2, …, n ij )，其中y ijk表示因变量， μ是均值， a是 BDE-209（玉米油，BDE-209 溶解在玉米油中）的影响， b是饮食（ND、HFD）的影响， (ab) ij是 BDE-209 与饮食之间的相互作用，以及e ijk是误差项。此后，进行单向方差分析，然后进行邓肯检验，以确定所有四组之间 P < 0.05 的差异显着性。

3. Results  3. 结果

3.1. BDE-209 did not change mouse growth performance
3.1. BDE-209 不会改变小鼠的生长性能

Four groups had similar BWs at week 0 (P > 0.05). During the experimental period, BDE-209 did not alter BW, but HFD caused an increase to a higher final BW than ND (P < 0.05), as expected. Neither BDE-209 nor HFD changed the daily food intake (Figure S1).
四组在第 0 周时的 BW 相似 (P > 0.05)。在实验期间，BDE-209 没有改变BW ，但 HFD 导致最终 BW 比 ND 更高 (P < 0.05)，正如预期的那样。 BDE-209 和 HFD 均未改变每日食物摄入量（图 S1 ）。

3.2. BDE-209 exacerbated liver damage in the HFD group
3.2. BDE-209 加剧了 HFD 组的肝损伤

3.2.1. Liver organ index  3.2.1.肝脏指数

The BDE-209 * diet interaction was not detected in liver organ index. However, BDE-209 could cause an increase of the index with the assistant of HFD (0.0395 ± 0.0024 vs 0.0456 ± 0.0012) (P < 0.05), which was not observed in ND (P > 0.05) (Fig. 1A).
肝器官指数中未检测到 BDE-209 * 饮食相互作用。然而，BDE-209 在 HFD 的辅助下会导致指数增加 (0.0395 ± 0.0024 vs 0.0456 ± 0.0012) (P < 0.05)，而在 ND 中未观察到这一点 (P > 0.05)（图 1 A） ）。
Fig. 1
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Fig. 1. BDE-209 exacerbated liver damage in HFD group. Mice were divided into normal diet control group (NDC), NDC +100 mg/kg body weight (BW) of BDE-209 group (ND209), high-fat diet control group (HFDC) and HFDC +100 mg/kg BW of BDE-209 group (HFD209) for 8 weeks. (A) Liver organ index; (B) Serum ALT and AST levels; (C) Hepatic TG and TC levels; (D) Area under curve of blood glucose; (E) Hepatic proinflammatory cytokines levels; (F) Hepatic anti-inflammatory cytokines levels; (G) Representative images of HE-stained hepatic section. The Gray box was the legend for Fig. 1B, C, E, and F. Data were expressed as mean ± SD. Bars with different letters differed significantly (P < 0.05). Asterisk represented the significant BDE-209 * diet interaction (P < 0.05).
图1 . BDE-209 加剧了 HFD 组的肝损伤。将小鼠分为正常饮食对照组(NDC)、BDE-209 NDC+100 mg/kg体重(BW)组(ND209)、高脂饮食对照组(HFDC)和HFDC+100 mg/kg BW组。 BDE-209 组 (HFD209) 持续 8 周。 (A)肝器官指数； (B) 血清 ALT 和 AST 水平； (C) 肝脏 TG 和 TC 水平； (D) 血糖曲线下面积； (E) 肝脏促炎细胞因子水平； (F) 肝脏抗炎细胞因子水平； (G) HE 染色肝切片的代表性图像。灰色框是图 1 B、C、E 和 F 的图例。数据表示为平均值±SD。不同字母的条形差异显着（P < 0.05）。星号代表显着的 BDE-209 * 饮食相互作用 (P < 0.05)。

3.2.2. Serum ALT and AST content
3.2.2.血清 ALT 和 AST 含量

A significant interaction between BDE-209 and HFD appeared on both serum ALT and AST levels (P < 0.05). ND209 administered mice had significantly higher serum AST content than NDC (P < 0.05) while ALT did not change obviously (P > 0.05). However, when eating BDE-209 at the basis of HFD, both ALT and AST activities were further enhanced to 62.97 ± 3.39 from 53.93 ± 1.66 U/L and to 140.73 ± 1.66 from 126.40 ± 1.17 U/L, respectively (P < 0.05) (Fig. 1B).
BDE-209 和 HFD 之间在血清ALT和AST 水平上均出现显着的相互作用(P < 0.05)。 ND209给药小鼠的血清AST含量显着高于NDC（P％3C 0.05），而ALT没有明显变化（P％3E 0.05）。然而，在 HFD 的基础上摄入 BDE-209 时，ALT 和 AST 活性分别从 53.93 ± 1.66 U/L 进一步增强至 62.97 ± 3.39，从 126.40 ± 1.17 U/L 进一步增强至 140.73 ± 1.66 (P < 0.05）（图1B ）。

3.2.3. Lipid accumulation in liver
3.2.3.肝脏内脂质堆积

There was an obvious BDE-209 * diet interaction in hepatic TG content (P < 0.05) which notably rose with the addition of BDE-209 only in the HFD group (281.19 ± 24.86 μmol/gprot) (P < 0.05). Regarding TC level in the liver, there was no influence of BDE-209 observed between NDC and ND209 or between HFDC and HFD209 group (P > 0.05) (Fig. 1C).
肝脏TG含量存在明显的 BDE-209 * 饮食相互作用 (P < 0.05)，仅在 HFD 组中添加 BDE-209 后，肝脏 TG 含量显着上升 (281.19 ± 24.86 μmol/gprot) (P < 0.05)。关于肝脏中的TC水平，在NDC和ND209之间或HFDC和HFD209组之间没有观察到BDE-209的影响（P％3E 0.05）（图1C ）。

3.2.4. Glucose tolerance  3.2.4.葡萄糖耐量

The alternation of serum glucose at 30-min intervals from −30 min to 120 min was plotted on Figure S2, and the calculated area under the curve was drawn in Fig. 1D. As shown, regardless of eating with ND or HFD, BDE-209 did not influence the glucose tolerance (P > 0.05).
将-30分钟至120分钟之间每隔30分钟的血糖变化绘制在图S2上，计算出的曲线下面积绘制在图1D中。如图所示，无论与ND或HFD一起进食，BDE -209不影响糖耐量(P>0.05)。

3.2.5. Hepatic inflammatory factors
3.2.5.肝脏炎症因子

IL-1 and TNF-α are the typical proinflammatory factors. Although there was not BDE-209 * diet interaction observed for IL-1 and TNF-α (P > 0.05), the combination of BDE-209 and HFD brought the highest levels of IL-1 and TNF-α among the four groups (P < 0.05) (Fig. 1E). Conversely, IL-4, IL-6, and IL-10 showed an antagonistic effect on inflammation. BDE-209 tended to make a reduction in their contents in ND groups or HFD groups, although there was not a statistical difference (P > 0.05) (Fig. 1F).
IL-1和TNF-α是典型的促炎因子。尽管没有观察到 BDE-209 * IL-1 和 TNF-α 的饮食相互作用 (P > 0.05)，但 BDE-209 和 HFD 的组合带来了四组中最高水平的 IL-1 和 TNF-α (P < 0.05) (图 1 E)。相反，IL-4、IL-6和IL-10对炎症表现出拮抗作用。 BDE-209在ND组或HFD组中倾向于降低其含量，尽管没有统计学差异（P>0.05）（图1F ）。

3.2.6. Hepatic HE staining
3.2.6.肝脏HE染色

Micrographs of HE-stained hepatic tissues were displayed in Fig. 1G. The liver in the NDC group presented normal tissue structure. However, it could be observed that focal infiltration of inflammatory cells (black arrow) in both ND209 and HFDC groups, and that necrotic degeneration and nuclear fragmentation in ND209 group (red arrow) and that steatosis in HFDC group (yellow arrow). Further, the simultaneous administration of BDE-209 and HFD aggravated the liver injury to the greatest extent, accompanied by all the above characteristics.
HE染色肝组织的显微照片如图1G所示。NDC组的肝脏呈现正常组织结构。然而，ND209和HFDC组均可见炎症细胞局灶性浸润（黑色箭头），ND209组（红色箭头）可见坏死变性和核碎裂，HFDC组（黄色箭头）可见脂肪变性。此外，BDE-209和HFD同时给药最大程度地加重了肝损伤，并伴有上述所有特征。

