Ethanol-Induced Hepatic Ferroptosis Is Mediated by PERK-Dependent MAMs Formation: Preventive Role of Quercetin
乙醇诱导的肝铁死亡是由 PERK 依赖性 MAM 形成介导的：槲皮素的预防作用
首次发布： 2024 年 3 月 19 日
Abstract 抽象的
Scope 范围
Iron deposition is frequently observed in alcoholic liver disease (ALD), which indicates a potential role of ferroptosis in its development. This study aims to explore the effects of quercetin on ferroptosis in ALD and elucidates the underlying mechanism involving the formation of mitochondria-associated endoplasmic reticulum membranes (MAMs) mediated by protein kinase RNA-like endoplasmic reticulum kinase (PERK).
在酒精性肝病（ALD）中经常观察到铁沉积，这表明铁死亡在其发展中具有潜在作用。本研究旨在探讨槲皮素对 ALD 中铁死亡的影响，并阐明蛋白激酶 RNA 样内质网激酶 (PERK) 介导的线粒体相关内质网膜 (MAM) 形成的潜在机制。
Methods and results 方法和结果
C57BL/6J mice are fed either a regular or an ethanol-containing liquid diet (with 28% energy form ethanol) with or without quercetin supplementation (100 mg kg−1 BW) for 12 weeks. Ethanol feeding or treatment induced ferroptosis in mice and AML12 cells, which is associated with increased MAMs formation and PERK expression within MAMs. Quercetin attenuates these changes and protects against ethanol-induced liver injury. The antiferroptotic effect of quercetin is abolished by ferroptosis inducers, but mimicked by ferroptosis inhibitors and PERK knockdown. The study demonstrates that PERK structure, rather than its kinase activity (transfected with the K618A site mutation that inhibits kinase activity-ΔK plasmid or protein C terminal knockout-ΔC plasmid of PERK), mediates the enhanced MAMs formation and ferroptosis during the ethanol exposure.
C57BL/6J小鼠喂食常规饮食或含乙醇液体饮食（28%能量来自乙醇），添加或不添加槲皮素（100 mg kg -1 BW），持续12周。乙醇喂养或治疗会诱导小鼠和 AML12 细胞铁死亡，这与 MAM 形成和 MAM 内 PERK 表达增加有关。槲皮素可以减轻这些变化并防止乙醇引起的肝损伤。槲皮素的抗铁死亡作用被铁死亡诱导剂消除，但被铁死亡抑制剂和 PERK 敲低所模仿。研究表明，在乙醇暴露期间，PERK 结构而不是其激酶活性（用抑制激酶活性的 K618A 位点突变 - ΔK 质粒或蛋白 C 末端敲除 - PERK 的 ΔC 质粒转染）介导了增强的 MAM 形成和铁死亡。
Conclusion 结论
Quercetin ameliorates ethanol-induced liver injury by inhibiting ferroptosis via modulating PERK-dependent MAMs formation.
槲皮素通过调节 PERK 依赖性 MAM 的形成来抑制铁死亡，从而改善乙醇引起的肝损伤。
1 Introduction 1 简介
Liver disease is a global health problem that causes about two million deaths annually and is on the rise.[1] The prevalence of alcoholic liver disease (ALD) is the most common type of liver diseases in Europe and the United States.[2] Research shows that almost all heavy drinkers develop fatty liver; about one-third of them develop hepatitis and fibrosis; 10–20% progress to cirrhosis; and 3–10% develop hepatocellular carcinoma.[3] While the health effects of moderate drinking are still debated, the health and social harms of alcohol abuse are undeniable. Therefore, exploring the mechanisms of ALD and implementing feasible early intervention strategies are of paramount importance.
肝病是一个全球性健康问题，每年导致约 200 万人死亡，而且死亡人数还在增加。 1酒精性肝病 (ALD) 的患病率是欧洲和美国最常见的肝脏疾病类型。 2研究表明，几乎所有酗酒者都会患上脂肪肝；其中约三分之一出现肝炎和纤维化； 10-20%进展为肝硬化； 3-10% 发展为肝细胞癌。 3虽然适量饮酒对健康的影响仍有争议，但酗酒对健康和社会的危害是不可否认的。因此，探索ALD的发病机制并实施可行的早期干预策略至关重要。
Ferroptosis, a newly identified iron-dependent and lipid peroxidation-mediated cell death mode.[4] Iron functions as an essential cofactor of cytochrome P450 oxidoreductase and arachidonic acid lipoxygenase to initiate the Fenton reaction and promote lipid peroxidation. Ferroptosis occurs when lipid peroxidation overwhelms the antioxidant buffering capacity of the ferroptosis defense system.[5] ALD is frequently associated with iron overload,[6] high labile iron pool, disrupted lipid metabolism and peroxidation,[7-9] depleted hepatic glutathione (GSH) levels, and inactivated various antioxidative enzymes, including glutathione peroxidase 4 (Gpx4), which suggests that hepatocytes are more susceptible to ferroptosis due to alcohol exposure.[10, 11] A growing body of research has demonstrated that ferroptosis is linked to various liver diseases, including ALD, nonalcoholic steatohepatitis, hepatitis C virus, and hepatocellular carcinoma.[12, 13] One study found that the ALD model constructed by the 10-day alcohol binge showed the occurrence of ferroptosis.[14] However, the precise molecular mechanisms and signaling pathways involved in the development of ferroptosis in ALD are yet to be elucidated.
铁死亡，一种新发现的铁依赖性和脂质过氧化介导的细胞死亡模式。 4铁作为细胞色素 P450 氧化还原酶和花生四烯酸脂氧合酶的重要辅助因子，启动芬顿反应并促进脂质过氧化。当脂质过氧化压倒铁死亡防御系统的抗氧化缓冲能力时，就会发生铁死亡。 5 ALD 通常与铁超载有关， 6高不稳定铁池，脂质代谢和过氧化破坏， 7 - 9肝谷胱甘肽 (GSH) 水平耗尽，以及各种抗氧化酶失活，包括谷胱甘肽过氧化物酶 4 (Gpx4)，这表明肝细胞由于酒精暴露，更容易出现铁死亡。 10 , 11越来越多的研究表明，铁死亡与多种肝脏疾病有关，包括 ALD、非酒精性脂肪性肝炎、丙型肝炎病毒和肝细胞癌。 12 , 13一项研究发现，连续10天酗酒构建的ALD模型出现了铁死亡的发生。 14然而，ALD 中铁死亡发生的确切分子机制和信号通路尚未阐明。
Mitochondria-associated endoplasmic reticulum membranes (MAMs) are structural/functional tethers between mitochondria and the endoplasmic reticulum (ER) that play multifunctional roles in various biological pathways, including Ca2+ homeostasis regulation, lipid metabolism, and mitochondrial dynamics.[15-17] The close contact between these two important organelles serves as an essential hub for direct and rapid signal transduction and crosstalk in response to various physiological and pathological stresses. Both organelles are vulnerable to iron overload, ferroptosis, and Fenton-activated labile iron.[18] MAMs are critical for synthesizing and intracellularly transporting phospholipids such as phosphatidylethanolamine, which is the lipidic source of lethal L-ROS related to ferroptosis. They also play a critical role of scaffold-like platforms with highly metabolical activity.[19] Additionally, death signals generated by MAMs may induce apoptosis and ferroptosis. One study reported that acyl-CoA synthetase long chain family member 4 (ACSL4), which catalyzes arachidonoyl biosynthesis and promotes phospholipid esterification and ferroptosis, accumulates in MAMs.[20] All these studies suggest that MAMs could be a candidate target for regulating ferroptosis in ALD prevention and treatment. Redox-regulated or misfolded proteins lead to the unfolded protein response (UPR).[21] Protein kinase RNA-like endoplasmic reticulum kinase (PERK) is one of the UPR transmembrane sensors triggered by ER stress (ERS) that is also known as one of the physical link proteins of MAMs.[22, 23] Some studies have shown that ERS can regulate mitochondrial function by mediating the number and function of MAMs.[24, 25] Arsenic exposure has been shown to cause abnormal MAMs and ferroptosis in pulmonary epithelial cells, along with increased PERK phosphorylation.[26] However, the role of the PERK-MAMs axis in alcohol-induced ferroptosis is still unknown.
