药学学报

	Small molecule	Structure	Mechanism
Inducer	RSL3		Inhibit GPX4 activity
ML162		Inhibit GPX4 activity
ML210		Inhibit GPX4 activity
FIN56		Decrease GPX4 levels
FINO₂		Induce lipid peroxidation
Erastin		Inhibit system X_c^-
Sorafenib		Inhibit system X_c^-
BSO		Deplete GSH
FeCl₂	–	Iron overload
(NH₄)₂Fe(SO₄)₂	–	Iron overload
Inhibitor	Deferoxamine (DFO)		Chelate iron
Vitamin E		Inhibit propagation of lipid peroxidation
Ferrostain-1		Inhibit lipid peroxidation
Liproxstatin-1		Inhibit lipid peroxidation
CoQ₁₀		Inhibit lipid peroxidation
Monounsaturated fatty acid		Inhibit lipid peroxidation

	Small molecule	Structure	Mechanism
Inducer	RSL3		Inhibit GPX4 activity
ML162		Inhibit GPX4 activity
ML210		Inhibit GPX4 activity
FIN56		Decrease GPX4 levels
FINO₂		Induce lipid peroxidation
Erastin		Inhibit system X_c^-
Sorafenib		Inhibit system X_c^-
BSO		Deplete GSH
FeCl₂	–	Iron overload
(NH₄)₂Fe(SO₄)₂	–	Iron overload
Inhibitor	Deferoxamine (DFO)		Chelate iron
Vitamin E		Inhibit propagation of lipid peroxidation
Ferrostain-1		Inhibit lipid peroxidation
Liproxstatin-1		Inhibit lipid peroxidation
CoQ₁₀		Inhibit lipid peroxidation
Monounsaturated fatty acid		Inhibit lipid peroxidation

Type of cell death	Typical morphological feature
Ferroptosis	Increased mitochondrial membrane densities Small size of mitochondria Mitochondrial crista vanished
Apoptosis	Membrane blebbing Pseudopod retraction Cell shrinkage Chromatin condensation and margination
Necrosis	Membrane rupture Cytoplasmic and organelle swelling
Necroptosis	Dilation of the perinuclear space Cell rounding and swelling Membrane rupture
Pyroptosis	Cell swelling Membrane rupture Deformation of organelles Chromatin condensation
Autophagy	Formation of double membrane structures

Type of cell death	Typical morphological feature
Ferroptosis	Increased mitochondrial membrane densities Small size of mitochondria Mitochondrial crista vanished
Apoptosis	Membrane blebbing Pseudopod retraction Cell shrinkage Chromatin condensation and margination
Necrosis	Membrane rupture Cytoplasmic and organelle swelling
Necroptosis	Dilation of the perinuclear space Cell rounding and swelling Membrane rupture
Pyroptosis	Cell swelling Membrane rupture Deformation of organelles Chromatin condensation
Autophagy	Formation of double membrane structures

Assay and tool	Method	Advantage	Disadvantage
LDH assay	Detects cell death based on chemiluminescence reaction	Easy to operate and low equipment requirements	Not specific for detecting ferroptosis
PI	A fluorescent probe for detecting cell death	Easy to operate and achieve quantitative and visual analysis of cell death	Not specific for detecting ferroptosis
SYTOX Green	A fluorescent probe for detecting cell death	Easier to operate and achieve quantitative and visual analysis of cell death than PI	Not specific for detecting ferroptosis
TEM	Detects morphological feature of ferroptosis	Directly observe the morphological features of ferroptosis	Subjectivity, complex to operate
C11-BODIPY^581/591	Lipid sensor for detecting lipid ROS in living cells	Highly lipophilic, easy for membrane entry, low toxicity	Not specific for detecting lipid peroxidation
Click-iT LAA	Detects lipid peroxidation-derived protein modifications in fixed cells	Localizes and quantifies lipid peroxidation in cell membranes	Linoleic acid dependence, not specific for detecting lipid peroxidation
Liperfluo	A fluorescent probe for relatively specific detecting lipid peroxidation in living cells	Specifically detects and quantifies lipid peroxidation in the membrane of living cells	Unable to distinguish different types of lipid peroxides
TBARS assay	Detects MDA product of lipid peroxidation	Easy to operate and low equipment requirements	Less specific for detecting lipid peroxidation, prone to false positives
DNPH assay	Detects 4-HNE product of lipid peroxidation	Easy to operate and low equipment requirements	Less specific for detecting lipid peroxidation, unable to distinguish different types of aldehydes
Immunoassays for lipid peroxidation products	Detects MDA- and 4-HNE-protein adduct	Easy to operate, strong specific for the detection of MDA- and 4-HNE-protein adduct	Unable to distinguish the specific modification of MDA- and 4-HNE-protein adduct
Oxidative lipidomic based on LC-MS	Identification and structural characterization of oxidatively modified lipids	High specificity, sensitivity, accuracy for detecting ferroptosis related lipid peroxidation	Complex to operate, require special equipment and technician
ICP-MS	Detects total iron level	Accurate determination of iron levels	Only for the total iron level detection
Prussian blue staining	Detects the distribution and amount of iron deposits in tissues	Good stability	Not suitable for the detection of free iron levels in living cells
Calcein-AM	A fluorescent probe detects iron level based on iron chelation	Sensitive	Not specific
RhoNox-1	A fluorescent probe detects Fe²⁺ level in living cells	Sensitive, high specificity for Fe²⁺	Not suitable for fixed cell and paraffin slices
FerrOrrange	A fluorescent probe detects Fe²⁺ level in living cells	Sensitive, high specificity for Fe²⁺, low toxicity	Not suitable for fixed cell and paraffin slices
Mito-FerroGreen	A fluorescent probe detects Fe²⁺ level in mitochondria	Sensitive, high specificity for Fe²⁺, low toxicity	Not suitable for fixed cell and paraffin slices
FRET iron probe 1	Detects Fe²⁺ level based on FRET	High specificity for Fe²⁺	Not suitable for fixed cell and paraffin slices
GSH assay kit	Detects GSH content	Sensitive, easy to operate and low equipment requirements	Unable to visualize the content of GSH
LC-MS	Detects GSH/GSSG content	High specificity, sensitivity, accuracy for detecting GSH/GSSG	Complex to operate, require special equipment and technician
BioTracker cystine-FITC	Detects cystine uptake	Achieve quantitative and visual analysis of cystine uptake	Prone to false positives
Radioisotope labeled cystine	Detects cystine uptake	High specificity, achieve tracking the cystine	Complex to operate, require special equipment
Immunoassays for key regulators of ferroptosis	Detects key regulators of ferroptosis	Sensitive, strong specific for detecting key regulators of ferroptosis	Subjectivity, prone to false positives
PCR analysis for key regulators of ferroptosis	Detects key regulators of ferroptosis	Sensitive, easy to operate, specific for detecting key regulators of ferroptosis	Relative expensive