3.3. BDE-209 exacerbated mitochondrial-dependent hepatocyte apoptosis in the HFD group
3.3. BDE-209 加剧了 HFD 组线粒体依赖性肝细胞凋亡

3.3.1. Hepatic tunel staining
3.3.1.肝小管染色

Tunel staining was utilized to determine the adverse effect of BDE-209 on in-situ apoptosis in the liver (Fig. 2A–a), and the apoptosis rate represented as the ratio of green and blue fluorescence was displayed in Fig. 2A-b. As shown, the apoptosis rate increased to 1.20 ± 0.11% in the ND209 group from 0.24 ± 0.05% in the NDC group (P < 0.05). Moreover, with the assistant of HFD, BDE-209 had a more obvious promotion effect on hepatocyte apoptosis rate, from 1.39 ± 0.24% (HFDC) to 3.44 ± 0.29% (HFD209), confirmed by the significant BDE-209 * diet interaction (P < 0.05).
利用Tunel染色测定BDE-209对肝脏原位细胞凋亡的不良影响（图2A -a），细胞凋亡率以绿色和蓝色荧光的比率表示，如图2Ab所示。如图所示，ND209组的细胞凋亡率从NDC组的0.24±0.05%增加到1.20±0.11%（P<0.05）。此外，在HFD的辅助下，BDE-209对肝细胞凋亡率有更明显的促进作用，从1.39±0.24％（HFDC）到3.44±0.29％（HFD209），这由显着的BDE-209*饮食相互作用证实（ P<0.05）。
Fig. 2
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Fig. 2. BDE-209 exacerbated mitochondrial-dependent hepatocyte apoptosis in HFD group. Mice were divided into normal diet control group (NDC), NDC +100 mg/kg body weight (BW) of BDE-209 group (ND209), high-fat diet control group (HFDC) and HFDC +100 mg/kg BW of BDE-209 group (HFD209) for 8 weeks. (A) Representative Tunel Staining (a) and Apoptosis rate (b) of hepatocyte; (B) Hepatic mitochondrial function: MMP (a), ATP content (b) and MtDNA copy number (c); (C) Western bolting strip of Bcl-2, Bax, caspase-3, c-caspase-3 and β-actin (a), and relative protein expression of Bax/Bcl-2 (b) and c-caspase-3/caspase-3 (c) in liver tissue. The data were expressed as the means ± SD. Bars with different letters differed significantly (P < 0.05). Asterisk represented the significant BDE-209 * diet interaction (P < 0.05).
图2 . BDE-209 加剧了 HFD 组线粒体依赖性肝细胞凋亡。将小鼠分为正常饮食对照组(NDC)、BDE-209 NDC+100 mg/kg体重(BW)组(ND209)、高脂饮食对照组(HFDC)和HFDC+100 mg/kg BW组。 BDE-209 组 (HFD209) 持续 8 周。 (A) 肝细胞的代表性隧道染色(a)和凋亡率(b)； (B) 肝线粒体功能：MMP (a)、ATP 含量 (b) 和 MtDNA 拷贝数 (c)； (C) Bcl-2、Bax、caspase-3、c-caspase-3 和 β-肌动蛋白的蛋白质印迹条 (a)，以及 Bax/Bcl-2 (b) 和 c-caspase-3/ 的相对蛋白表达肝组织中的 caspase-3 (c)。数据表示为平均值±SD。不同字母的条形差异显着（P < 0.05）。星号代表显着的 BDE-209 * 饮食相互作用 (P < 0.05)。

3.3.2. Hepatic mitochondrial function
3.3.2.肝线粒体功能

JC-1 is a cell-permeable cationic dye that accumulates in mitochondria, and the ratio of JC-1 aggregates (red fluorescence) to JC-1 mono (green fluorescence) is usually used to measure MMP. The visible photos of MMP took by the Operetta CLSTM High Content Analysis System were supplemental in Figure S3 and the quantitative results detected by a microplate reader were listed in Fig. 2B–a. As shown, BDE-209 could reduce MMP in both ND and HFD groups (P < 0.05), but the combination of BDE-209 and HFD weakened the harmfulness of BDE-209 (P < 0.05 of BDE-209 * diet). The alternation of ATP content was consistent with that of MMP, while no effect of diet on BDE-209 was observed (P > 0.05) (Fig. 2B–b). There was no obvious reduction of hepatic MtDNA copy number from NDC to NDC209 groups (P > 0.05), whereas HFD enabled BDE-209 to depress this parameter (P < 0.05), consistent with the significant interaction effect of HFD and BDE-209 (P < 0.05) (Fig. 2B-c).
JC-1是一种可渗透细胞的阳离子染料，在线粒体中积累，JC-1聚集体（红色荧光）与JC-1单体（绿色荧光）的比率通常用于测量MMP 。 Operetta CLSTM高内涵分析系统拍摄的 MMP 可见照片补充在图 S3中，酶标仪检测到的定量结果列在图 2 B-a 中。如图所示，BDE-209可以降低ND和HFD组的MMP（P < 0.05），但BDE-209和HFD的组合减弱了BDE-209的危害性（BDE-209*饮食的P < 0.05）。 ATP含量的变化与MMP的变化一致，而饮食对BDE-209没有影响（P>0.05）（图2B -b）。从NDC组到NDC209组，肝脏MtDNA拷贝数没有明显减少（P > 0.05），而HFD使BDE-209降低了该参数（P < 0.05），这与HFD和BDE-209的显着交互作用一致（ P < 0.05）（图 2 Bc）。

3.3.3. Expression of proteins and mRNA involving in the mitochondrial apoptosis pathway
3.3.3.线粒体凋亡途径相关蛋白和mRNA的表达

WB strips presented that the protein expression of B-cell lymphoma-2 (Bcl-2) decreased from NDC to HFD209 while the trend of Bcl-2-associated X (Bax) was inverse (Fig. 2C–a). The semiquantitative results (Fig. 2C–b) showed that Bax/Bcl-2 ratio, a more appropriate index corresponding with the onset of mitochondrial apoptosis, was significantly increased by 20%, 142%, and 207% of NDC following the treatment with ND209, HFDC, and HFD209, respectively (P < 0.05). Obviously, the increase of Bax/Bcl-2 ratio from HFDC to HFD209 was more than from NDC to ND209 (P < 0.05 of BDE-209 * diet). As for the gene expression patterns of Bax and Bcl-2, no alteration of Bax/Bcl-2 was observed among four groups (P > 0.05) (Table 1).
WB条带显示B细胞淋巴瘤-2（Bcl-2）的蛋白表达从NDC到HFD209下降，而Bcl-2相关X（Bax）的趋势相反（图2C -a）。半定量结果（图2 C-b）显示，Bax/Bcl-2比值（与线粒体凋亡开始相对应的更合适指标）在治疗后显着增加了NDC的20%、142%和207%分别与 ND209、HFDC 和 HFD209 (P < 0.05)。显然，从 HFDC 到 HFD209，Bax/Bcl-2 比率的增加量大于从 NDC 到 ND209（BDE-209 * 饮食的 P < 0.05）。至于Bax和Bcl-2的基因表达模式，四组中没有观察到Bax/Bcl-2的改变(P％3E 0.05)(表1 )。

Table 1. Effects of BDE-209 on mRNA expression in liver of mice eating ND and HFD.
表 1 . BDE-209 对食用 ND 和 HFD 的小鼠肝脏 mRNA 表达的影响。