线粒体相关内质网膜 (MAM) 是线粒体和内质网 (ER) 之间的结构/功能系绳，在多种生物途径中发挥多功能作用，包括 Ca 2+稳态调节、脂质代谢和线粒体动力学。 15 - 17这两个重要细胞器之间的密切联系是直接、快速信号转导和串扰的重要枢纽，以应对各种生理和病理应激。这两种细胞器都容易受到铁过载、铁死亡和芬顿激活的不稳定铁的影响。 18 MAM 对于磷脂酰乙醇胺等磷脂的合成和细胞内运输至关重要，磷脂酰乙醇胺是与铁死亡相关的致命 L-ROS 的脂质来源。它们还在具有高代谢活性的支架类平台中发挥着关键作用。 19此外，MAM 产生的死亡信号可能会诱导细胞凋亡和铁死亡。一项研究报道，酰基辅酶 A 合成酶长链家族成员 4 (ACSL4) 在 MAM 中积聚，该酶催化花生四烯酰生物合成并促进磷脂酯化和铁死亡。 20所有这些研究表明，MAM 可能成为 ALD 预防和治疗中调节铁死亡的候选靶点。氧化还原调节或错误折叠的蛋白质会导致未折叠蛋白质反应 (UPR)。21蛋白激酶 RNA样内质网激酶 (PERK) 是内质网应激 (ERS) 触发的 UPR 跨膜传感器之一，也被称为 MAM 的物理连接蛋白之一。 22 , 23一些研究表明 ERS 可以通过介导 MAM 的数量和功能来调节线粒体功能。 24 , 25砷暴露已被证明会导致肺上皮细胞中 MAM 异常和铁死亡，同时 PERK 磷酸化增加。 26然而，PERK-MAMs 轴在酒精诱导的铁死亡中的作用仍不清楚。
Quercetin is a natural flavonoid with antioxidative and anti-inflammatory effects. It also acts as an iron chelator that regulates intracellular iron content, particularly in the liable iron pool (LIP).[27] Previous studies have demonstrated that quercetin can protect against diabetic encephalopathy and peripheral nerve damage by reducing PERK expression and cellular apoptosis.[28, 29] However, there is no evidence to suggest that quercetin regulates ERS by modulating PERK expression in ALD. Similarly, there is no indication that quercetin regulates MAMs through PERK. Therefore, our study aims to investigate whether the regulation of PERK expression affects MAMs structure and mediates ferroptosis during ALD development. Additionally, we aim to evaluate the protective effect of quercetin against ALD.
槲皮素是一种天然黄酮类化合物，具有抗氧化和抗炎作用。它还充当铁螯合剂，调节细胞内铁含量，特别是责任铁库 (LIP) 中的铁含量。 27先前的研究表明，槲皮素可以通过减少 PERK 表达和细胞凋亡来预防糖尿病脑病和周围神经损伤。 28 , 29然而，没有证据表明槲皮素通过调节 ALD 中的 PERK 表达来调节 ERS。同样，没有迹象表明槲皮素通过 PERK 调节 MAM。因此，我们的研究旨在探讨 PERK 表达的调节是否影响 MAM 结构并介导 ALD 发展过程中的铁死亡。此外，我们的目的是评估槲皮素对 ALD 的保护作用。
In this study, we demonstrated that chronic alcohol-induced ferroptosis in C57BL/6J mice and AML12 cells. Furthermore, we showed that PERK enhances MAMs formation through its cytoplasmic domain rather than its kinase activity, which is implicated in ethanol-induced ferroptosis. Quercetin played a protective role by attenuating the PERK-mediated increase in MAMs and consequently reducing ethanol-induced ferroptosis.
在这项研究中，我们证明了 C57BL/6J 小鼠和 AML12 细胞中慢性酒精诱导的铁死亡。此外，我们发现 PERK 通过其胞质结构域而不是其激酶活性来增强 MAM 的形成，而激酶活性与乙醇诱导的铁死亡有关。槲皮素通过减弱 PERK 介导的 MAM 增加而发挥保护作用，从而减少乙醇诱导的铁死亡。
2 Experimental Section 2 实验部分
2.1 Animals 2.1 动物
Forty male C57BL/6J mice (18–20 g) purchased from Beijing Vital Rival Laboratory Animal Technology Co., Ltd. (Beijing, China) were randomly divided into four groups: CON (normal control group); EtOH (ethanol group; 28% energy replacement); EQ (ethanol plus quercetin group; quercetin, 100 mg kg−1 bw); and Q (quercetin group).
40只雄性C57BL/6J小鼠（18-20 g）购自北京维锐实验动物技术有限公司（中国北京），随机分为四组：CON组（正常对照组）；CON组（正常对照组）； EtOH（乙醇基团；28%能量替代）； EQ（乙醇加槲皮素组；槲皮素，100 mg kg -1 bw）；和Q（槲皮素组）。
Animals were cared for according to the Guiding Principles in the Care and Use of Laboratory Animals published by the US National Institutes of Health. This study was approved by the Experimental Animal Medicine Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology (IACUC Number: 432).
根据美国国立卫生研究院出版的实验动物护理和使用指导原则来护理动物。本研究经华中科技大学同济医学院实验动物医学伦理委员会批准（IACUC号：432）。
2.2 Reporting Dose and Administration Details
2.2 报告剂量和给药细节
According to the chronic and binge ethanol feeding protocol,[30] mice were pair-fed with regular or ethanol-containing Lieber De Carli liquid diets for 12 weeks, and quercetin was dissolved in the liquid diets (100 mg kg−1 bw). The intervention dose of quercetin in mice was based on previous research and other studies conducted by the research group.[31, 32] The recommended daily quercetin supplement dose for humans was 200–1200 mg.[33] The study calculated the intervention dose of quercetin for mice based on the equivalent dose ratio converted by body surface area. The formula was mouse dose = human dose /60 × 12.3, resulting in an intervention dose of 41–246 mg kg-1 bw for mice. This dose was consistent with the quercetin dose used in the study. At the end of 12 weeks, mice were orally administered with either ethanol or maltose solutions and were sacrificed after 9 h.
根据慢性和暴食乙醇喂养方案，将30只小鼠与常规或含乙醇的Lieber De Carli液体饮食配对喂养12周，并将槲皮素溶解在液体饮食中（100 mg kg -1 bw）。槲皮素对小鼠的干预剂量是基于之前的研究和研究组进行的其他研究。 31 , 32人类每日推荐的槲皮素补充剂剂量为 200–1200 毫克。 33该研究根据体表面积换算的当量剂量比计算了槲皮素对小鼠的干预剂量。公式为小鼠剂量=人剂量/60×12.3，得出小鼠的干预剂量为41–246 mg kg -1 bw。该剂量与研究中使用的槲皮素剂量一致。 12 周结束时，小鼠口服乙醇或麦芽糖溶液，9 小时后处死。
2.3 Cell Culture and Treatment
2.3 细胞培养和处理
AML12 cells were purchased form the Stem Cell Bank, Chinese Academy of Sciences (Shanghai, China). Cells were maintained in DM/F12 medium (Gibco, USA) supplemented with 10% fetal bovine serum, 100 U mL−1 penicillin/streptomycin (Gibco, USA), 40 ng mL−1 dexamethasone (MCE, HY-14648), and liquid media supplement (ITS) at 37 °C in a humidified atmosphere containing 5% CO2. The cells were exposed to ethanol (200 mM) for 24 h, and RSL-3 (MCE, HY-100218A), Erastin (MCE, HY-15763), Ferrostatin-1 (MCE, HY-100579), DFO (MCE, HY-D0903), Necrostatin-1s (MCE, HY-15760), Z-VAD-FMK (MCE, HY-16658), and quercetin were added at 1, 2.5, 2, 20, 50, 50, and 20 µM, respectively.
AML12细胞购自中国科学院干细胞库（中国上海）。将细胞维持在补充有10%胎牛血清、100 U mL -1青霉素/链霉素（Gibco，美国）、40 ng mL -1地塞米松（MCE，HY-14648）和液体培养基补充剂 (ITS)，温度为 37 °C，潮湿气氛中含有 5% CO 2 。将细胞暴露于乙醇 (200 mM) 24 小时，以及 RSL-3 (MCE, HY-100218A)、Erastin (MCE, HY-15763)、Ferrostatin-1 (MCE, HY-100579)、DFO (MCE,添加 1、2.5、2、20、50、50 和 20 µM 的 Necrostatin-1s（MCE、HY-15760）、Z-VAD-FMK（MCE、HY-16658）和槲皮素，分别。
2.4 Transfection and RNA Interference
2.4 转染和RNA干扰
Cells were transfected with 100 nM PERK or control siRNA (Ribobio, Guangzhou, China) mixed with Lipofectamine 3000 Reagent for 24 h in 70–90% confluent, the siRNA sequences were listed in Table 1. Cells were further transfected with either PERK-ΔC (Addgene plasmid 21815) or PERK-ΔK (Addgene plasmid 21816) plasmids depending on the experimental objectives.