Assay and tool	Method	Advantage	Disadvantage
LDH assay	Detects cell death based on chemiluminescence reaction	Easy to operate and low equipment requirements	Not specific for detecting ferroptosis
PI	A fluorescent probe for detecting cell death	Easy to operate and achieve quantitative and visual analysis of cell death	Not specific for detecting ferroptosis
SYTOX Green	A fluorescent probe for detecting cell death	Easier to operate and achieve quantitative and visual analysis of cell death than PI	Not specific for detecting ferroptosis
TEM	Detects morphological feature of ferroptosis	Directly observe the morphological features of ferroptosis	Subjectivity, complex to operate
C11-BODIPY^581/591	Lipid sensor for detecting lipid ROS in living cells	Highly lipophilic, easy for membrane entry, low toxicity	Not specific for detecting lipid peroxidation
Click-iT LAA	Detects lipid peroxidation-derived protein modifications in fixed cells	Localizes and quantifies lipid peroxidation in cell membranes	Linoleic acid dependence, not specific for detecting lipid peroxidation
Liperfluo	A fluorescent probe for relatively specific detecting lipid peroxidation in living cells	Specifically detects and quantifies lipid peroxidation in the membrane of living cells	Unable to distinguish different types of lipid peroxides
TBARS assay	Detects MDA product of lipid peroxidation	Easy to operate and low equipment requirements	Less specific for detecting lipid peroxidation, prone to false positives
DNPH assay	Detects 4-HNE product of lipid peroxidation	Easy to operate and low equipment requirements	Less specific for detecting lipid peroxidation, unable to distinguish different types of aldehydes
Immunoassays for lipid peroxidation products	Detects MDA- and 4-HNE-protein adduct	Easy to operate, strong specific for the detection of MDA- and 4-HNE-protein adduct	Unable to distinguish the specific modification of MDA- and 4-HNE-protein adduct
Oxidative lipidomic based on LC-MS	Identification and structural characterization of oxidatively modified lipids	High specificity, sensitivity, accuracy for detecting ferroptosis related lipid peroxidation	Complex to operate, require special equipment and technician
ICP-MS	Detects total iron level	Accurate determination of iron levels	Only for the total iron level detection
Prussian blue staining	Detects the distribution and amount of iron deposits in tissues	Good stability	Not suitable for the detection of free iron levels in living cells
Calcein-AM	A fluorescent probe detects iron level based on iron chelation	Sensitive	Not specific
RhoNox-1	A fluorescent probe detects Fe²⁺ level in living cells	Sensitive, high specificity for Fe²⁺	Not suitable for fixed cell and paraffin slices
FerrOrrange	A fluorescent probe detects Fe²⁺ level in living cells	Sensitive, high specificity for Fe²⁺, low toxicity	Not suitable for fixed cell and paraffin slices
Mito-FerroGreen	A fluorescent probe detects Fe²⁺ level in mitochondria	Sensitive, high specificity for Fe²⁺, low toxicity	Not suitable for fixed cell and paraffin slices
FRET iron probe 1	Detects Fe²⁺ level based on FRET	High specificity for Fe²⁺	Not suitable for fixed cell and paraffin slices
GSH assay kit	Detects GSH content	Sensitive, easy to operate and low equipment requirements	Unable to visualize the content of GSH
LC-MS	Detects GSH/GSSG content	High specificity, sensitivity, accuracy for detecting GSH/GSSG	Complex to operate, require special equipment and technician
BioTracker cystine-FITC	Detects cystine uptake	Achieve quantitative and visual analysis of cystine uptake	Prone to false positives
Radioisotope labeled cystine	Detects cystine uptake	High specificity, achieve tracking the cystine	Complex to operate, require special equipment
Immunoassays for key regulators of ferroptosis	Detects key regulators of ferroptosis	Sensitive, strong specific for detecting key regulators of ferroptosis	Subjectivity, prone to false positives
PCR analysis for key regulators of ferroptosis	Detects key regulators of ferroptosis	Sensitive, easy to operate, specific for detecting key regulators of ferroptosis	Relative expensive