Item  物品NDHFDP value  P值
NDCND209HFDCHFD209Diet  饮食BDE-209Interaction  相互作用
Mitochondrial apoptosis  线粒体凋亡
Bax/Bcl-2  巴克斯/Bcl-21.00 ± 0.06 a  1.00±0.06a1.26 ± 0.18 a  1.26±0.18a1.17 ± 0.25 a  1.17±0.25a1.01 ± 0.16 a  1.01±0.16a0.8020.7220.248
caspase-3  胱天蛋白酶-31.02 ± 0.18 b  1.02±0.18b1.10 ± 0.05 b  1.10±0.05b1.26 ± 0.11 b  1.26±0.11b1.85 ± 0.25 a  1.85±0.25a0.0110.0500.117
Endoplasmic reticulum stress
内质网应激
PERK1.00 ± 0.06 c  1.00±0.06℃1.67 ± 0.29 b  1.67±0.29b0.94 ± 0.09 c  0.94±0.09℃2.52 ± 0.07 a  2.52±0.07a0.0320.0000.017
IRE11.01 ± 0.13 c  1.01±0.13℃2.13 ± 0.07 b  2.13±0.07b2.02 ± 0.13 b  2.02±0.13b2.79 ± 0.03 a  2.79±0.03a0.0000.0000.051
ATF61.00 ± 0.03 a  1.00±0.03a0.96 ± 0.16 a  0.96±0.16a0.94 ± 0.10 a  0.94±0.10a0.93 ± 0.11 a  0.93±0.11a0.6490.8280.866
CHOP1.00 ± 0.06 d  1.00±0.06天1.54 ± 0.13 c  1.54±0.13℃4.42 ± 0.45 b  4.42±0.45b6.49 ± 0.60 a  6.49±0.60a0.0000.0100.064
Transfer of Ca2+
Ca 2+的转移
IP3R11.00 ± 0.05 d  1.00±0.05天1.45 ± 0.08 c  1.45±0.08℃3.49 ± 0.11 b  3.49±0.11b4.40 ± 0.19 a  4.40±0.19a0.0000.0000.027
Sig-1R1.00 ± 0.01 a  1.00±0.01a1.64 ± 0.39 a  1.64±0.39a1.54 ± 0.11 a  1.54±0.11a1.81 ± 0.55 a  1.81±0.55a0.2630.1670.543
Endoplasmic reticulum-mitochondrial contact
内质网-线粒体接触
Mfn21.01 ± 0.14 a  1.01±0.14a1.14 ± 0.17 a  1.14±0.17a1.22 ± 0.06 a  1.22±0.06a0.99 ± 0.12 a  0.99±0.12a0.7770.5970.083
PACS-21.01 ± 0.14 b  1.01±0.14b1.17 ± 0.20 b  1.17±0.20b1.76 ± 0.10 a  1.76±0.10a1.89 ± 0.29 a  1.89±0.29a0.0000.2650.878
After normalized to caspase-3, the relative intensity of cleaved caspase-3 (c-caspase-3) was drawn in Fig. 2C–c. As demonstrated, the single effect of BDE-209 was slight (P > 0.05, compared with NDC), but when combining BDE-209 with HFD c-caspase-3 protein expression was appreciably higher than HFDC (P < 0.05), up to 2.11 ± 0.25 times that of NDC. The interaction effect between BDE-209 and diet was significant (P < 0.05). Different from protein expression, the transcription of caspase-3 gene did not change between NDC and ND209 groups (P > 0.05) but increased notably from HFDC to HFD209 groups (P < 0.05) (Table 1).
对 caspase-3 进行归一化后，裂解的 caspase-3 (c-caspase-3) 的相对强度如图 2 C-c 所示。正如所证明的，BDE-209 的单一效应轻微（与 NDC 相比，P > 0.05），但当 BDE-209 与 HFD 联合使用时，c-caspase-3 蛋白表达明显高于 HFDC（P < 0.05），高达NDC的2.11±0.25倍。 BDE-209 与饮食之间的交互作用显着（P < 0.05）。与蛋白表达不同的是，Caspase-3基因的转录在NDC组和ND209组之间没有变化(P>0.05)，但从HFDC组到HFD209组显着增加(P<0.05)(表1 )。

3.4. Over-transfer of Ca2+ from endoplasmic reticulum to mitochondria mediated the hepatocyte apoptosis
3.4. Ca 2+从内质网过度转移至线粒体介导肝细胞凋亡

3.4.1. Expression of proteins and mRNA involving in endoplasmic reticulum stress
3.4.1.内质网应激相关蛋白和mRNA的表达

Fig. 3A presented that BDE-209 significantly activated the protein expression of activating transcription factor 6 (ATF6) in both diets (P < 0.05), reaching 1.38 ± 0.06 times that of NDC in ND209 and increasing from 1.39 ± 0.16 to 1.71 ± 0.20 that times of NDC in mice eating HFD. The alteration of phosphorylated inositol-requiring enzyme 1 (p-IRE1)/IRE1, phosphorylated protein kinase RNA-like ER kinase (p-PERK)/PERK, and C/EBP-homologous protein (CHOP) protein expression was similar. Besides, under the effect of BDE-209 * diet interaction (P < 0.05), a bigger increment of CHOP protein expression induced by BDE-209 in HFD groups than ND groups was achieved (from 1.00 ± 0.17 to 1.69 ± 0.10 in ND vs. from 1.60 ± 0.07 to 2.85 ± 0.18 in HFD). Similarly, the mRNA transcription of CHOP could be increased by both BDE-209 and HFD (P < 0.05), and there was a trend for the interaction of BDE-209 * diet (P = 0.064) (Table 1).
图3A显示，BDE-209显着激活两种日粮中激活转录因子6（ATF6）的蛋白表达（P< 0.05），达到ND209中NDC的1.38±0.06倍，并从1.39±0.16增加到1.71 ± 0.20 是食用 HFD 的小鼠 NDC 的倍数。磷酸化肌醇需要酶 1 (p-IRE1)/IRE1、磷酸化蛋白激酶 RNA 样 ER 激酶 (p-PERK)/PERK 和 C/EBP 同源蛋白 (CHOP) 蛋白表达的变化相似。此外，在 BDE-209 * 饮食相互作用的影响下 (P < 0.05)，HFD 组中 BDE-209 诱导的 CHOP 蛋白表达量比 ND 组有更大的增量（ND 中从 1.00 ± 0.17 增加到 1.69 ± 0.10）对比 HFD 中的 1.60 ± 0.07 至 2.85 ± 0.18）。同样，BDE-209和HFD均可增加CHOP的mRNA转录（P< 0.05），并且BDE-209*饮食有相互作用的趋势（P=0.064）（表1 ）。
Fig. 3
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Fig. 3. Expression of proteins involving in ER stress and the transfer of Ca2+ from ER to mitochondria. Mice were divided into normal diet control group (NDC), NDC +100 mg/kg body weight (BW) of BDE-209 group (ND209), high-fat diet control group (HFDC) and HFDC +100 mg/kg BW of BDE-209 group (HFD209) for 8 weeks. (A) Western bolting strip of p-IRE1, IRE1, p-PERK, PERK, ATF6, CHOP and β-actin (a), and relative protein expression of p-IRE1/IRE1 (b), p-PERK/PERK (c), ATF6 (d) and CHOP (e) in liver tissue; (B) Western bolting strip of IP3R, Sig-1R, GAPDH and β-actin (a), and relative protein expression of IP3R1 (b) and Sig-1R (c) in liver tissue. The data were expressed as the means ± SD. Bars with different letters differed significantly (P < 0.05). Asterisk represented the significant BDE-209 * diet interaction (P < 0.05).
图3 .参与 ER 应激和 Ca 2+从 ER 转移至线粒体的蛋白质表达。将小鼠分为正常饮食对照组(NDC)、BDE-209 NDC+100 mg/kg体重(BW)组(ND209)、高脂饮食对照组(HFDC)和HFDC+100 mg/kg BW组。 BDE-209 组 (HFD209) 持续 8 周。 (A) p-IRE1、IRE1、p-PERK、PERK、ATF6、CHOP 和 β-肌动蛋白 (a) 的 Western 抽苔条带，以及 p-IRE1/IRE1 (b)、p-PERK/PERK ( c)、肝组织中的 ATF6 (d) 和 CHOP (e)； (B) IP3R、Sig-1R、GAPDH 和 β-肌动蛋白 (a) 的 Western 螺栓条，以及肝组织中 IP3R1 (b) 和 Sig-1R (c) 的相对蛋白表达。数据表示为平均值±SD。具有不同字母的条形差异显着 (P < 0.05)。星号代表显着的 BDE-209 * 饮食相互作用 (P < 0.05)。

3.4.2. Expression of proteins and mRNA involving in the transfer of Ca2+ from endoplasmic reticulum to mitochondria
3.4.2.参与Ca 2+从内质网转移到线粒体的蛋白质和mRNA的表达

The abundance of IP3R1 and Sigma-1 receptor (Sig-1R) proteins in ND209 and HFD209 groups was significantly greater than the corresponding control (P < 0.05). Furthermore, BDE-209 and HFD have a positive superimposing effect on promoting the protein expression of IP3R1 (P < 0.05) (Fig. 3B). Changes in gene transcription of IP3R1 was consistent with that in protein expression, while of Sig-1R was not similar due to the no variation of Sig-1R among all four groups (Table 1).
ND209和HFD209组中IP3R1和Sigma-1受体(Sig-1R)蛋白的丰度显着高于相应的对照(P< 0.05)。此外，BDE-209和HFD对促进IP3R1蛋白表达具有正向叠加作用（P<<0.05）（图3B ）。 IP3R1基因转录的变化与蛋白质表达的变化一致，而Sig-1R的基因转录变化与蛋白质表达的变化一致，因为四组之间Sig-1R没有变化(表1 )。