细胞用 100 nM PERK 或对照 siRNA（Ribobio，广州，中国）与 Lipofectamine 3000 试剂混合转染 24 小时，汇合度为 70-90%，siRNA 序列列于表1 。根据实验目的，用 PERK-ΔC（Addgene 质粒 21815）或 PERK-ΔK（Addgene 质粒 21816）质粒进一步转染细胞。
表 1.三个 PERK siRNA 序列。
| Name 姓名 | sequence 顺序 |
|---|---|
| S1 | CGGGAAAACGGTTCTGAGA |
| S2 | CCACAGACATCATTGAAAA |
| S3 | GAGTTCATCTGGAACAAAA |
2.5 Western Blot Analysis
2.5 蛋白质印迹分析
Liver tissue and AML12 cells were homogenized and lysed in RIPA buffer at 4 °C and then quantified and denatured. The protein was separated by a 10% SDS-polyacrylamide gel and then transferred onto a PVDF membrane. The membrane was blocked and incubated with primary antibodies including anti-Gpx4 (abcam, ab125066), anti-ACSL4 (abcam, ab155282), antixCT (proteintech, 26864-1-AP), anti-PERK (Cell Signaling Technology, 3179s), anti-p-PERK (Cell Signaling Technology, 3192s), anti-XBP-1s (abcam, ab198999), anti-p-IRE (abcam, ab48187), anti-IRE (abcam, ab37073), anti-CHOP (proteintech, 15204-1-AP), anti-p-eIF2α (Cell Signaling Technology, 3398), antieIF2α (Cell Signaling Technology, 5324), anti-ATF-4 (proteintech, 60035-1-Ig), anti-p90-ATF6/p50-ATF6 (proteintech, 66563-1-Ig), and anti-GRP78 (abcam, ab21685) for 2 h at room temperature. The membrane was then incubated with the appropriate secondary antibodies and immunoreactive bands were detected using an ECL plus Western Blotting Detection System (GENE GNOME XRQ, Syngene, UK). The band densities were measured by ImageProplus software.
将肝组织和 AML12 细胞匀浆并在 4 °C 的 RIPA 缓冲液中裂解，然后进行定量和变性。通过 10% SDS-聚丙烯酰胺凝胶分离蛋白质，然后转移到 PVDF 膜上。将膜封闭并用一抗孵育，包括抗 Gpx4 (abcam, ab125066)、抗 ACSL4 (abcam, ab155282)、抗 xCT ( Proteintech, 26864-1-AP)、抗 PERK (Cell Signaling Technology, 3179s)、抗 p-PERK (Cell Signaling Technology, 3192s)、抗 XBP-1s (abcam, ab198999)、抗 p-IRE (abcam, ab48187)、抗 IRE (abcam, ab37073)、抗 CHOP ( Proteintech, 15204-1-AP)、抗 p-eIF2α (Cell Signaling Technology, 3398)、抗 eIF2α (Cell Signaling Technology, 5324)、抗 ATF-4 ( Proteintech, 60035-1-Ig)、抗 p90-ATF6/ p50-ATF6（ Proteintech，66563-1-Ig）和抗 GRP78（abcam，ab21685）在室温下 2 小时。然后将膜与适当的二抗一起孵育，并使用 ECL 加蛋白质印迹检测系统（GENE GNOME XRQ，Syngene，UK）检测免疫反应条带。通过ImageProplus软件测量条带密度。
2.6 Histochemical Analysis
2.6 组织化学分析
Sections (5 µm) obtained from formalin-fixed paraffin-embedded liver samples were stained with PERK (proteintech, 20582-1-AP) and VDAC1 (abcam, ab14734) for the colocalization analysis of immunofluorescence (IF) or hematoxylin-eosin (H&E). Lipid accumulation was observed by Oil-red O staining of frozen liver tissue sections. The amount of MAMs was detected by proximity ligation assay (PLA, Sigma-Aldrich, DUO92101) with primary antibodies against IP3R1 (Invitrogen, PA5-85753) and VDAC1 (abcam, ab14734) by using paraffin-embedded liver samples. In AML12 cells, IF analysis was used to measure the amount of MAMs under various treatments. Fixing and permeabilizing the cells, then incubated with primary antibodies against IP3R1 and VDAC1 or PERK and VDAC1. Furthermore, AML12 cells were incubated with the appropriate secondary antibodies and the images were monitored by microscopy.
从福尔马林固定石蜡包埋的肝脏样本中获得的切片 (5 µm) 用 PERK ( Proteintech, 20582-1-AP) 和 VDAC1 (abcam, ab14734) 染色，用于免疫荧光 (IF) 或苏木精-伊红 (H&E) 的共定位分析）。通过冷冻肝组织切片的油红O染色观察脂质积累。使用石蜡包埋的肝脏样品，通过邻位连接测定（PLA，Sigma-Aldrich，DUO92101）和针对 IP3R1（Invitrogen，PA5-85753）和 VDAC1（abcam，ab14734）的一抗来检测 MAM 的量。在 AML12 细胞中，IF 分析用于测量各种处理下的 MAM 量。固定并透化细胞，然后与针对 IP3R1 和 VDAC1 或 PERK 和 VDAC1 的一抗一起孵育。此外，将 AML12 细胞与适当的二抗一起孵育，并通过显微镜监测图像。
2.7 Transmission Electron Microscopy
2.7 透射电子显微镜
The ultrastructure of hepatocytes was detected by transmission electron microscopy (TEM) according to protocol.[34] In briefly, ultrathin sections (80–100 nm) were prepared by fixing fresh liver in 2.5% glutaraldehyde and storing it at 4 °C. These sections were then scanned using a TEM (Tecnai G 2 20 TWIN, USA).
根据方案通过透射电子显微镜（TEM）检测肝细胞的超微结构。 34简而言之，超薄切片（80–100 nm）的制备方法是将新鲜肝脏固定在 2.5% 戊二醛中并储存在 4 °C 下。然后使用 TEM（Tecnai G 2 20 TWIN，美国）扫描这些切片。
2.8 Measurement of Liver Damage
2.8 肝损伤的测量
The study measured the levels of serum aspartate aminotransferase (AST) and alanine aminotransferases (ALT) were using the AST (Nanjing Jiancheng, China) and ALT assay kit (Nanjing Jiancheng, China), respectively. The study detected the levels of GSH and malondialdehyde (MDA) in liver tissue using the GSH (Nanjing Jiancheng, China) and the MDA assay kit (Nanjing Jiancheng, China), respectively.
本研究分别使用AST（南京建成，中国）和ALT检测试剂盒（南京建成，中国）测量血清天冬氨酸转氨酶（AST）和丙氨酸转氨酶（ALT）水平。本研究分别使用GSH（南京建成，中国）和MDA检测试剂盒（南京建成，中国）检测肝组织中GSH和丙二醛（MDA）的水平。
2.9 Real-Time Quantitative PCR
2.9 实时定量PCR
The study extracted total RNA was extracted from live tissue using the TRIzol reagent (TaKaRa BIO INC, Dalian, China). Prostaglandin Endoperoxide Synthase 2 (PTGS2) mRNA expressions were quantified using the SYBR green-based qRT-PCR kit (TaKaRa BIO INC, Dalian, China) and the 7900HT PCR machine (Applied Biosystems, Forster, CA, USA). The study calculated the relative mRNA expression using the comparative 2−ΔΔct method. The mRNA level of GAPDH was quantified as an endogenous control. The primer sequences were listed in Table 2.
本研究提取的总RNA是使用TRIzol试剂（TaKaRa BIO INC，大连，中国）从活组织中提取的。使用基于 SYBR green 的 qRT-PCR 试剂盒（TaKaRa BIO INC，大连，中国）和 7900HT PCR 仪（Applied Biosystems，Forster，CA，USA）对前列腺素内过氧化物合酶 2 (PTGS2) mRNA 表达进行定量。该研究使用比较2 −ΔΔct方法计算了相对mRNA表达量。 GAPDH 的 mRNA 水平作为内源性对照进行定量。引物序列列于表2中。
表 2.研究中使用的 qPCR 引物。
| Gene name 基因名称 | Primer sequence (5′→3′) 引物序列（5′→3′） | |
|---|---|---|
| Ptgs2 目标2 | Forward 向前 | TGGAGGCGAAGTGGGTTTTA |
| Reverse 撤销 | GAGTGGGAGGCACTTGCATT | |
| GAPDH | Forward 向前 | ATACGGCTACAGCAACAGGG |
| Reverse 撤销 | GCTTTGCACATGCCGGAGCC | |
2.10 Assay of Liver Total Iron, Liable Iron Pool
2.10 肝脏总铁、有效铁池的测定
Hepatic total iron and LIP were detected by flame atomic absorption spectrometry (FAAS).[35
采用火焰原子吸收光谱法（FAAS）检测肝脏总铁和LIP。 35]
2.11 Determination of Cellular Damage
2.11 细胞损伤的测定
Using the LDH assay kit (Beyotime, China) according to the manufacturer's instructions, the cellular damage was detected and evaluated.