铁死亡主要检测方法及其应用的研究进展

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欧阳淑桦 ¹^,²^,³ , 吴燕萍 ¹^,²^,³ , 孙万阳 ¹^,²^,³ , 闫昌誉 ¹^,²^,³ , 栗原博 ¹^,²^,³ , 李怡芳 ¹^,²^,³ , 何蓉蓉 ¹^,²^,³^,^*

药学学报 | 专题报道：疾病氧化应激损伤机制与药物干预研究 2022,57(6): 1544-1556

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药学学报 | 专题报道：疾病氧化应激损伤机制与药物干预研究 2022, 57(6): 1544-1556

铁死亡主要检测方法及其应用的研究进展

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欧阳淑桦¹^,²^,³, 吴燕萍¹^,²^,³, 孙万阳¹^,²^,³, 闫昌誉¹^,²^,³, 栗原博¹^,²^,³, 李怡芳¹^,²^,³, 何蓉蓉¹^,²^,³^,^*

作者信息

1.暨南大学, 广东省疾病易感性及中医药研发工程技术研究中心, 广东广州 510632

2.暨南大学, 中药及天然药物研究所, 广东广州 510632

3.暨南大学, 广东省中药药效物质基础及创新药物研究重点实验室, 广东广州 510632

通讯作者:

*何蓉蓉, Tel / Fax: 86-20-85221559, E-mail: rongronghe@jnu.edu.cn

Research progress on the detection methods and their application in ferroptosis

Shu-hua OUYANG¹^,²^,³, Yan-ping WU¹^,²^,³, Wan-yang SUN¹^,²^,³, Chang-yu YAN¹^,²^,³, Hiroshi KURIHARA¹^,²^,³, Yi-fang LI¹^,²^,³, Rong-rong HE¹^,²^,³^,^*

Affiliations

1. Guangdong Engineering Research Center of Chinese Medicine and Disease Susceptibility, Jinan University, Guangzhou 510632, China

2. Institute of Traditional Chinese Medicine and Natural Products, Jinan University, Guangzhou 510632, China

3. Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research, Jinan University, Guangzhou 510632, China

出版时间: 2022-06-12 doi: 10.16438/j.0513-4870.2022-0276

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摘要

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铁死亡是一种在形态学、生物化学、基因学等方面均不同于细胞凋亡、坏死、焦亡和自噬等传统细胞死亡途径的新型细胞死亡类型, 其主要特征为铁累积和脂质过氧化, 铁死亡的调控机制涉及铁代谢、谷胱甘肽代谢和脂质过氧化反应等方面, 并与肿瘤、衰老、神经退行性疾病、缺血再灌注损伤、心脑血管疾病、肾脏损伤、肝纤维化等疾病的病理过程密切相关。如何有效研究铁死亡调控机制治疗疾病已成为铁死亡研究领域的热点。近年来, 随着对铁死亡研究的深入, 铁死亡的识别、确认及机制等方面的研究进展显著, 用于检测铁死亡的方法不断发展且日趋成熟, 形态学、生物化学、分子生物学、基因学等方面的技术已广泛应用于铁死亡的检测与研究。为进一步加深读者对铁死亡及其检测方法的认识, 本文主要综述了目前常规使用的铁死亡检测方法及其应用, 并对其在铁死亡检测中的优缺点进行总结和讨论。这些方法将有助于更好地理解和研究铁死亡的诱导和调控过程及其在治疗疾病中的作用。