3.4.3. Knockdown of IP3R1 prevented BDE-209-induced mitochondrial Ca2+ overload and apoptosis
3.4.3. IP3R1 的敲低可阻止 BDE-209 诱导的线粒体 Ca 2+过载和细胞凋亡

The high dose of BDE-209, 25 μM, was used to give rise to the apparent apoptosis. As shown in Fig. 4, BDE-209 could cause the highest fluorescence intensity of Rhod-2AM (P < 0.05), as well as the highest percentage of cellular apoptosis (P < 0.05) with the treatment of siNC. IP3R1 is the key component for mediating ER-mitochondria Ca2+ crosstalk. Indeed, the knockdown of IP3R1 adjusted the BDE-209-induced overloaded mitochondrial Ca2+ to the normal level (P > 0.05, compared with the BDE-209 (−) siNC group), accompanied by the obvious reduced apoptotic rate (P < 0.05, compared with BDE-209(+) siNC group).
使用高剂量的 BDE-209（25 μM）引起明显的细胞凋亡。如图4所示，在siNC处理下，BDE-209可以引起Rhod-2AM的最高荧光强度(P<0.05)，以及最高的细胞凋亡百分比(P<0.05)。 IP3R1 是介导 ER-线粒体 Ca 2+串扰的关键组件。事实上，IP3R1的敲低将BDE-209诱导的线粒体Ca 2+过载调整至正常水平（与BDE-209 (−) siNC组相比，P > 0.05），同时细胞凋亡率明显降低（P < 0.05，与BDE-209(+)siNC组相比）。
Fig. 4
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Fig. 4. Knockdown of IP3R1 prevented BDE-209-induced mitochondrial Ca2+ overload and apoptosis. HepG2 Cells treated with 0.32% DMSO (NDC), 25 μM BDE-209 dissolved in 0.32% DMSO (ND209), 0.32% DMSO + sodium oleate/sodium palmitate (500/250 μM) (HFDC) and 25 μM BDE-209 dissolved in 0.32% DMSO + sodium oleate/sodium palmitate (500/250 μM) (HFD209). (A) Mitochondrial Ca2+ labeled by Rhod-2AM probe, took by an inverted fluorescence microscope (a) and Rhod-2AM fluorescence intensity (b); (B) Flow cytometric diagram (a) and the percent of apoptotic cells (b). The data were expressed as the means ± SD. Bars with different letters differed significantly (P < 0.05).
图4 . IP3R1 的敲低可阻止 BDE-209 诱导的线粒体 Ca 2+过载和细胞凋亡。用 0.32% DMSO (NDC)、溶解在 0.32% DMSO (ND209) 中的 25 μM BDE-209、0.32% DMSO + 油酸钠/棕榈酸钠(500/250 μM) (HFDC) 和溶解的 25 μM BDE-209 处理的 HepG2 细胞0.32% DMSO + 油酸钠/钠棕榈酸酯 (500/250 μM) (HFD209)。 (A) Rhod-2AM探针标记的线粒体Ca 2+ ，倒置荧光显微镜拍摄(a)和Rhod-2AM荧光强度(b)； (B) 流式细胞图 (a) 和凋亡细胞百分比(b)。数据表示为平均值±SD。具有不同字母的条形差异显着 (P < 0.05)。

3.4.4. BDE-209 exacerbated Ca2+ transfer in the HFD group
3.4.4. BDE-209 加剧了 HFD 组中的 Ca 2+转移

No cell cytotoxicity was found in HepG2 after treated with SIR or/and 5 μM BDE-209 (Fig. 5A). The results of Oil Red O Staining confirmed the effectiveness of SIR (Fig. 5B). Mag-Fluo-AM probe indicates the Ca2+ content in ER (Xie et al., 2020), while Rhod-2AM probe is widely used to label mitochondrial Ca2+ (Wei et al., 2020; Zhang et al., 2017). Inferred from the conclusion of Che et al. (2020), the increased ratio of Rhod-2AM to Mag-Fluo-AM fluorescence intensity represents the extended transfer content of Ca2+ from ER to mitochondria. As shown, BDE-209 caused a notable increase of Ca2+ transfer in ND209 and HFD209 groups compared with the corresponding control group (P < 0.05). Benefiting from the apparent BDE-209 * diet interaction (P < 0.05), the increment in HFD209 was higher than that in ND209 (Fig. 5C).
用SIR或/和5μM BDE-209处理后，在HepG2中未发现细胞毒性（图5A ）。油红O染色结果证实了SIR的有效性（图5B ）。 Mag-Fluo-AM探针可指示内质网中Ca 2+含量( Xie et al., 2020 )，而Rhod-2AM探针广泛用于标记线粒体Ca 2+ ( Wei et al., 2020 ； Zhang et al., 2017 ）。从Che等人的结论推断。 (2020) ，Rhod-2AM 与 Mag-Fluo-AM 荧光强度之比的增加代表 Ca 2+从内质网转移到线粒体的量增加。如图所示，与相应的对照组相比，BDE-209导致ND209和HFD209组的Ca 2+转移显着增加（P < 0.05）。受益于明显的 BDE-209 * 饮食相互作用（P < 0.05），HFD209 的增量高于 ND209（图 5 C）。
Fig. 5
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Fig. 5. BDE-209 exacerbated Ca2+ transfer in the HFD group. HepG2 Cells treated with 0.0.06% DMSO (NDC), 5 μM BDE-209 dissolved in 0.06% DMSO (ND209), 0.0.06% DMSO + sodium oleate/sodium palmitate (500/250 μM) (HFDC) and 5 μM BDE-209 dissolved in 0.0.06% DMSO + sodium oleate/sodium palmitate (500/250 μM) (HFD209). (A) Cytotoxicity of BDE-209 or/and HFD; (B) Oil Red O staining; (C) Mitochondrial Ca2+ labeled by Rhod-2AM probe and ER Ca2+ labeled by Mag-Fluo-AM probe (a) and Ratio of Rhod-2AM to Mag-Fluo-AM fluorescence intensity (b). The data were expressed as the means ± SD. Bars with different letters differed significantly (P < 0.05). Asterisk represented the significant BDE-209 * diet interaction (P < 0.05). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
图5 。 BDE-209 加剧了 HFD 组中的 Ca 2+转移。用 0.0.06% DMSO (NDC)、溶解在 0.06% DMSO (ND209) 中的 5 μM BDE-209、0.0.06% DMSO + 油酸钠/棕榈酸钠 (500/250 μM) (HFDC) 和 5 μM 处理的 HepG2 细胞BDE-209 溶于 0.0.06% DMSO + 钠油酸/棕榈酸钠 (500/250 μM) (HFD209)。 (A) BDE-209 或/和 HFD 的细胞毒性； (B) 油红O染色； (C) 由Rhod-2AM 探针标记的线粒体Ca 2+和由Mag-Fluo-AM 探针标记的ER Ca 2+ (a) 以及Rhod-2AM 与Mag-Fluo-AM 荧光强度的比率(b)。数据表示为平均值±SD。不同字母的条形差异显着（P < 0.05）。星号代表显着的 BDE-209 * 饮食相互作用 (P < 0.05)。 （为了解释该图例中对颜色的引用，读者可以参考本文的网络版本。）

3.5. HFD helped to increase endoplasmic reticulum-mitochondrial contact
3.5. HFD 有助于增加内质网-线粒体接触

3.5.1. Expression of proteins and mRNA involving in endoplasmic reticulum-mitochondrial contact
3.5.1.参与内质网-线粒体接触的蛋白质和 mRNA 的表达