使用LDH测定试剂盒（Beyotime，中国）按照制造商的说明，检测和评估细胞损伤。
2.12 Isolation of MAMs 2.12 MAM 的分离
The study extracted MAMs from liver tissue based on published protocols.[36] Brief, 300 mg of liver tissues were homogenized and centrifuging the tissue homogenate twice for 5 min each at 740 g. The study collected the supernatant and centrifuged it once more for 10 min at 9000 × g. The study resuspended the pellet and centrifuged it at 10,000 × g for 10 min twice. The study collected the resulting pellet and resuspended it with MRB buffer. The study layered MRB buffer in a 30% Percoll gradient and centrifuged it at 95,000 × g for 30 min using a Beckman ultracentrifuge in a 41.1 SW rotor. The study collected the MAMs fraction and diluted it 10-fold in MRB buffer. The study centrifuged the suspension at 100 000 × g in a Beckman instrument for 1 h before resuspended the pellet in 0.2 mL MRB buffer. Finally, the protein concentration was determined by BCA assay.
该研究根据已发布的方案从肝组织中提取 MAM。 36简而言之，将 300 mg 肝组织均质化，并将组织匀浆在 740 g 下离心两次，每次 5 分钟。该研究收集了上清液，并在 9000 × g下再次离心 10 分钟。该研究重悬沉淀并以 10,000 × g离心 10 分钟两次。该研究收集了所得沉淀并用 MRB 缓冲液将其重新悬浮。该研究将 MRB 缓冲液分层放入 30% Percoll 梯度中，并使用 Beckman 超速离心机在 41.1 SW 转子中以 95,000 × g离心 30 分钟。该研究收集了 MAM 组分，并在 MRB 缓冲液中将其稀释 10 倍。该研究在 Beckman 仪器中以 100 000 × g离心悬浮液 1 小时，然后将沉淀重新悬浮在 0.2 mL MRB 缓冲液中。最后，通过BCA测定测定蛋白质浓度。
2.13 Detection of Lipid Peroxidation
2.13 脂质过氧化的检测
After intervention, C11-bodipy (Invitrogen, D3861) was added to the medium and incubated with the cells at 37 °C for 30 min. The cells were then observed using fluorescence microscopy or detected them with a multifunctional enzyme label. The excitation/emission wavelength of nonoxidized lipids was 540 nm/620 nm, while that of oxidized lipids was 428 nm/528 nm.
干预后，将 C11-bodipy（Invitrogen，D3861）添加到培养基中，并在 37°C 下与细胞一起孵育 30 分钟。然后使用荧光显微镜观察细胞或使用多功能酶标记检测细胞。非氧化脂质的激发/发射波长为540 nm/620 nm，而氧化脂质的激发/发射波长为428 nm/528 nm。
2.14 Statistical Analysis
2.14 统计分析
Data from animal experiments and cell experiments were expressed as mean ± SD and mean ± SEM, respectively. By using IBM SPSS Statistics 21 software (Armonk, NY, USA), one-way ANOVA was used to compare the results, followed by LSD multiple-comparison analysis, and the value of p < 0.05 was shown as the statistical significance.
动物实验和细胞实验的数据分别表示为平均值±SD和平均值±SEM。采用IBM SPSS Statistics 21软件(Armonk, NY, USA)，采用单因素ANOVA对结果进行比较，再进行LSD多重比较分析，以p < 0.05的值为有统计学意义。
3 Results 3 个结果
3.1 Quercetin Attenuates Liver Damage in Chronic-Plus-Single-Binge Ethanol Feeding Mice
3.1 槲皮素减轻慢性加单次暴食乙醇喂养小鼠的肝损伤
To investigate how quercetin plays a protective role in ALD, we fed C57BL/6J male mice were administrated chronic-plus-single-binge ethanol to induce ALD. As shown in Figure 1A,B, chronic ethanol administration elevated the levels of ALT and AST, which are sensitive indicators of liver damage, compared with the control group. In Figure 1C, H&E staining of the liver revealed that, compared with the control group, chronic alcohol consumption caused liver structure disarrangement and lipid accumulation, which was further confirmed by oil red O staining (Figure 1D). Quercetin significantly ameliorated ethanol-induced serum ALT and AST elevations and morphological changes in liver tissue.
为了研究槲皮素如何在 ALD 中发挥保护作用，我们给 C57BL/6J 雄性小鼠喂食长期加单次暴食乙醇以诱导 ALD。如图1A、B所示，与对照组相比，长期服用乙醇会升高 ALT 和 AST 的水平，而 ALT 和 AST 是肝损伤的敏感指标。图1C中，肝脏H&E染色显示，与对照组相比，长期饮酒导致肝脏结构紊乱和脂质堆积，油红O染色进一步证实了这一点(图1D )。槲皮素显着改善乙醇引起的血清 ALT 和 AST 升高以及肝组织的形态变化。
槲皮素可减轻慢性加单次暴饮乙醇喂养小鼠的肝损伤。 A、B) 通过标准试剂盒测定血清 ALT 和 AST。数据显示为平均值±SD（ n = 8）。光学显微镜（400×）下用苏木精和伊红（C）和油红O（D）染色的小鼠肝组织切片。 CON，对照组； EtOH，乙醇（慢性加单次暴饮乙醇）组； EQ：乙醇+槲皮素组；问：槲皮素组。 a：与对照相比， p < 0.05； b：相对于乙醇， p < 0.05。
3.2 Quercetin Decreases Hepatocytes Ferroptosis in Chronic Ethanol-Treated Mice
3.2 槲皮素减少慢性乙醇治疗小鼠的肝细胞铁死亡
To examine the effect of quercetin on ethanol-induced ferroptosis, a process characterized by dramatic mitochondrial morphology changes and lipid peroxidation-related lethal ROS originating from iron metabolism.[4] As shown in Figure 2A, chronic ethanol treatment induced mitochondrial damage, such as reduced cristae, denser mitochondrial membranes, and destroyed mitochondrial membranes, compared with the control group. Furthermore, we measured the total iron and LIP content of liver tissue were measured by FAAS (Figure 2B,C). The results showed that chronic ethanol treatment significantly increased the average liver total iron and LIP contents in the ethanol group compared to the control group, and quercetin supplementation reversed this effect. In addition, chronic ethanol treatment depleted GSH by 17% and increased MDA by 28% compared with the control group (Figure 2D,E). Moreover, we assessed the level of PTGS2 mRNA and the expression of Gpx4, xCT, and ACSL4, which are involved in ferroptosis regulation.[1, 37] As shown in Figure 2F,J, chronic ethanol treatment significantly increased the PTGS2 mRNA and ACSL4 protein levels, and decreased the Gpx4 and xCT protein levels in the liver tissue compared with the control group, whereas quercetin intervention restored these levels. There was no significant difference in the levels of ferroptosis-related proteins (Gpx4, xCT, and ACSL4) between the quercetin alone and control groups. These data indicated that quercetin protected hepatocytes from ethanol-induced ferroptosis.