关键词

铁死亡 / 检测方法 / 荧光探针 / 液相色谱-质谱联用 / 脂质过氧化

Abstract

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Ferroptosis is a novel type of cell death, which is distinguished from the traditional cell death pathways such as apoptosis, proptosis, necrosis and autophagy in terms of morphology, biochemistry and genetics. The main features of ferroptosis are the iron accumulation and lipid peroxidation. The regulation mechanism of ferroptosis involves glutathione metabolism, lipid peroxidation reactions and iron metabolism, which are closely related to the pathological process of tumor, aging, neurodegenerative diseases, ischemia reperfusion injury, cardiovascular and cerebrovascular diseases, kidney injury, hepatic fibrosis and so on. How to effectively study the role of ferroptosis regulation mechanism in the treatment of diseases becomes the hot spot and focus of the ferroptosis research. In recent years, with the in-depth study of ferroptosis, the identification, confirmation and the mechanism of ferroptosis have been developed significantly and have come forth continuously, in the meantime, techniques based on the morphology, biochemistry, molecular biology and genetics have been widely applied in the detection of ferroptosis. In order to deepen readers' understanding of ferroptosis and its detection methods, this paper will mainly review the current research progress on the detection methods and their application in ferroptosis, summarize and discuss their advantages and disadvantages in the detection of ferroptosis, this knowledge are crucial for better understanding and studying the biological function of ferroptosis.

Key words

ferroptosis / detection method / fluorescence probe / liquid chromatography-mass spectrometry / lipid peroxidation

引用本文

欧阳淑桦, 吴燕萍, 孙万阳, 闫昌誉, 栗原博, 李怡芳, 何蓉蓉. 铁死亡主要检测方法及其应用的研究进展. 药学学报, 2022 , 57 (6) : 1544 -1556 . DOI: 10.16438/j.0513-4870.2022-0276

Shu-hua OUYANG, Yan-ping WU, Wan-yang SUN, Chang-yu YAN, Hiroshi KURIHARA, Yi-fang LI, Rong-rong HE. Research progress on the detection methods and their application in ferroptosis[J]. Acta Pharmaceutica Sinica, 2022 , 57 (6) : 1544 -1556 . DOI: 10.16438/j.0513-4870.2022-0276

正文

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铁死亡(ferroptosis) 于2012年被Stockwell团队发现, 是一种在形态学、基因学以及生物化学等方面与凋亡、坏死、自噬、焦亡等不同的铁依赖的细胞死亡方式^[1], 主要由铁或酶介导的磷脂过氧化引起细胞发生死亡。大量的研究显示, 铁死亡与肿瘤^[2]、衰老^[3]、神经退行性疾病^[4]、缺血再灌注损伤^[5]、肾脏损伤^[6]、心脑血管疾病^[4]、肝纤维化^[7]等疾病的病理过程密切相关, 干预铁死亡可能作为一种潜在的疾病治疗策略。目前, 铁死亡也已经成为了生命科学领域的研究热点和焦点^{[8, 9]}。细胞发生铁死亡主要表现为一系列特征性变化, 包括线粒体嵴消失、线粒体变小、膜密度增加、细胞变小变圆且相互之间分离等细胞形态的改变^[1]以及铁代谢、谷胱甘肽代谢、脂质过氧化反应、磷脂重塑等方面的基因学及生物化学特征的变化^[10] (图 1)。基于铁死亡的这些特征性变化, 形态学、生物化学、分子生物学、基因学等方面的检测方法得到了广泛应用, 这些检测方法结合铁死亡工具药物^[11] (表 1) 的使用, 在铁死亡细胞识别、确认、调控机制等方面的研究发挥重要作用。为进一步加深读者对铁死亡及其检测方法的认识, 本文将对铁死亡相关检测方法的原理与应用进行综述, 并对其优点和局限性进行讨论, 希望能够为相关领域的科研工作者提供参考。

1 细胞死亡的检测方法

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细胞的铁死亡可以使用一些常用的细胞死亡检测方法进行研究, 如乳酸脱氢酶(lactate dehydrogenase, LDH) 检测法和碘化丙啶(propidium iodide, PI)、SYTOX Green等细胞核荧光染色标记法。它们在铁死亡的早期检识和筛选过程中发挥重要的作用。

1.1 LDH法检测细胞死亡情况

细胞死亡伴随的细胞膜结构破坏会导致细胞质中的内容物释放到培养液中, 其中包括活性较为稳定的LDH, 因此检测LDH的释放可用于死细胞和受损细胞的定量分析^[12]。LDH的相对水平通过其体外酶活力来反映, 即在LDH催化乳酸脱氢的作用下, NAD⁺被还原生成NADH, 而NADH和四唑盐类化合物被硫辛酰胺脱氢酶催化反应生成NAD⁺和红色甲臜, 生成的红色甲臜在490 nm下产生吸收峰, 通过比色法可以定量乳酸脱氢酶的活性, 进而测定死细胞和受损细胞的数量。该方法操作简单, 对仪器设备要求低, 但特异性不高, 不能用于区分不同的细胞死亡方式。因此, 这种方法一般作为铁死亡研究的早期筛选手段, 如Doll等^[13]运用该方法发现Acsl4是促发铁死亡的关键基因, 他莫昔芬诱导Gpx4条件性敲除的细胞同时敲除Acsl4能够显著抑制LDH的释放; Wang等^[14]通过检测LDH的释放联合铁死亡工具药及其他铁死亡检测方法共同证明了(+)-黄皮酰胺能够抑制对乙酰氨基酚和铁死亡诱导剂erastin诱导的肝细胞铁死亡。