BDE-209 and HFD did not exert influence on the mitofusin 2 (Mfn2) protein and gene level (P > 0.05). The content of phosphofurin acidic cluster sorting protein 2 (PACS-2) protein increased in the two HFD groups at both the mRNA and protein levels compared with ND groups (P < 0.05), while BDE-209 did not perform any change (Fig. 6A & Table 1).
BDE-209 和 HFD 对线粒体融合蛋白 2 (Mfn2) 蛋白和基因水平没有影响 (P > 0.05)。与ND组相比，两个HFD组的磷酸呋喃酸性簇分选蛋白2（PACS-2）蛋白含量在mRNA和蛋白水平上均有所增加（P<<0.05），而BDE-209则没有任何变化（图1） 。 . 6 A 和表 1 )。
Fig. 6
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Fig. 6. HFD helped to increase ER-mitochondrial contact. Mice were divided into normal diet control group (NDC), NDC +100 mg/kg body weight (BW) of BDE-209 group (ND209), high-fat diet control group (HFDC) and HFDC +100 mg/kg BW of BDE-209 group (HFD209) for 8 weeks. (A) Western bolting strip of Mfn2, PACS-2, and β-actin (a), and relative protein expression of Mfn2 (b) and PACS-2 (c) in liver tissue; (B) Representative TEM images of hepatic tissue, top-2500× magnification, middle-10000× magnification, bottom-illustration of the measure of distance between ER and mitochondria and ER width (a), the number of mitochondria in the 2500× flied (b), the width of ER (c) and the distance between ER and mitochondria (d). The data were expressed as the means ± SD. Bars with different letters differed significantly (P < 0.05). Asterisk represented the significant BDE-209 * diet interaction (P < 0.05).
图6 . HFD 有助于增加内质网与线粒体的接触。将小鼠分为正常饮食对照组(NDC)、BDE-209 NDC+100 mg/kg体重(BW)组(ND209)、高脂饮食对照组(HFDC)和HFDC+100 mg/kg BW组。 BDE-209 组 (HFD209) 持续 8 周。 (A) Mfn2 、PACS-2和β-肌动蛋白(a)的Western抽苔条，以及肝组织中Mfn2(b)和PACS-2(c)的相对蛋白表达； (B) 肝组织的代表性 TEM 图像，顶部 2500× 放大倍数，中间 10000× 放大倍数，底部图示 ER 和线粒体之间的距离以及 ER 宽度的测量值 (a)，2500× 切片中的线粒体数量(b)、ER 宽度 (c) 以及 ER 和线粒体之间的距离 (d)。数据表示为平均值±SD。不同字母的条形差异显着（P < 0.05）。星号代表显着的 BDE-209 * 饮食相互作用 (P < 0.05)。

3.5.2. Transmission electron microscope
3.5.2.透射电子显微镜

There were abundant number of mitochondria (top), normal ER structures (black arrow), neatly arranged mitochondrial cristae (red arrow), intact mitochondrial membrane (blue arrow) and a suitable gap between ER and mitochondria (bottom) in the liver of mice without any treatment. After fed with BDE-209 or/and HFD, there was the obvious status of mitochondrial membrane rupture and disordered arrangement of mitochondrial cristae in the order of NDC, ND209, HFDC and HFD209. Besides, the quantitative analysis showed that BDE-209 and HFD could reduce the number of mitochondria (P < 0.05), and the combined effect of the two was significant (P < 0.05), leading to the minimal mitochondrial amount (Fig. 6B–b). Expanded ER was observed, with the same variance trend as the mitochondria number (Fig. 6B-c). BDE-209 did not change the distance between ER and mitochondria while HFD had an obvious effect on shortening it (P < 0.05) (Fig. 6B–d).
小鼠肝脏中线粒体数量丰富（上），内质网结构正常（黑色箭头），线粒体嵴排列整齐（红色箭头），线粒体膜完整（蓝色箭头），内质网与线粒体之间有合适的间隙（下）无需任何治疗。饲喂BDE-209或/和HFD后，线粒体膜出现明显破裂，线粒体嵴排列紊乱，顺序为NDC、ND209、HFDC、HFD209。此外，定量分析表明，BDE-209和HFD均可减少线粒体数量（P < 0.05），且两者联合作用显着（P < 0.05），导致线粒体数量最小（图 1）。 6 B–b)。观察到扩大的 ER，其方差趋势与线粒体数量相同（图 6 Bc）。 BDE-209没有改变ER和线粒体之间的距离，而HFD有明显缩短距离的作用（P<0.05）（图6B -d）。