研究槲皮素对乙醇诱导的铁死亡的影响，该过程的特征是线粒体形态发生显着变化和源自铁代谢的脂质过氧化相关的致死性活性氧。 4如图2A所示，与对照组相比，慢性乙醇治疗引起线粒体损伤，例如嵴减少、线粒体膜致密以及线粒体膜被破坏。此外，我们还通过 FAAS 测量了肝组织的总铁和 LIP 含量（图2B、C ）。结果表明，与对照组相比，长期乙醇治疗显着增加了乙醇组的平均肝脏总铁和LIP含量，而补充槲皮素则逆转了这种效应。此外，与对照组相比，长期乙醇治疗使 GSH 减少 17%，MDA 增加 28%（图2D、E ）。此外，我们评估了参与铁死亡调节的 PTGS2 mRNA 水平以及 Gpx4、xCT 和 ACSL4 的表达。 1 , 37如图2F,J所示，与对照组相比，长期乙醇治疗显着增加了肝组织中的PTGS2 mRNA和ACSL4蛋白水平，并降低了Gpx4和xCT蛋白水平，而槲皮素干预恢复了这些水平。单独使用槲皮素组和对照组之间铁死亡相关蛋白（Gpx4、xCT 和 ACSL4）的水平没有显着差异。这些数据表明槲皮素可以保护肝细胞免受乙醇诱导的铁死亡。
槲皮素可减少长期加单次暴饮乙醇治疗的小鼠的肝细胞铁死亡。 A) TEM 观察到的肝细胞超微结构（箭头：线粒体萎缩）。 B, C) 通过 FAAS 测量肝脏总铁和 LIP， n = 8。D, E) 肝脏 GSH 和 MDA， n = 6。F) 肝脏中 PTGS2 mRNA 水平， n = 6。G–J) Gpx4，xCT ，以及肝脏中的 ACSL4 蛋白。数据显示为平均值±标准差。 CON，对照组； EtOH：乙醇（慢性加单次暴饮乙醇）组； EQ，乙醇+槲皮素组； Q，槲皮素组。 a：与对照相比， p < 0.05； b：相对于乙醇， p < 0.05。
3.3 Quercetin Decreases Ethanol-Induced Ferroptosis in AML12 Cells
3.3 槲皮素减少 AML12 细胞中乙醇诱导的铁死亡
To investigate the occurrence of ferroptosis induced by ethanol, we treated AML12 cells, a nontransformed mouse hepatocyte cell line, with ethanol and various death inhibitors. We assessed cell damage by measuring LDH release, as shown in Figure 3A. Ethanol administration (200 mM) significantly increased LDH release. The ferroptosis inhibitor ferrostaitin-1 (2 µM), the apoptosis inhibitor Z-VAD-FMK (50 µM), and the necrosis inhibitor necrostatin-1 (50 µM) all significantly reduced LDH release (P < 0.05), indicating that these three forms of cell death coexist in the case of ethanol exposure. To further explore the role and mechanism of ferroptosis under ethanol exposure, we measured the expression of ferroptosis-related proteins (ACSL4, xCT, and Gpx4) and LDH release in AML12 cells treated with ethanol and either ferropotosis inducers (RSL3, 1 µM; erastin, 2.5 µM) or inhibitors (ferrostatin-1, 2 µM; DFO, 20 µM). The results showed that ethanol and both inducers further increased ACSL4 (p < 0.05) and decrease xCT (p < 0.05) compared with the ethanol group (Figure 3F). RSL3, but not erastin, significantly decreased Gpx4 (p < 0.05). LDH release further increased after treatment with RSL3 (p < 0.05) but not erastin under ethanol administration (Figure 3B,C). Therefore, we used RSL3 in the subsequent experiments. In contrast, treatment with ferrostatin-1 or DFO plus ethanol administration significantly increased the expression of xCT (p < 0.05) and Gpx4 (p < 0.05) compared with the ethanol group (Figure 3H,I). DFO plus ethanol decreased (p < 0.05) but ferrostatin-1 plus ethanol increased (p < 0.05) the expression of ACSL4. Treatment with ferropotosis inhibitors significantly decreased (p < 0.05) LDH release in ethanol-treated cells (Figure 3D,E). We then measured the lipid peroxidation of AML12 cells under ethanol administration by loading C11-BODIPY and observing with a fluorescence inverted microscope or quantifying with a fluorometric plate reader (Figure 3J–N). The results showed that ethanol treatment significantly increased (p < 0.05) the ratio of oxidized/nonoxidized lipids. After coprocessing with RSL3, the increase was further induced, and treatment with ferrostatin-1 could counteract the increase.
为了研究乙醇诱导的铁死亡的发生，我们用乙醇和各种死亡抑制剂处理 AML12 细胞（一种非转化的小鼠肝细胞系）。我们通过测量 LDH 释放来评估细胞损伤，如图3A所示。施用乙醇 (200 mM) 显着增加 LDH 释放。铁死亡抑制剂ferrostatin-1 (2 µM)、凋亡抑制剂Z-VAD-FMK (50 µM) 和坏死抑制剂necrostatin-1 (50 µM) 均显着减少LDH 释放（ P < 0.05），表明这三种药物在乙醇暴露的情况下，多种形式的细胞死亡同时存在。为了进一步探讨乙醇暴露下铁死亡的作用和机制，我们测量了用乙醇和铁死亡诱导剂（RSL3，1 µM；erastin）处理的 AML12 细胞中铁死亡相关蛋白（ACSL4、xCT 和 Gpx4）的表达和 LDH 释放。 ，2.5 µM）或抑制剂（ferrostatin-1，2 µM；DFO，20 µM）。结果显示，与乙醇组相比，乙醇和两种诱导剂进一步增加了ACSL4（ p <0.05）并降低了xCT（ p <0.05）（图3F ）。 RSL3（而非erastin）显着降低了Gpx4 ( p < 0.05)。在使用 RSL3 治疗后，LDH 释放进一步增加（ p < 0.05），但在乙醇给药下，erastin 没有增加（图3B，C ）。因此，我们在后续实验中使用RSL3。相反，与乙醇组相比，用ferrostatin-1或DFO加乙醇治疗显着增加了xCT（ p <0.05）和Gpx4（ p <0.05）的表达（图3H，I ）。 DFO 加乙醇减少 ( p < 0.05)，但 Ferrostatin-1 加乙醇增加 ( p < 0.05)ACSL4的表达。在乙醇处理的细胞中，用铁磷酸化抑制剂治疗显着减少（ p < 0.05）LDH 释放（图3D，E ）。然后，我们通过加载 C11-BODIPY 并用荧光倒置显微镜观察或用荧光板读数器定量来测量乙醇施用下 AML12 细胞的脂质过氧化（图3J-N ）。结果表明，乙醇处理显着增加了氧化/非氧化脂质的比率（ p < 0.05）。与RSL3共处理后，进一步诱导增加，并且用ferrostatin-1处理可以抵消这种增加。
乙醇诱导 AML12 细胞铁死亡。 A) AML12 细胞用乙醇 (200 mM) 和 Z-VAD (50 µM)、Nec-1 (50 µM) 或 Ferrostatin-1 (2 µM) 处理 24 小时。 LDH 释放按细胞损伤计算（ n = 3）。 B–E) 使用 RSL-3 (1 µM)、erastin (2.5 µM)、ferrostatin-1 (2 µM) 或 DFO (20 µM) 的乙醇组中 LDH 释放， n = 3。FI) ACSL4、xCT、和 Gpx4 表达， n = 6。 J) C11-BODIPY 染色的脂质过氧化作用。红色信号和绿色信号分别表示未氧化和氧化的脂质。 K–N) 通过荧光板读数器测定负载 C11-BODIPY 和脂质过氧化的细胞， n = 3。数据显示为平均值±SEM。 CON，对照组； EtOH，乙醇基团； E+RSL-3，乙醇+RSL-3组； E+Era，乙醇+erastin组； E+Fer-1，乙醇+铁他汀-1组； ED，乙醇+DFO组。 a：与对照相比， p < 0.05； b：相对于乙醇， p < 0.05。
We then explored how quercetin protected AML12 cells form the ethanol-induced ferroptosis. Quercetin significantly reduced the level of ferroptosis induced by ethanol or ethanol plus RSL3, as shown in Figure 4A–D. This was evidenced by the increased expression of xCT and Gpx4 (p < 0.05) and the decreased expression of ACSL4 (p < 0.05), as well as the reduced level of oxidized/nonoxidized lipids and LDH release.
然后我们探讨了槲皮素如何保护 AML12 细胞免受乙醇诱导的铁死亡。槲皮素显着降低乙醇或乙醇加RSL3诱导的铁死亡水平，如图4A-D所示。 xCT 和 Gpx4 表达增加 ( p < 0.05)、ACSL4 表达减少 ( p < 0.05) 以及氧化/非氧化脂质和 LDH 释放水平降低证明了这一点。
槲皮素可改善 AML12 细胞中乙醇诱导的铁死亡。 A–D) ACSL4、xCT 和 Gpx4 蛋白， n = 3。E) LDH 释放， n = 3。F) 通过荧光板读数器计算脂质过氧化， n = 3。数据显示为平均值±SEM。 CON，对照组； EtOH，乙醇基团； E+RSL-3，乙醇+RSL-3组； EQ，乙醇+槲皮素组； EQR、乙醇+RSL-3和槲皮素组。 a：与对照相比， p < 0.05； b：相对于乙醇， p < 0.05； c：与 E+RSL-3 相比， p < 0.05； d：与 EQ 相比， p < 0.05。
3.4 MAMs Are Associated with Ethanol-Induced Ferroptosis In Vivo and In Vitro and the Effect of Quercetin
3.4 MAM 与体内外乙醇诱导的铁死亡相关以及槲皮素的作用
To investigate the involvement of MAMs in ethanol-induced ferroptosis, we measured the level of MAMs in the liver of mice and in AML12 cells. We used in situ PLA[38] to detect the interaction of VDAC1 (mitochondrial marker) and IP3R1 (ER marker), which indicated the amount of MAMs, and TEM further confirmed the structure of MAMs. The amount of MAMs was significantly increased in the liver of mice treated with ethanol compared with the control group, as shown in Figure 5A–C. Quercetin intervention restored the amount of MAMs. In AML12 cells, we used immunofluorescence co-localization of VDAC1 and IP3R1 to measure MAMs. Ethanol increased MAMs, which were further increased by the ferroptosis agonist RSL3, and reduced by the ferroptosis inhibitor ferrostatin-1 (Figure 5D). Quercetin treatment reduced the MAMs increased by either ethanol or ethanol plus RSL3 (Figure 5E).