1.2 荧光探针法

PI是一种常用于检测细胞死亡的荧光染料, 当细胞膜破损后, 它可以进入细胞嵌入双链DNA, 形成PI-DNA复合物释放出红色荧光(激发和发射波长分别为535和615 nm)。PI常与钙黄绿素乙酰氧基甲酯(Calcein-AM) 等活细胞染料联用, 借助流式细胞仪、荧光显微镜等仪器广泛用于活细胞和死细胞的定量分析^[15]。近年来, 许多研究也将PI应用于铁死亡的检测, 如Gao等^[16]使用PI荧光探针检测细胞的死亡率, 发现了线粒体三羧酸循环或电子传递链能够促进半胱氨酸剥夺导致的细胞死亡, 结合其他实验共同证明了线粒体在半胱氨酸剥夺诱导的铁死亡中起关键作用。该研究团队^[17]也在多种细胞进行PI荧光染色标记死细胞, 发现自噬抑制剂BafA1和氯喹能够抑制erastin引起的细胞铁死亡, 辅助证明了自噬能够正向调控铁死亡的过程。

SYTOX Green是另外一种用于检测细胞死亡的高亲和力核酸染料, 它与PI的作用原理相似, 可轻松穿透细胞膜受损的死亡细胞, 而不会穿透活细胞。死细胞在与SYTOX Green短暂孵育后, 会被激发出亮绿色的荧光(激发和发射波长分别为502和523 nm)^[18]。与PI相比, SYTOX Green与核酸结合的能力更强, 操作更为简单(染色后无需清洗), 这也使得其在铁死亡研究中的应用更具优势。如Magtanong等^[19]运用SYTOX Green对不同类型脂肪酸处理的稳定表达mKate的HT1080细胞进行了标记, 并通过延时荧光拍摄发现油酸等单不饱和脂肪酸能够抑制细胞发生铁死亡; Riegman等^[20]则运用SYTOX Green对不同铁死亡诱导剂刺激的几种细胞进行活细胞成像, 发现了铁死亡细胞以波状形式在细胞中进行传播的现象, 揭示了新的铁死亡执行机制。

值得注意的是, 这些荧光探针虽然操作简便, 可用作定量和可视化分析, 但它们存在检测特异性不高的缺点。因此, 在实际应用的时候, 应结合使用铁死亡及其他细胞死亡方式的工具药来帮助鉴定铁死亡的发生。

2 形态学检测—透射电子显微镜(transmission electron microscope, TEM)

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细胞发生不同形式的死亡时, 会出现一系列特异的形态学特征, 因此, 通过形态学检测可简单直观地区分不同的细胞死亡方式(表 2)^{[1, 21, 22]}。TEM是形态学检测最为直观的方法, 通过TEM可以观察到铁死亡的形态学变化: 线粒体变小、线粒体嵴消失、线粒体膜密度增加、细胞变小变圆且相互之间分离^[1]。Klöditz等^[23]采用TEM观察到Junket细胞发生不同死亡方式时的细胞形态变化, 并通过比较它们的细胞形态差异成功区分出发生铁死亡的细胞; Yoshida等^[24]借助TEM确认了烟雾暴露能够诱导HUVECs细胞发生铁死亡; Bao等^[25]通过TEM观察到铁通道蛋白Ferroportin缺失的小鼠及阿尔茨海默病模型小鼠的海马神经元均出现了铁死亡的细胞形态学特征。形态学检测方法因直观、清晰等优点被广泛应用于鉴别铁死亡, 但是其仍然存在很多不足之处, 如实际应用中实验操作人员对于样品制备、镜下细胞及亚细胞形态的判定等过程均可能存在主观的偏差; 不能满足定量的要求; 前处理较为复杂; 实验周期较长等, 由于形态学检测具有较大的局限性, 因而常作为其他技术的基础, 与其他检测方法结合使用。

3 脂质过氧化的检测方法

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细胞膜磷脂的过氧化在铁死亡过程中发挥核心作用^[11]。累积的磷脂过氧化物会断裂代谢为多种脂质过氧化终末产物^[26], 针对这些脂质过氧化物及其代谢产物的常用检测方法为荧光探针法、化学发光法、免疫分析法及基于高分辨液相色谱-质谱联用技术开发的氧化脂质组学分析(liquid chromatography-mass spectrometry, LC-MS, based oxidative lipidomics) 等方法, 其中基于LC-MS的氧化脂质组学技术可准确检测和鉴定氧化磷脂及其代谢产物的类型。