4. Discussion  4. 讨论

Emerging evidence has proven that BDE-209 had potential hepatotoxicity through altering tissue morphology, disturbing glycolipid metabolism, inducing oxidative stress and inflammation, and so on ( Sun et al., 2020; Zhu et al., 2021), while no research reported the obese-induced reinforced effect which was theoretically existent when considering the lipophilicity of BDE-209, leading to the underestimated hepatotoxicity of BDE-209. Not surprisingly, our results testified that there was a significant interaction of HFD and BDE-209 on elevating serum AST and ALT levels (Fig. 1B), and the liver swelled in the HFD209 group (Fig. 1A), implying the more serious liver dysfunction in the obese subjects. Specifically, lipid degeneration and inflammation occurred in the liver, reflected by the increasing hepatic TG (Fig. 1C) and inflammatory factors contents (Fig. 1E&F) as well as the HE staining (Fig. 1G). However, no alteration of glycometabolism of BDE-209 was detected (Fig. 1D), different from the result of Zhu et al. (2021), which might be caused by differences in mouse species, BDE-209 dosage, raising conditions, etc.
新证据证明BDE-209通过改变组织形态、扰乱糖脂代谢、诱导氧化应激和炎症等具有潜在的肝毒性( Sun et al., 2020 ; Zhu et al., 2021 )，但尚无研究报道。考虑到 BDE-209 的亲脂性，理论上存在肥胖诱导的强化效应，导致低估了 BDE-209 的肝毒性BDE-209。毫不奇怪，我们的结果证明，HFD 和 BDE-209 在升高血清 AST和ALT 水平方面存在显着的相互作用（图 1B ），并且 HFD209 组的肝脏肿胀（图 1A），这意味着 HFD209 组的肝脏肿胀（图 1A ），这意味着肥胖者有严重的肝功能障碍。具体来说，肝脏发生了脂质变性和炎症，这通过肝脏TG（图1C ）和炎症因子含量（图1E &F）以及HE染色（图1G ）的增加反映出来。然而，与Zhu等人的结果不同，没有检测到BDE-209的糖代谢发生改变（图1D ）。 (2021) ，这可能是由于小鼠种类、BDE-209剂量、饲养条件等差异造成的。
Apoptosis is a form of physiological cell death, important in controlling the normal cell renewal, proliferation, and regeneration of liver tissue under physiological conditions. However, dysregulated or excessive apoptosis results in severe liver failure (Guicciardi & Gores, 2005). Unfortunately, the tunnel staining suggested that BDE-209 aggravated hepatocyte apoptosis (Fig. 2A). There are three pathways well recognized to regulate the cell apoptosis, that is, the extrinsic death receptor pathway and the intrinsic mitochondrial pathway as well as the perforin/granzyme pathway (Elmore, 2007). Here, the mitochondrial-dependent intrinsic apoptosis induced by ER stress, where the transfer of Ca2+ signal plays a key role (Pinton et al., 2008), was demonstrated and discussed in detail below.
细胞凋亡是生理性细胞死亡的一种形式，对于控制生理条件下肝组织的正常细胞更新、增殖和再生非常重要。然而，失调或过度的细胞凋亡会导致严重的肝衰竭（ Guicciardi & Gores，2005 ）。不幸的是，隧道染色表明BDE-209加剧了肝细胞凋亡（图2A ）。公认的调节细胞凋亡的途径有三种，即外源性死亡受体途径、内源性线粒体途径以及穿孔素/颗粒酶途径( Elmore，2007 )。在这里，由 ER 应激诱导的线粒体依赖性内在细胞凋亡，其中 Ca 2+信号的传递起着关键作用（ Pinton 等人，2008 ），在下面进行了详细论证和讨论。
The ER is the key Ca2+ storage and release system. During the chronic ER stress, three UPR pathways of PERK, IRE1 and, ATF6 would be partially or fully persistently activated, which apparently happened in the present research (Fig. 3A–a, b, c&d). Going through different signal molecules, the ends of these three pathways converge at CHOP (Malhotra & Kaufman, 2011). As a result, the expression of CHOP protein and gene was significantly higher in BDE-209-treated mice than in normal mice (Fig. 3A-a&e and Table 1). Next, CHOP opened the calcium channel built by IP3R1, supported by the incremental IP3R1 protein and gene level (Fig. 3B-a&b) and the discovery by Li et al. (2009). Besides, the increased expression of Sig-1R could chaperone and stabilize IP3R1 to prolong Ca2+ release from ER (Fig. 3B- a&c) (Hayashi & Su, 2007).
ER 是关键的 Ca 2+储存和释放系统。在慢性 ER 应激期间，PERK、IRE1 和ATF6三个 UPR 通路将部分或完全持续激活，这在本研究中显然发生了（图 3 A-a、b、c&d）。经过不同的信号分子，这三个途径的末端在 CHOP 处汇聚（ Malhotra & Kaufman，2011 ）。结果，BDE-209处理的小鼠中CHOP蛋白和基因的表达显着高于正常小鼠(图3Aa &e和表1 )。接下来，CHOP 打开了 IP3R1 构建的钙通道，这得到了增量 IP3R1 蛋白和基因水平的支持（图 3 Ba&b）以及Li 等人的发现。 （2009） 。此外，Sig-1R表达的增加可以陪伴和稳定IP3R1以延长Ca 2+从ER的释放（图3B -a&c）（ Hayashi & Su, 2007 ）。
The mitochondria are the primary effectors of Ca2+ absorption. The mitochondrial-mediated internal apoptosis pathway is controlled by the Bcl-2 protein family, regulated by mitochondria, and ultimately executed by c-caspase-3 (Elmore, 2007), where the Bax and Bcl-2 were associated with the manipulation of Ca2+ transfer from the ER to mitochondria (Marchi et al., 2018). In generally, in ER, Bax binds with IP3R1 to promote ER-Ca2+ release while Bcl-2 reduces IP3R channel activity, and in mitochondria, Bax binds the voltage-dependent anion channel (VDAC), the entry of Ca2+ into mitochondria, to keep the channel sustained open while bcl-2 could close this channel (Lewis et al., 2014; Morris et al., 2021). Thus, western blotting analysis showed that the Bax/Bcl-2 protein expression was promoted by BDE-209 (Fig. 2C-a&b), resulting in the mitochondrial Ca2+ overload. Indeed, cell experiments clearly presented the extended Ca2+ shift from the ER to the mitochondria in HepG2 cells treated with BDE-209 (Fig. 5C). Next, the redundant Ca2+ elicited mitochondrial dysfunction (Toledo et al., 2014), reflected by the impairment of MMP, MtDNA copy number, and ATP content (Fig. 2B). Consistent with the drop of MMP and MtDNA copy number, TEM technology clearly presented the broken mitochondrial membrane and fewer mitochondria numbers in the liver of mice eating BDE-209 (Fig. 6B-a&b). Specially, the decline of MMP has been solidly proven to involve in mitochondrial-mediated apoptosis (Liu et al., 2020). Finally, the increment of c-caspase-3/caspase-3 protein expression initiated the apoptosis program (Fig. 2C-a&c). Similarly, the mitochondrial-dependent pathway played a role in the apoptosis induced by other BDE congeners, such as BDE-47, BDE-99, and BDE-100 (Chen et al., 2020; Pereira et al., 2013; Souza et al., 2013).
线粒体是Ca 2+吸收的主要效应器。线粒体介导的内部凋亡途径由 Bcl-2 蛋白家族控制，受线粒体调节，最终由 c-caspase-3 执行（ Elmore，2007 ），其中Bax和 Bcl-2 与 Ca 的操纵有关。 2+从 ER 转移至线粒体（ Marchi 等人，2018 ）。一般来说，在 ER 中，Bax 与 IP3R1 结合，促进 ER-Ca 2+释放，而 Bcl-2 降低 IP3R 通道活性，在线粒体中，Bax 与电压依赖性阴离子通道 (VDAC) 结合，Ca 2+进入线粒体，以保持通道持续开放，而 bcl-2 可以关闭该通道（ Lewis et al., 2014 ； Morris et al., 2021 ）。因此，蛋白质印迹分析表明BDE-209促进Bax/Bcl-2蛋白表达（图2Ca &b），导致线粒体Ca 2+过载。事实上，细胞实验清楚地表明，在用 BDE-209 处理的 HepG2 细胞中，Ca 2+从内质网转移到线粒体的时间延长了（图 5 C）。接下来，多余的Ca 2+引起线粒体功能障碍( Toledo et al., 2014 )，反映为MMP、MtDNA拷贝数和ATP含量的受损(图2B )。 与 MMP 和 MtDNA 拷贝数下降一致，TEM 技术清楚地显示了食用 BDE-209 的小鼠肝脏中线粒体膜破裂和线粒体数量减少（图 6 Ba&b）。特别是，MMP 的下降已被证实与线粒体介导的细胞凋亡有关（ Liu et al., 2020 ）。最后，c-caspase-3/caspase-3蛋白表达的增加启动了细胞凋亡程序（图2 Ca&c）。同样，线粒体依赖性途径在其他 BDE 同系物（例如 BDE-47、BDE-99 和 BDE-100）诱导的细胞凋亡中也发挥了作用（ Chen 等，2020 ； Pereira 等，2013 ； Souza 等）等，2013 ）。
As discussed above, it was believed that the flow of Ca2+ carried the apoptotic signal from ER to mitochondria. Besides, IP3R1 accompanied by Sig-1R was a major Ca2+ release channel, which has opened in the BDE-209 treated groups, contributing to the increased mitochondrial Ca2+ level. Thus, to solidly confirm the role of Ca2+ in BDE-209 induced apoptosis, the crucial gene of IP3R1 was knocked down using siRNA technology. Fortunately, the knockdown counteracted mitochondrial Ca2+ overload induced by BDE-209 treatment (Fig. 4A), in line with a previous study (Zhao et al., 2017). Further, the ameliorate of apoptosis (Fig. 4B) appearing at the same time illustrated that BDE-209-induced apoptosis was dependent of mitochondrial Ca2+. Such result has also been reported by other research where the depletion of GRP75, the bright of IP3R and VDAC1, provided protection against glutamate and superparamagnetic iron oxide nanoparticles-induced toxicity by reducing mitochondrial Ca2+ (Che et al., 2020; Honrath et al., 2017).
如上所述，据信Ca 2+的流动将细胞凋亡信号从ER携带至线粒体。此外，IP3R1 与 Sig-1R 一起是主要的 Ca 2+释放通道，该通道在 BDE-209 处理组中打开，导致线粒体 Ca 2+水平增加。因此，为了切实证实Ca 2+在BDE-209诱导的细胞凋亡中的作用，使用siRNA技术敲低了IP3R1的关键基因。幸运的是，敲低抵消了 BDE-209 治疗引起的线粒体 Ca 2+超载（图 4 A），与之前的研究一致（ Zhao 等，2017 ）。此外，同时出现的细胞凋亡的改善(图4B )说明BDE-209诱导的细胞凋亡依赖于线粒体Ca 2+ 。其他研究也报道了这样的结果，其中 GRP75 的消耗、IP3R 和VDAC1的明亮，通过减少线粒体Ca 2+提供针对谷氨酸和超顺磁性氧化铁纳米粒子诱导的毒性的保护（ Che 等人，2020 ； Honrath 等人）等，2017 ）。