为了研究 MAM 在乙醇诱导的铁死亡中的作用，我们测量了小鼠肝脏和 AML12 细胞中的 MAM 水平。我们使用原位PLA 38检测VDAC1（线粒体标记）和IP3R1（ER标记）的相互作用，这表明了MAM的数量，TEM进一步证实了MAM的结构。与对照组相比，用乙醇处理的小鼠肝脏中MAM的量显着增加，如图5A-C所示。槲皮素干预恢复了 MAM 的数量。在 AML12 细胞中，我们使用 VDAC1 和 IP3R1 的免疫荧光共定位来测量 MAM。乙醇增加了MAM，铁死亡激动剂RSL3进一步增加了MAM，铁死亡抑制剂ferrostatin-1则减少了MAM（图5D ）。槲皮素处理减少了乙醇或乙醇加RSL3增加的MAM（图5E ）。
MAMs 的增加与乙醇诱导的体内外铁死亡和槲皮素作用有关。 A) 在石蜡包埋的肝脏中通过原位 PLA 测量的 VDAC1/IP3R1 相互作用的代表性图像， n = 3 (400×)。 B) 肝脏中 MAM（红色箭头）的 TEM 图像， n = 4 (400×)。 C) 小鼠中 MAM 与线粒体的比率。数据显示为平均值±标准差。 D,E) 在不同处理的 AML12 细胞中，用针对 IP3R1（绿色）和 VDAC1（红色）的抗体对免疫荧光进行染色（400×）。 CON，对照组； EtOH，乙醇基团； E+RSL-3，乙醇+RSL-3组； E+Fer-1，乙醇+铁他汀-1组； EQ，乙醇+槲皮素组； EQR、乙醇+RSL-3和槲皮素组。 a：与对照相比， p < 0.05； b：相对于乙醇， p < 0.05。
3.5 PERK Regulates the Change of MAMs in Mice with Ethanol Administration
3.5 PERK 调节乙醇给药小鼠 MAM 的变化
MAMs were originally linked to lipid metabolism and UPR.[39-41] We explored the UPR-MAMs relationship in ALD. UPR, the response to ERS, has three branches: IRE1, PERK, and ATF6 (Figure 6F).[42] We measured the changes in these pathways, respectively. The results showed that ethanol increased the expression of glucose-regulated protein 78 (GRP78, Figure 6A,B) (p < 0.05), which was reversed by quercetin. The results of the UPR-associated proteins showed that ethanol did not affect the IRE1 pathway (although consistent with a recent study,[43] 100 mg kg−1 BW quercetin intervention showed a tendency to decrease p-IRE-1 and XBP-1s compared with controls, but there was no statistical difference between the groups, as shown in Figure 6A,D), but activated the PERK pathway (Figure 6A,C). The expression of P-PERK, P-eIF2α/ eIF2α, and CHOP increased significantly, and ATF4 showed an increasing trend. In contrast, the ATF6 pathway (Figure 6A,E) was downregulated by ethanol. This might be due to impaired ER function and the ATF6 decompensation by chronic ethanol. Previous studies have shown that PERK, besides being an ERS sensor, also regulates MAMs stability by acting as a physical ligand in MAMs.[44, 45] Therefore, we examined PERK modulated the effect of MAMs on ferroptosis in ALD. We isolated the MAMs structure from liver tissue and measured the expression of PERK and ACSL4 in the MAMs structure (Figure 6G–I). The results show that ethanol significantly increased PERK and ACSL4 in the MAMs structure (p < 0.05), which was reversed by quercetin. The immunofluorescence co-localization of PERK and VDAC1 in the liver further indicated that ethanol increased the MAMs structure (Figure 6J). Further, we explored the regulatory role of PERK in MAMs and ferroptosis.
MAM 最初与脂质代谢和 UPR 有关。 39 - 41我们探讨了 ALD 中的 UPR-MAM 关系。 UPR 是对 ERS 的响应，具有三个分支：IRE1、PERK 和 ATF6（图6F ）。 42我们分别测量了这些途径的变化。结果表明，乙醇增加了葡萄糖调节蛋白 78（GRP78，图6A，B ）的表达（ p < 0.05），而槲皮素可逆转这一现象。 UPR相关蛋白的结果表明，乙醇不会影响IRE1途径（尽管与最近的一项研究一致，与相比， 43 100 mg kg -1 BW槲皮素干预显示出减少p-IRE-1和XBP-1s的趋势）对照组，但组间无统计学差异，如图6A，D ），但激活了PERK通路（图6A，C ）。 P-PERK、P-eIF2α/eIF2α、CHOP的表达显着增加，ATF4呈增加趋势。相反，ATF6途径（图6A，E ）被乙醇下调。这可能是由于长期乙醇导致的 ER 功能受损和 ATF6 失代偿所致。先前的研究表明，PERK 除了作为 ERS 传感器外，还通过充当 MAM 中的物理配体来调节 MAM 稳定性。 44 , 45因此，我们检查了 PERK 调节 MAM 对 ALD 中铁死亡的影响。我们从肝组织中分离出MAMs结构，并测量MAMs结构中PERK和ACSL4的表达（图6G-I ）。 结果表明，乙醇显着增加了 MAM 结构中的 PERK 和 ACSL4 ( p < 0.05)，而槲皮素可逆转这一现象。 PERK 和 VDAC1 在肝脏中的免疫荧光共定位进一步表明乙醇增加了 MAM 结构（图6J ）。此外，我们探讨了 PERK 在 MAM 和铁死亡中的调节作用。
PERK 调节暴露于乙醇的肝脏和细胞中 MAM 的变化。 A–E) ERS 相关蛋白（GRP78、p-/PERK、p-/eIF2α、ATF4、CHOP、p-IRE、XBP-1s、p90/p50-ATF6）在肝脏中的表达， n = 3。F) 插图慢性乙醇处理小鼠中 UPR 的三个经典途径的研究。 G–I) MAM 中的 PERK 和 ACSL4 表达， n = 8。J) PERK 和 VDAC1 共染色。肝脏中的 PERK（红色）和线粒体标记物 VDAC1（绿色）免疫荧光。 ata 显示为平均值±SD。 CON，对照组； EtOH，乙醇（慢性加单次暴饮乙醇）组； EQ，乙醇+槲皮素组； Q，槲皮素组。 a：与对照相比， p < 0.05； b：相对于乙醇， p < 0.05。
3.6 The Cytoplasmic Domain of PERK Is Responsible for Ferroptosis in Cells Exposed to Ethanol
3.6 PERK 的胞质结构域导致暴露于乙醇的细胞中的铁死亡
PERK has the ER lumen domain and the cytoplasmic domain.[46] We established two plasmids with different sites mutated: ΔC (protein C terminal knockout) and ΔK (K618A site mutation that inhibits PERK kinase activity). We transfected these plasmids into AML12 cells (Figure 8B). The results of ferroptosis-related protein and lipid peroxidation show that ΔC plasmid transfection (Figure 7A–D,K) reduced ACSL4 and lipid peroxidation, and increased xCT and Gpx4, compared to ethanol administration. In contrast, ΔK plasmid transfection worsened the ferroptosis induced by ethanol, as shown in Figure 7E–H, Gpx4 expression decreased significantly compared to ethanol alone, and xCT expression tended to decrease. The expression of ACSL4 and lipid peroxidation (Figure 7K) did not change significantly compared to ethanol, but the LDH release increased further by ΔK plasmid transfection (Figure 7J). Remarkably, ΔC plasmid transfection also increased the LDH release compared to ethanol alone (Figure 7I). Figure 7L showed that the Z-VAD and Nec-1, but not ferrostatin-1, partially inhibited the LDH release in AML12 cells transfected with ΔC plasmid and exposed to ethanol. This suggests that ΔC plasmid transfection decreased ferroptosis, but PERK as an apoptotic transmitter[47] enhanced the susceptibility to other forms of death such as apoptosis and necrosis. We then measured the MAMs change by immunofluorescence co-localization after transfecting with ΔC or ΔK. The results (Figure 7M) show that ΔK but not ΔC increased the MAMs. This implies that the cytosolic domain of PERK, not its kinase activity, was involved in the MAMs increase.