3.1 基于荧光探针的脂质过氧化物检测方法

脂质过氧化荧光探针借助流式细胞仪、荧光显微镜、激光共聚焦显微镜成像系统等仪器, 可实现在单个细胞或者亚细胞结构上对脂质过氧化水平进行监测与定量。

C11-BODIPY^581/591是一种亲脂性的活细胞染料, 可用于检测细胞中的脂质过氧化水平, 近年来也开始被广泛用于指示铁死亡细胞内脂质过氧化物的累积^[27]。Gao等^[16]使用C11-BODIPY^581/591对细胞染色并用流式细胞术检测定量, 发现阻断三羧酸循环和电子传递链能够有效抑制细胞脂质过氧化物的积累和铁死亡。Lei等^[28]检测了电离辐射处理后细胞的C11-BODIPY^581/591阳性细胞率, 帮助证明了电离辐射能够诱导细胞发生铁死亡。C11-BODIPY^581/591在正常状态时显示为亮红色荧光, 但当其多不饱和丁二烯部分被氧化后, 会致使其荧光基团的荧光发射峰从590 nm转移到510 nm, 从而使荧光颜色从红色转变为绿色^[29] (图 2A)。这种荧光探针的优势在于它对O₂·、一氧化氮(NO)、铁离子和过氧化氢(H₂O₂) 不敏感, 具有较高的稳定性和敏感度, 且毒性小。基于C11-BODIPY^581/591的检测优势, 其也被用作很多其他检测技术的分子指示剂, 如Wang等^[30]利用了C11-BODIPY^581/591作为脂质过氧化指示剂, 开发了一种对正常和肿瘤原位组织的铁死亡敏感性进行分级的快速成像技术。不足的是, C11-BODIPY^581/591对脂溶性膜内各种活性氧(reactive oxygen species, ROS) 和过氧亚硝酸盐(ONOO^-) 也较为敏感, 因此它对脂质过氧化物的检测不具有特异性。

一种基于亚油酸酯的Click-iT LAA (亚油酰胺炔) 探针也被应用于铁死亡相关脂质过氧化水平的检测^{[31, 32]} (图 2B)。这种探针主要是利用铜催化的Click反应和可以嵌入细胞膜的亚油酰胺炔试剂, 在固定细胞中对脂质过氧化衍生的修饰蛋白质进行检测, 借助荧光显微镜和流式细胞仪可对细胞膜的脂质过氧化水平进行定位和定量。不足的是, 这种探针对脂质过氧化物的检测同样不具有特异性^[33]。

Spy-LHP是一种用于检测脂质氢过氧化物(LOOHs) 的荧光染料^[34]。Spy-LHP具有的2个烷基链提高了与脂质双层膜的结合能力, 并且能够选择性地与LOOHs反应形成带荧光的氧化Spy-LHP (激发波长和发射波长分别为524和535 nm)。基于Spy-LHP的特性, 也有一些研究人员在研究铁死亡的过程中使用该探针对细胞的脂质过氧化进行检测^[13]。但不足的是, Spy-LHP不易溶于甲醇和二甲基亚砜等低细胞毒性的溶剂, 这也使得它在活细胞检测中的应用存在较大的局限性。

随后, Yamanaka等^[35]优化了Spy-LHP的溶解性问题, 开发出了一种Spy-LHP的衍生物—Liperfluo。Liperfluo的反应原理与GPX4还原脂质氢过氧化物的方式类似, 它在与LOOHs作用后会产生带绿色荧光的氧化型Liperfluo, 氧化型Liperfluo在水溶液中几乎不带荧光, 但在细胞膜等脂溶性高的部位中会产生较强的绿色荧光(激发波长和发射波长分别为524和535 nm)^[36] (图 2C)。因此, 通过荧光显微镜、流式细胞仪等仪器可对活细胞中细胞膜部分的脂质过氧化物进行检测和定量。目前, Liperfluo已成为检测铁死亡相关脂质过氧化的一种重要探针, 并广泛应用在铁死亡的相关研究中^{[36, 37]}, 利用Liperfluo探针与亚细胞定位探针共标记还能实现在亚细胞水平指示脂质过氧化情况^[38]。然而, Liperfluo同样也存在有不足之处: ①这种探针检测的是细胞中总的脂质过氧化物水平; ②不能区分不同类别的脂质过氧化物, 如不饱和脂肪酸过氧化物、磷脂过氧化物等。

3.2 基于化学发光反应的脂质过氧化产物检测方法

铁死亡相关的脂质过氧化反应过程能够产生大量的脂质过氧化产物, 其中LOOHs作为脂质过氧化反应的初级产物, 会在氧化反应过程中逐渐分解成一系列复杂的醛类化合物, 包括丙二醛(malondialdehyde, MDA)、4-羟基壬烯醛(4-hydroxynonenal, 4-HNE)、丙醛和己醛等, 这些活性醛类物质会攻击蛋白等生物大分子, 形成加合物引起细胞发生进一步损伤^[26]。对这些脂质过氧化产物含量的准确测定, 在一定程度上可以反映铁死亡样品中脂质过氧化的程度, 体现机体及细胞受损伤的程度。