Identically, there was a notable interaction effect between BDE-209 and HFD on the induction of hepatocyte apoptosis via the pathway of ER/Ca2+/mitochondria. The climb of apoptosis rate was steeper from HFDC to HFD209 than from NDC to ND209 (Fig. 2A), in parallel with the decline of the number of mitochondria (Fig. 2B–c, Fig. 6B-a&b), the enhancive protein expression of Bax/Bcl-2 (Fig. 2C-a&b), c-caspase-3/caspase-3 (Fig. 2C-a&c), CHOP (Fig. 3A-a&e) and IP3R1 (Fig. 3B-a&b), and the increasing transfer of Ca2+ from the ER to the mitochondria (Fig. 5C-a&b). However, learning from the phenomenon that the amplification of apoptosis rate from HFDC to HFD209 even exceeded the increasing range in ND-fed mice eating from 100 to 500 mg/kg BW BDE-209 (Figure S3), the simple increase of BDE-209 content by HFD cannot fully explain such a high level of apoptosis in HFD209. Previous researchers have found that the ER and mitochondria interacted both physiologically and functionally via mitochondria-associated ER membranes (MAM), which is important for the flow of Ca2+ (Marchi et al., 2017; Rizzuto et al., 1998). MAM contains several proteins such as IP3R1, Sig-1R, Mfn-2 and PACS-2, and the latter two control the apposition of mitochondria with the ER (Zhao et al., 2017). Further, the effect of HFD on MAM has been explored by Zhao et al. (2017) and Arruda et al. (2014), drawing the conclusion that the enrichment of MAM via PACS-2 by HFD could accelerate Ca2+ flux into mitochondria, resulting in mitochondrial dysfunction and apoptosis. Similarly, the higher PACS-2 protein and gene expression in the HFD groups than ND groups was detected in the current study (Fig. 6A-a&c). Besides, the induced ER stress activated the UPR (discussed above), which determined ER volume expansion (Fig. 6B-a&c) with consequent increscent in the ER-mitochondria interaction. Accordingly, there was the closer distance between the two displayed in the TEM images in the HFD groups (Fig. 6B-a&d). As a result, it was speculated that HFD exacerbated BDE-209-induced hepatocyte apoptosis through providing a shortcut for the rapid movement of Ca2+. In fact, it is not the first time that HFD provided convenience for poisons to exert their toxicity. Yang et al. (2019) found that BDE47-induced liver fibrosis was tightly associated with oxidative stress, so the hepatic destruction was more serious in the obese mice whose oxidation level was uplifted by HFD.
同样，BDE-209 和 HFD 之间通过 ER/Ca 2+ /线粒体途径诱导肝细胞凋亡存在显着的相互作用。与从 NDC 到 ND209 相比，从 HFDC 到 HFD209 的细胞凋亡率上升幅度更大（图 2 A），同时增强蛋白线粒体数量的下降（图 2 B-c，图 6 Ba&b）。 Bax/Bcl-2（图 2 Ca&b）、c-caspase-3/caspase-3（图 2 Ca&c）、CHOP（图 2）的表达3 Aa&e）和 IP3R1（图 3 Ba&b），以及 Ca 2+从 ER 向线粒体转移的增加（图 5 Ca&b）。然而，从HFDC到HFD209的细胞凋亡率放大甚至超过了喂食100至500mg/kg BW BDE-209的ND喂养小鼠的增加范围的现象得知（图S3 ），BDE-209的简单增加HFD 含量不能完全解释 HFD209 中如此高水平的细胞凋亡。先前的研究人员发现，内质网和线粒体通过线粒体相关内质网膜（MAM）在生理和功能上相互作用，这对于Ca 2+的流动很重要（ Marchi 等，2017 ； Rizzuto 等，1998 ）。 MAM含有IP3R1、Sig-1R、Mfn-2和PACS-2等多种蛋白，后两者控制线粒体与ER的并置( Zhao et al., 2017 )。此外， Zhao 等人还探讨了 HFD 对 MAM 的影响。 （2017）和阿鲁达等人。 (2014) ，得出的结论是HFD通过PACS-2富集MAM可以加速Ca 2+流入线粒体，导致线粒体功能障碍和细胞凋亡。同样，在本研究中检测到 HFD 组中的 PACS-2 蛋白和基因表达高于 ND 组（图 6 Aa&c）。此外，诱导的内质网应激激活了UPR（如上所述），这决定了内质网体积膨胀（图6 Ba和c），从而增加了内质网-线粒体相互作用。因此，HFD 组的 TEM 图像中显示的两者之间的距离更近（图 6 Ba&d）。因此，推测HFD通过为Ca 2+的快速移动提供捷径而加剧BDE-209诱导的肝细胞凋亡。事实上，HFD为毒物发挥毒性提供了便利，这并不是第一次。杨等人。 （2019）发现BDE47诱导的肝纤维化与氧化应激密切相关，因此HFD升高氧化水平的肥胖小鼠的肝脏破坏更为严重。
Here, a high dose of BDE-209, 100 mg/kg bw, was applied. The main consideration was that the primary aim of our research was to explore the underlying mechanism of the hepatotoxicity induced by BDE-209, so a dose proven to be toxic to the liver was preferred (Sun et al., 2020; Zhu et al., 2021). The high exposure to BDE-209 is, however, the situation in real life. Workers at electronic waste recycling facility and brominated flame retardants production enterprise are occupational exposure to BDE-209. As reported, the median of BDE-209 concentration in the blood of general population worldwide was 2.81 ng/g lipid weight (lw), while in occupational population, the mean concentration reached up to be 66.8 ng/g lw (more than 20 times) and the highest value was even to be 521 ng/g lw (more than 180 times) (Meng et al., 2021), suggesting a higher health risk. Wang et al. (2019) estimated that the daily intake level of BDE-209 for manufacturing workers was in the range of 0.67–18.7 μg/kg bw/day. Thus, 100 mg/kg bw was also equal to 18.7 * dose conversion coefficients (12) * 100-fold uncertainty factor * 5-fold amplification factor, and these results provided a theoretical foundation for assessing the risk the high exposure groups suffering.
这里使用了高剂量的 BDE-209，即 100 毫克/千克体重。主要考虑是我们研究的主要目的是探索BDE-209引起肝毒性的潜在机制，因此优先选择已证明对肝脏有毒的剂量（ Sun et al., 2020 ； Zhu et al., 2020）。 ，2021 ）。然而，现实生活中却存在大量接触 BDE-209 的情况。电子废物回收设施和溴化阻燃剂生产企业的工人是BDE-209的职业接触者。据报道，全球普通人群血液中BDE-209浓度的中位数为2.81纳克/克脂重（lw），而职业人群的平均浓度高达66.8纳克/克脂重（lw）（是人体血液中BDE-209浓度的20倍以上）。 ），最高值甚至达到521 ng/g lw（超过180倍）（ Meng et al., 2021 ），表明健康风险较高。王等人。 (2019)估计制造业工人每日 BDE-209 摄入量在 0.67–18.7 微克/公斤体重/天之间。因此，100mg/kg体重也等于18.7*剂量换算系数(12)*100倍不确定因子*5倍放大因子，这些结果为评估高暴露人群遭受的风险提供了理论基础。
In addition to high levels of exposure, the biotransformation of BDE-209 into the debrominated, hydroxylated (OH), and methoxylated (MeO) metabolites might also magnify the damage of BDE-209, because these metabolites have the greater toxicity than the parent compound (Mi et al., 2017). Learning from these researches (Huwe & Smith, 2007; Kortenkamp et al., 2013; Zhang et al., 2011), there were many metabolites predictably existing in mice treated with BDE-209 for 8 weeks, mainly including nona-BDEs (BDE-208, BDE-207, BDE-206) and octa-BDEs (BDE-201, BDE-197, BDE-196), although the measure has not really been finished in the present study. DE-79 is an octa-BDE mixture (Alvarez-Gonzalez et al., 2020), and Zhou et al. (2001) have found that the liver wight/dw was larger in the rats fed with 10–100 mg/kg bw DE-79 for 4 days than those with DE-83R containing 98% deca-BDE. Recently, whether mammals could metabolize BDE-209 to OH- and MeO-metabolites is controversial. However, it is confirmed that fishes have such capacity, where MeO-BDE-100, MeO-BDE-68, MeO-BDE-47, OH-BDE-42 and OH-BDE-28 were detected in rainbow trout dosed by intraperitoneal injection for 22-day exposure (Feng et al., 2015). Through food chain, these toxic metabolites would no doubt appear in the human finally (Fujii et al., 2014). Although few studies have involved the liver toxicity, the more severe thyroid disruption was found after the treatment of OH-metabolites due to their higher affinity to thyroid hormone transport protein than the corresponding PBDEs (Hamers et al., 2008). Taking the prevalence of BDE-209 into consideration together, the toxicological rank of BDE-209 is on top of all PBDEs, as a result (Chain, 2011; Tait et al., 2017). Therefore, the process of thoroughly banning the use of BDE-209 needs to be accelerated.
除了高水平暴露外，BDE-209生物转化为脱溴、羟基化 (OH) 和甲氧基化 (MeO) 代谢物也可能放大 BDE-209 的损害，因为这些代谢物比母体化合物具有更大的毒性（米等人，2017 ）。从这些研究中（ Huwe & Smith, 2007 ； Kortenkamp et al., 2013 ； Zhang et al., 2011 ）可知，在用 BDE-209 治疗 8 周的小鼠体内，预计存在多种代谢物，主要包括九溴二苯醚 (BDE) -208、BDE-207、BDE-206）和八-BDE（BDE-201、 BDE-197、BDE-196），尽管该措施在本研究中尚未真正完成。 DE-79 是一种八溴二苯醚混合物（ Alvarez-Gonzalez 等人，2020 ）， Zhou 等人。 (2001)发现，饲喂 10-100 mg/kg bw DE-79 4 天的大鼠的肝脏重量/干重比饲喂含有 98% 十溴二苯醚的 DE-83R 的大鼠更大。最近，哺乳动物是否可以将 BDE-209 代谢为 OH 和 MeO 代谢物存在争议。然而，已证实鱼类具有这种能力，在腹腔注射的虹鳟鱼中检测到MeO-BDE-100、MeO-BDE-68、MeO-BDE-47、OH-BDE-42和OH-BDE-28 22 天暴露（ Feng et al., 2015 ）。通过食物链，这些有毒代谢物最终无疑会出现在人体中（ Fujii et al., 2014）。，2014 ）。尽管很少有研究涉及肝脏毒性，但由于 OH 代谢物与甲状腺激素转运蛋白的亲和力比相应的PBDE更高，因此在治疗后发现更严重的甲状腺破坏（ Hamers 等，2008 ）。综合考虑 BDE-209 的流行程度，BDE-209 的毒理学排名位居所有 PBDE 之首（ Chain，2011 ； Tait 等，2017 ）。因此，彻底禁止使用BDE-209的进程需要加快。