PERK 具有 ER 腔结构域和细胞质结构域。 46我们建立了两种具有不同位点突变的质粒：ΔC（蛋白 C 末端敲除）和 ΔK（抑制 PERK 激酶活性的 K618A 位点突变）。我们将这些质粒转染到 AML12 细胞中（图8B ）。铁死亡相关蛋白和脂质过氧化的结果表明，与乙醇给药相比，ΔC质粒转染（图7A-D，K ）减少了ACSL4和脂质过氧化，并增加了xCT和Gpx4。相反， ΔK质粒转染加剧了乙醇诱导的铁死亡，如图7E-H所示，与单独乙醇相比，Gpx4表达显着下降，xCT表达趋于下降。与乙醇相比，ACSL4 和脂质过氧化的表达（图7K ）没有显着变化，但通过 ΔK 质粒转染进一步增加了 LDH 释放（图7J ）。值得注意的是，与单独的乙醇相比，ΔC质粒转染还增加了LDH释放（图7I ）。图7L显示Z-VAD和Nec-1，但不是铁他汀-1，部分抑制用ΔC质粒转染并暴露于乙醇的AML12细胞中的LDH释放。这表明 ΔC 质粒转染减少了铁死亡，但 PERK 作为细胞凋亡递质47增强了对其他形式死亡（如细胞凋亡和坏死）的易感性。然后，我们通过免疫荧光共定位测量了转染 ΔC 或 ΔK 后 MAM 的变化。结果（图7M ）表明 ΔK 而非 ΔC 增加了 MAM。 这意味着 PERK 的胞质结构域（而不是其激酶活性）参与了 MAM 的增加。
PERK 细胞质结构域负责乙醇暴露细胞中的铁死亡。 A–D) AML12 细胞中的 xCT、Gpx4 和 ACSL4 表达， n = 6。E–H) AML12 细胞中的 xCT、Gpx4 和 ACSL4 表达， n = 6。IJ) LDH 释放， n = 3。K) 脂质通过荧光板读数器计算过氧化， n = 3。L) LDH 释放， n = 3。M) IP3R1（绿色）和 VDAC1（红色）在乙醇暴露的 AML12 细胞中使用 ΔC 或 ΔK 质粒进行免疫荧光， n = 3（ 400×）。数据显示为平均值±SEM。 E+△C：乙醇+C端缺失-PERK组； E+△K：乙醇+激酶死亡-PERK组； E+siPERK：乙醇加siPERK； E+△C+Z-VAD/Nec-1/Fer-1：乙醇+C末端缺失-PERK与Z-VAD、Nec-1或ferrostatin-1。 a：与对照相比， p < 0.05； b：(L)中相对于乙醇或E+△C， p < 0.05； c：相对于（B，K）中的E+△C，相对于（F，G，J）中的E+△K，相对于（L）中的E+△C + Nec-1， P < 0.05； d：与 E+△C +Z-VAD 相比，P < 0.05。
3.7 PERK Deficiency Protectes Ethanol-Treated Cells from Ferroptosis and the Effect of Quercetin
3.7 PERK 缺乏保护乙醇处理的细胞免于铁死亡和槲皮素的作用
We investigate the role of PERK in ferroptosis induced by ethanol using the ferroptosis inducer RSL3 or the inhibitor ferrostatin-1 in AML12 cells (Figure 8A). Ethanol plus RSL3 increased PERK-related MAMs, while ethanol plus ferrostatin-1 decreased them, suggesting that PERK-related MAMs are involved in ferroptosis in ALD. We transfected AML12 cells with the siRNA of PERK, and showed the knockdown effect in Figure 8B. The PERK knockdown abolished ferroptosis induced by ethanol (Figure 8C–H, decreased the expression of ACSL4, LDH release, and lipid peroxidation, which were increased by ethanol, and increased the expression of xCT and GPx4, which were reduced by ethanol). We also showed that quercetin, by modulating the PERK-related MAMs (yellow fluorescence), protected against ferroptosis induced by ethanol (Figure 8I). We concluded that PERK elevation in ALD induced ferroptosis and increased MAMs, and quercetin modulated ferroptosis by modulating the PERK-related MAMs.
我们使用铁死亡诱导剂RSL3或抑制剂ferrostatin-1在AML12细胞中研究PERK在乙醇诱导的铁死亡中的作用（图8A ）。乙醇加RSL3增加了PERK相关的MAM，而乙醇加ferrostatin-1则减少了它们，这表明PERK相关的MAM与ALD中的铁死亡有关。我们用PERK的siRNA转染AML12细胞，并在图8B中显示了敲低效果。 PERK敲除消除了乙醇诱导的铁死亡（图8C-H ，减少了ACSL4、LDH释放和脂质过氧化的表达，这些表达被乙醇增加，并且增加了xCT和GPx4的表达，这些表达被乙醇减少）。我们还表明，槲皮素通过调节 PERK 相关的 MAM（黄色荧光），可以防止乙醇诱导的铁死亡（图8I ）。我们得出结论，ALD 中 PERK 升高会诱导铁死亡并增加 MAM，而槲皮素通过调节 PERK 相关的 MAM 来调节铁死亡。
槲皮素和 PERK 对乙醇暴露细胞铁死亡的影响。 A) 在不同处理的 AML12 细胞中使用针对 PERK（绿色）和 VDAC1（红色）的抗体进行免疫荧光染色 (400×)。 B) 用三种 PERK siRNA、ΔC 质粒或 ΔK 质粒转染的 AML12 细胞中的 PERK 表达。 CF) 使用或不使用 siPERK 的乙醇暴露细胞中 ACSL4、xCT 和 Gpx4 的表达， n = 6。G) LDH 释放， n = 3。H) 通过荧光板读数器计算脂质过氧化， n = 3。I ) 在用 RSL-3 和/或槲皮素处理的暴露于乙醇的 AML12 细胞中，使用针对 PERK（绿色）和 VDAC1（红色）的抗体进行免疫荧光染色。所有数据均以平均值±SEM 表示。 CON，对照组； EtOH，乙醇组（200 mM）； E+siPERK，乙醇+siPERK（siPERK，100 nM）； ΔC：C末端缺失-PERK组(PERK-ΔC，1.0μgmL -1 )； ΔK：激酶死亡-PERK组（PERK-ΔK，0.75 μg mL -1 ）； siPERK：siPERK（siPERK，100 nm）。 a：与对照相比， p < 0.05； b：相对于乙醇， p < 0.05； c：与 E+siPERK 相比， p < 0.05。
4 Discussion 4 讨论
In this study, we showed that ethanol induces ferroptosis by forming MAMs through the structure of PERK, independently of its kinase activity under ethanol treatment. Quercetin reduces the expression of PERK in MAMs, increases MAMs instability, and prevents hepatic ferroptosis and injury form chronic ethanol exposure.
在这项研究中，我们发现乙醇通过 PERK 结构形成 MAM 来诱导铁死亡，而与乙醇处理下的激酶活性无关。槲皮素可降低 MAM 中 PERK 的表达，增加 MAM 的不稳定性，并防止肝铁死亡和慢性乙醇暴露造成的损伤。
ALD is a common liver disease worldwide. Besides steatosis, iron accumulation is a characteristic pathology in ALD.[48] The liver iron level is associated with fibrosis and cirrhosis.[49] Ethanol-induced oxidative stress and innate immunity trigger programmed cell death (apoptosis, necroptosis, pyroptosis, and ferroptosis), which is central to ALD progression.[50] Ferroptosis, defined in 2012, is driven by iron-dependent lipid peroxidation.[4] Previous studies have identified increased of PTGS2 and ACSL4 and decreased GSH system, CoQ10 system, and NRF2 transcription pathway as ferroptosis biomarkers.[20, 51, 52] Recently, DHODH was found to acts as a novel ferroptosis defense mechanism in the mitochondrial inner membrane, parallel to cytoplasmic GPx4 or FSP1 defense mechanisms.[53] Studies have shown that ferroptosis can be induced by elevated LIP due to anomalies in the ferroportin and transferrin receptor,[54, 55] and that ferroptosis is closely related to ALD progression.[14, 56] In line with previous studies,[48] our study showed increased total iron and LIP under ethanol administration in our study. Moreover, our results showed decreased GPx4 and xCT and increased ACSL4 protein levels in the ethanol group, indicating ferroptosis occurrence. Ferroptosis inhibitors and quercetin prevented ethanol-induced changes in ACSL4, GPx4, and xCT protein levels and reduced cell damage. This is consistent with previous findings that quercetin alleviates ferroptosis in diabetes and acute kidney injury,[57, 58] confirming ferroptosis in ALD and the quercetin's protective effect. This is related to quercetin's ability to change iron absorption function as an iron chelator and affect iron homeostasis,[59] highlighting quercetin's clinical value in ALD treatment.