硫代巴比妥酸(TBA) 反应物法(TBARS assay) 是目前用于检测脂质过氧化水平最为常用的一种方法^[39]。MDA在较高温度及酸性条件下, 易与TBA发生亲核加成反应, 形成红色的MDA-TBA加合物。该加合物在535 nm处产生最大吸收峰, 据此可以通过比色法来定量反映脂质过氧化水平。该方法操作简单, 在铁死亡的研究中常被用于对样品的脂质过氧化水平进行检测^{[40, 41]}。但由于TBA缺乏反应的专一性, 因此, 使用该方法容易在实验过程中出现假阳性。2, 4-二硝基苯肼(2, 4-dinitrophenylhydrazine, DNPH) 等醛反应探针可实现对另一种脂质过氧化产物—4-HNE的定量检测^[42]。DNPH可以与4-HNE反应, 形成稳定的2, 4-二硝基苯腙, 形成的产物同样可以通过比色法进行检测定量。不足的是, 这些探针本身无法区分不同的醛类化合物, 因此, 同样具有特异性差的缺点。

值得注意的是, MDA和4-HNE并不是在铁死亡过程中特异性产生的脂质过氧化产物, 它们在其他的各种氧化应激状态下均可能产生。因此, 把它们作为铁死亡相关脂质过氧化的指标物质仍然缺乏一定的特异性和代表性。在实际应用过程中, 研究人员应需结合其他的检测方法相互验证。

3.3 基于免疫反应的脂质过氧化产物检测方法

由于MDA、4-HNE具有高反应性, 很大一部分MDA和4-HNE可以与生物分子如蛋白质、DNA或氨基磷脂结合形成大分子加合物^[43]。目前, 免疫检测法是针对MDA、4-HNE蛋白质加合物的一种重要检测手段。酶联免疫吸附法(enzyme-linked immunosorbent assay, ELISA) 是一种将抗原抗体免疫反应的特异性和酶高催化作用结合的用于定量分析的新型免疫测定技术, 能够定量分析铁死亡过程产生的MDA、4-HNE-蛋白质加合物^{[44, 45]}。ELISA检测技术具有一定的优势, 最大的特点就是操作简单、检测速度快、测定成本低等特点。同样, 基于抗原-抗体结合反应原理, 蛋白质免疫印迹法(Western blot) 和免疫染色法利用MDA、4-HNE特异性的抗体, 也可以用于检测和定量MDA、4-HNE蛋白质加合物^[46]。此类方法虽然特异性强, 但它们仅能反映蛋白质加合物的整体变化的情况, 而不能确定修饰的蛋白质种类及修饰的准确位置。

3.4 LC/MS技术在氧化脂质组学中的应用

铁依赖的脂质过氧化是铁死亡的重要特征, 揭示铁死亡过程中特异性脂质过氧化物及过氧自由基的代谢有助于理解其生物学基础。脂质氧化产物的化学结构众多, 含量差异极大, 给相关的生物学研究过程带来了巨大挑战^[33]。免疫分析、化学或荧光光度、NMR、MS等方法在氧化脂质分析中起到了重要作用, 但是在定性和定量分析生物样品中复杂的脂质氧化产物时具有较大局限性。在过去30年里, LC-ESI-MS/MS已发展成为定性定量分析细胞和组织中氧化磷脂的最常用方法之一^[47]。LC-MS结合了LC的分离性能和MS的检测能力, 相比于传统的高效薄层色谱、LC-UV、GC-MS等检测方法, 具有灵敏度高和特异性强的优点。随着质谱技术的飞速发展, 质谱的灵敏度、分辨率和分析效率都得到了极大的提升。2017年Kagan等^[36]发现, 多不饱和磷脂而非多不饱和脂肪酸的过氧化能够引起细胞发生铁死亡, 并采用正相LC-MS/MS建立了氧化磷脂组学方法, 在数百种过氧化磷脂中发现PE-AA和PE-AdA的氧化产物, 即PE-AA-OOH和PE-AdA-OOH, 是细胞铁死亡的执行分子。随后Luo等^[48]利用这种能够快速和准确分析细胞中不同氧化磷脂类型的氧化脂质组学方法, 揭示了SAPE-OOH是巨噬细胞吞噬铁死亡细胞的独特“eat-me”信号。此外, Sun等^[49]还发现了铁死亡信号分子SAPE-OOH为磷脂重塑关键蛋白iPLA₂β所选择性识别, 帮助阐明了iPLA₂β的突变或活性下降, 会导致多巴胺能神经元中过氧化磷脂SAPE-OOH堆积, 进而引起多巴胺能神经元损伤和丢失的生化基础与分子机制。