5. Conclusion  5. 结论

In summary, the BDE-209-induced hepatic dysfunction and hepatocyte apoptosis could be exacerbated by HFD. The pro-apoptotic mechanism of BDE-209 was dependent on the ER-mitochondrial intrinsic pathway. Based on the role of Ca2+ flux in the conduction of apoptotic signals from the ER to mitochondria, the acceleration of Ca2+ flow by HFD via enriched ER-mitochondria interaction explained the promotion of HFD on BDE-209 hepatotoxicity. To the best of our knowledge, this research was the first to shed light on the hepatotoxicity of BDE-209 in the obese model and comprehensively demonstrate the underlying mechanism. It could be expected that these results provided the theoretical support for re-evaluating the toxicity of PBDEs and their analogs facing the worldwide epidemic of obesity.
总之，HFD 可能会加剧 BDE-209 诱导的肝功能障碍和肝细胞凋亡。 BDE-209 的促凋亡机制依赖于 ER 线粒体内在途径。基于Ca 2+流在​​细胞凋亡信号从内质网传导至线粒体中的作用，HFD 通过富集内质网-线粒体相互作用加速 Ca 2+流解释了 HFD 对 BDE-209 肝毒性的促进作用。据我们所知，这项研究首次揭示了 BDE-209 在肥胖模型中的肝毒性，并全面论证了其潜在机制。可以预期，这些结果为重新评估PBDEs及其类似物面对全球肥胖流行的毒性提供了理论支持。

Author statement  作者声明

Sunni Chen: Conceptualization, Investigation, Data curation, Writing – original draft preparation. Siyan Che: Investigation. Shiqi Li: Investigation. Jin Wan: Writing – review & editing. Zheng Ruan: Conceptualization, Supervision.
Sunni Chen：概念化、调查、数据管理、写作——原稿准备。车思彦：调查。李世奇：调查。金万：写作——审稿和编辑。阮郑：构思、监督。

Declaration of competing interest
竞争利益声明

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
作者声明，他们没有已知的可能影响本文报告工作的相互竞争的经济利益或个人关系。

Acknowledgments  致谢

The project was financially supported by the National Key R&D Program of China (2017YFC1600302), the Key R&D Program of Jiangxi Province of China (20192ACB60006) and the Key R&D Program of Nanchang Metropolis of China (2019-NCZDSY-002).
该项目得到国家重点研发计划（ 2017YFC1600302 ）、江西省重点研发计划（ 20192ACB60006 ）和南昌市重点研发计划（ 2019-NCZDSY-002 ）的资助。

Appendix A. Supplementary data
附录 A 补充数据

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Multimedia component 1.


以下是本文的补充数据：下载：下载Word文档(10MB)

多媒体组件 1 ．

Abbreviations  缩写

    (BDE-209)
    decabromodiphenyl ether  十溴二苯醚
    (PBDE)  (多溴联苯醚)
    polybrominated diphenyl ether
    多溴二苯醚
    (HFD)  （高频FD）
    high-fat diet  高脂肪饮食
    (ND)  （ND）
    normal diet  正常饮食
    (ER)  （急诊室）
    endoplasmic reticulum  内质网
    UPR  普遍定期审议
    unfold protein response  展开蛋白反应
    (TEM)  （透射电镜）
    transmission electron microscope
    透射电子显微镜
    (TG)
    triacylglycerol  三酰甘油
    (TC)  （TC）
    total cholesterol  总胆固醇
    (AST)  (谷草转氨酶)
    aspartate transaminase  天冬氨酸转氨酶
    (ALT)  （丙氨酸）
    alanine transaminase  丙氨酸转氨酶
    (TNF-α)  (肿瘤坏死因子-α)
    tumor necrosis factor-α  肿瘤坏死因子-α
    (IL-1)
    interleukin 1  白细胞介素1
    (IL-4)
    interleukin 4  白细胞介素4
    (IL-6)
    interleukin 6  白细胞介素6
    (IL-10)
    interleukin 10  白细胞介素10
    (DMSO)  (二甲基亚砜)
    dimethyl sulfoxide  二甲亚砜
    (NDC)  （国家数据中心）
    normal diet control group
    正常饮食对照组
    (ND209)
    NDC +100 mg/kg body weight (BW) of BDE-209 group
    BDE-209 组的 NDC +100 mg/kg 体重 (BW)
    (HFDC)  （高频直流）
    high-fat diet control group
    高脂饮食对照组
    (HFD209)
    HFDC +100 mg/kg BW of BDE-209 group
    HFDC + 100 mg/kg BW BDE-209 组
    (BW)  （体重）
    body weight  体重
    (PBS)  （公共广播公司）
    phosphate buffered saline
    磷酸盐缓冲盐水
    (MMP)  （基质金属蛋白酶）
    mitochondrial membrane potential
    线粒体膜电位
    (MtDNA)  （线粒体DNA）
    mitochondrial DNA  线粒体DNA
    (qRT-PCR)
    quantitative real-time polymerase chain reaction
    实时定量聚合酶链反应
    (GAPDH)
    glyceraldehyde 3 phosphoric acid dehydrogenase
    甘油醛3磷酸脱氢酶
    (HE)  （他）
    hematoxylin and eosin  苏木精和伊红
    (DMEM)
    Dulbecco's modified Eagle's medium
    杜尔贝科改进的伊格尔培养基
    (FBS)  （胎牛血清）
    fetal bovine serum  胎牛血清
    (DMSO)  (二甲基亚砜)
    dimethyl sulfoxide  二甲亚砜
    (SIR)  （先生）
    steatosis-induced reagent
    脂肪变性诱导试剂
    (IP3R1)
    inositol 1,4,5-trisphosphate receptor 1
    肌醇1,4,5-三磷酸受体1
    (NC)
    negative control  阴性对照
    (Bcl-2)
    B-cell lymphoma-2  B细胞淋巴瘤2
    (Bax)  （巴克斯）
    Bcl-2-associated X  Bcl-2相关X
    (c-caspase-3)
    cleaved caspase-3  裂解的 caspase-3
    (p-IRE1)
    phosphorylated inositol-requiring enzyme 1
    磷酸化肌醇需要酶1
    (p-PERK)
    phosphorylated protein kinase RNA-like ER kinase
    磷酸化蛋白激酶 RNA 样 ER 激酶
    (CHOP)  （劈）
    C/EBP-homologous protein  C/EBP-同源蛋白
    (Sig-1R)
    sigma-1 receptor  西格玛-1受体
    (Mfn2)  （最惠国2）
    mitofusin 2  丝裂霉素2
    (PACS-2)
    phosphofurin acidic cluster sorting protein 2
    磷酸呋喃酸性簇分选蛋白2
    (VDAC)  （VDAC）
    voltage-dependent anion channel
    电压依赖性阴离子通道
    (MAM)  (美安美)
    mitochondria-associated ER membranes
    线粒体相关内质网膜
    (OH)  （哦）
    hydroxylated  羟基化的
    (MeO)  (甲醇)
    methoxylated  甲氧基化
    (lw)  （长）
    lipid weight  脂质重量

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