ALD 是世界范围内常见的肝病。除了脂肪变性之外，铁积累也是 ALD 的特征性病理。 48肝铁水平与纤维化和肝硬化有关。 49乙醇诱导的氧化应激和先天免疫会触发程序性细胞死亡（细胞凋亡、坏死性凋亡、细胞焦亡和铁死亡），这是 ALD 进展的核心。 50 2012 年定义的铁死亡是由铁依赖性脂质过氧化引起的。 4先前的研究已将 PTGS2 和 ACSL4 的增加以及 GSH 系统、CoQ10 系统和 NRF2 转录途径的减少确定为铁死亡生物标志物。 20 , 51 , 52最近，人们发现 DHODH 在线粒体内膜中充当一种新型的铁死亡防御机制，与细胞质 GPx4 或 FSP1 防御机制平行。 53研究表明，由于铁转运蛋白和转铁蛋白受体异常，LIP 升高可诱导铁死亡， 54 , 55并且铁死亡与 ALD 进展密切相关。 14 , 56与之前的研究一致， 48我们的研究表明，在我们的研究中，乙醇给药后总铁和 LIP 有所增加。此外，我们的结果显示，乙醇组中 GPx4 和 xCT 降低，ACSL4 蛋白水平升高，表明铁死亡的发生。 铁死亡抑制剂和槲皮素可防止乙醇诱导的 ACSL4、GPx4 和 xCT 蛋白水平的变化，并减少细胞损伤。这与之前的研究结果一致，即槲皮素可减轻糖尿病和急性肾损伤中的铁死亡， 57 , 58证实了 ALD 中的铁死亡和槲皮素的保护作用。这与槲皮素作为铁螯合剂改变铁吸收功能并影响铁稳态的能力有关， 59突出了槲皮素在 ALD 治疗中的临床价值。
MAMs are structures between mitochondria and ER at 10–25 nm distance, providing a platform for lipid metabolism, Ca2+ exchange, mitochondrial dynamics, autophagy, apoptosis, and ER stress.[60] MAMs increase in ATP import under ERS but switch to apoptosis.[61] Cellular lipid imbalance activates UPR by SREBP, which enhances MAMs.[62, 63] PERK localizes in MAMs and associated with UPR-dead forms and MAMs growth.[47] It forms a complex with S1R or Mfn2 that participates in MAMs composition.[64] A recent study reported that MAMs mediated arsenic-induced acute lung injury and ferroptosis and that MAMs dysfunction resulted from reduced PERK and Mf2 interaction, suggesting PERK-MAMs as potential ferroptosis targets, but their role in ALD is unclear.[26] We found increased MAMs associated with ferroptosis and activated PERK pathway of UPR in the ethanol group, while the ATF6 pathway was downregulated, possibly due to decompensation in this ALD stage. Quercetin improved PERK-MAMs-mediated ferroptosis and modulated UPR activation by lowering PERK phosphorylation in the ethanol group. This agrees with the previous finding that quercetin accumulates in mitochondria and protects mitochondrial function by regulating ERS.[65
MAM 是线粒体和内质网之间距离为 10-25 nm 的结构，为脂质代谢、Ca 2+交换、线粒体动力学、自噬、细胞凋亡和内质网应激提供平台。 60 MAM 在 ERS 下增加 ATP 导入，但转为细胞凋亡。 61细胞脂质失衡通过 SREBP 激活 UPR，从而增强 MAM。 62 , 63 PERK 位于 MAM 中，并与 UPR 死亡形式和 MAM 生长相关。 47它与 S1R 或 Mfn2 形成复合物，参与 MAM 的组成。 64最近的一项研究报告称，MAM 介导砷诱导的急性肺损伤和铁死亡，并且 MAM 功能障碍是由于 PERK 和 Mf2 相互作用减少所致，表明 PERK-MAM 是潜在的铁死亡靶标，但其在 ALD 中的作用尚不清楚。 26我们发现，乙醇组中与铁死亡相关的 MAM 增加，并且 UPR 的 PERK 通路激活，而 ATF6 通路下调，可能是由于该 ALD 阶段的失代偿。槲皮素改善了 PERK-MAM 介导的铁死亡，并通过降低乙醇组中的 PERK 磷酸化来调节 UPR 激活。这与之前的发现一致，即槲皮素在线粒体中积累，并通过调节ERS来保护线粒体功能。 65]
PERK consists of ER lumen domain and the cytoplasmic domain, and the cytoplasmic domain includes serine/threonine protein kinase activity, which autophosphorylates and activates UPR by phosphorylating eIF2α at Ser51 site.[66] We transfected AML12 cells with mutant PERK plasmids, ΔC and ΔK, and found that ΔC reduced ethanol-induced ferroptosis but increased LDH release and was inhibited by apoptosis and necrosis inhibitors, while ΔK worsened ethanol-induced ferroptosis. This suggests that PERK induces ferroptosis through its cytoplasmic domain, related to MAMs formation, while its kinase activity reduced ferroptosis but increased apoptosis and necrosis, which possibly due to P62-keap1-Nrf2 antioxidant pathway activation.[67] Thus, whether quercetin reduces ferroptosis under ethanol by regulating ERS or acting on PERK needs further investigated.
PERK由ER腔结构域和胞质结构域组成，胞质结构域包含丝氨酸/苏氨酸蛋白激酶活性，通过磷酸化Ser51位点的eIF2α来自身磷酸化并激活UPR。 66我们用突变型 PERK 质粒 ΔC 和 ΔK 转染 AML12 细胞，发现 ΔC 减少了乙醇诱导的铁死亡，但增加了 LDH 的释放，并被细胞凋亡和坏死抑制剂抑制，而 ΔK 则恶化了乙醇诱导的铁死亡。这表明 PERK 通过其细胞质结构域诱导铁死亡，与 MAM 形成相关，而其激酶活性减少铁死亡，但增加细胞凋亡和坏死，这可能是由于 P62-keap1-Nrf2 抗氧化途径激活所致。 67因此，槲皮素是否通过调节 ERS 或作用于 PERK 来减少乙醇条件下的铁死亡，需要进一步研究。
In conclusion, we showed that quercetin regulated PERK expression on MAMs to normalize MAMs and reduce ethanol-induced ferroptosis. PERK's cytoplasmic domain enhanced MAMs formation, involved in ethanol-induced ferroptosis regardless of kinase activity. We suggest PERK-MAMs as a potentially therapeutic target to mitigate ALD progression by inhibiting ferroptosis in hepatocytes. Quercetin improves alcoholic liver damage by blocking ferroptosis via PERK-MAMs, highlighting new light on the prophylactic value of iron-chelating phytochemicals for ALD.
总之，我们发现槲皮素调节 MAM 上的 PERK 表达，使 MAM 正常化并减少乙醇诱导的铁死亡。 PERK 的胞质结构域增强了 MAM 的形成，无论激酶活性如何，都参与乙醇诱导的铁死亡。我们建议 PERK-MAM 作为潜在的治疗靶点，通过抑制肝细胞铁死亡来减缓 ALD 进展。槲皮素通过 PERK-MAM 阻断铁死亡来改善酒精性肝损伤，这凸显了铁螯合植物化学物质对 ALD 的预防价值。
Acknowledgements 致谢
This work was supported by the National Natural Science Foundation of China (No. 81673164， 8187120776， and 82273628), and National Outstanding Youth Science Fund Project of National Natural Science Foundation of China (No. 82003448). The authors appreciated for supporting and helping of the teachers and students form Huazhong University of Science and Technology Tongji Medical College, and the equipment supporting of the SHARED CENTER FOR LARGE INSTRUMENT platform of the School of Public Health.
该工作得到了国家自然科学基金项目（No. 81673164、8187120776、82273628）和国家自然科学基金杰出青年科学基金项目（No. 82003448）的资助。感谢华中科技大学同济医学院师生的支持和帮助，以及公共卫生学院大型仪器共享中心平台的设备支持。
Conflict of Interest 利益冲突
The authors declare no conflict of interest.
作者声明不存在利益冲突。
Author Contributions 作者贡献
H.K.L., P.Y., J.S.Y., X.P.G., J.J.L., Y.H.T., H.M.C., L.C., Y.Z., L.L.W., and H.X.L. performed experimental analysis and discussion. P.Y. and J.S.Y. provided valuable suggestions and help in experiment revision. H.K.L., X.P.G., and J.J.L. performed all experiments. X.P.G., H.K.L. designed experiments and H.K.L. wrote the manuscript with advice from all other authors.
HKL、PY、JSY、XPG、JJL、YHT、HMC、LC、YZ、LLW、HXL 进行了实验分析和讨论。 PY和JSY为实验修改提供了宝贵的建议和帮助。 HKL、XPG 和 JJL 进行了所有实验。 XPG、HKL 设计了实验，HKL 在所有其他作者的建议下撰写了手稿。