LC-MS分析还可以将氧化磷脂自由基与蛋白质结合形成的加合物的信息表达得更为丰富。以4-HNE蛋白加合物为例, 利用MS/MS碎片分析可推断氨基酸序列中发生4-HNE羰基化修饰的位点, 通过氨基酸位点突变则可进一步确证羰基化修饰的位置。LC-MS技术与蛋白质组学、Western blot等技术的连用帮助许多研究团队成功鉴定出多种4-HNE羰基化修饰蛋白, 并实现了对这些4-HNE羰基化修饰蛋白的含量测定, 如Chen等^[50]开发了特异性苯胺探针m-APA, 结合LC-MS蛋白质组学技术, 在RSL3诱导的铁死亡细胞模型中鉴定了超过400种羰基化蛋白; Zhang等^[51]采用aminoxyTMT标记技术开展了LC-MS蛋白质组学研究, 在细胞样品中鉴定了1 121个4-HNE修饰的蛋白。总而言之, LC-MS方法在铁死亡相关新型氧化磷脂及其衍生物的发现、鉴定和测定工作中发挥着重要作用。

此外, 一些基于质谱发展起来的前沿检测技术也开始被开发和应用于探索生物样品的脂质种类及变化规律。其中, 质谱成像技术(mass spectrometry imaging, MSI) 受到广泛关注且发展迅速。MSI技术以质谱为基础, 通过质谱对生物样品进行直接的扫描成像, 实现对生物组织切片中包括脂质在内等数百种分子的空间分布特征的同步分析^{[52, 53]}。目前, 已有研究利用气体团簇离子束二次离子质谱成像技术(GCIB-SIMS), 描绘出铁死亡心肌细胞及缺血再灌注模型中的皮层/海马神经元中氧化型磷脂酰乙醇胺的空间分布^[54]。MSI技术具有极高的灵敏度和特异性, 未来有望成为揭示铁死亡脂质过氧化反应分子机制的前沿技术。

4 铁含量变化的检测方法

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铁离子在驱动细胞内脂质过氧化反应和铁死亡执行中起着不可或缺的作用^[55], 因此, 细胞铁含量或者Fe²⁺与Fe³⁺的比值的测定可作为铁死亡监测的重要指标。目前, 针对铁含量检测的方法主要为荧光探针法(Calcein-AM)、化学反应法(普鲁士蓝染色法) 和电感耦合等离子体质谱法(inductively coupled plasma mass spectrometry, ICP-MS) 等。Fe²⁺的特异性检测方法主要涉及一些荧光探针如RhoNox-1、FerroOrange、FRET iron probe 1 (FIP-1) 等的应用。

ICP-MS作为一种以电感耦合等离子体为离子源, 采用质谱进行检测的无机多元素分析技术, 是目前用于量化生物体内总铁离子含量最为准确的技术之一, 具有灵敏度高的优点。目前, 这一技术也被应用在铁死亡的相关研究中, 如Yoshida等^[24]运用ICP-MS和Calcein-AM荧光探针检测了细胞中的铁含量, 发现香烟烟雾暴露显著增加细胞中铁含量, 而敲低铁自噬相关蛋白核受体共激活因子4 (nuclear receptor coactivator 4, NCOA4) 能够逆转香烟烟雾暴露引起的细胞铁累积, 证明了铁自噬参与了香烟烟雾诱导的铁死亡过程。当然, 这种方法也存在一些检测局限性, 如复杂的前处理过程、对仪器和检测人员的要求高等。普鲁士蓝染色法是显示组织内三价铁的一种敏感、优良的经典方法, Yu等^[56]运用该方法, 发现肝脏特异性敲除转铁蛋白会引起肝脏发生铁过载, 进而增加了对铁死亡诱导的肝损伤的易感性。但该方法仅适用于检测动物组织中与蛋白紧密结合或者松散结合的铁离子含量, 不适用于完全检测游离的铁离子。荧光探针则是检测铁离子含量更为常用的检测技术, Calcein-AM和Phen Green SK等这类具有铁离子螯合基团的荧光探针在正常情况下能够被激发产生较强的荧光, 而螯合铁离子后能够使自身的荧光淬灭, 通过对荧光变化的监测实现活细胞中铁含量的动态检测^[57] (图 3A)。这类探针使用比较简便, 不足的是它们的结合能力相对较弱, 对不同类型铁离子的选择性差, 并可能与其他的二价金属离子进行反应。为了克服Calcein-AM等探针的不足之处, 研究人员又开发了一系列新的用于特异检测Fe²⁺含量的荧光探针。其中, 一类含有氮氧化物结构的荧光探针如RhoNox-1^[58]能够与Fe²⁺特异反应, 从而使自身还原成能被激发成强荧光表达的结构(图 3B)。通过对荧光强度进行检测, 可实现在活细胞或者冰冻组织切片上对Fe²⁺进行半定量分析。Kremer等^[59]运用该探针检测细胞铁离子的含量, 发现了抑制天冬氨酸氨基转移酶能够促进铁离子的释放, 诱导胰腺癌细胞发生铁死亡。FerroOrange和Mito-FerroGreen等探针也可以用于特异性检测不稳定Fe²⁺的含量。这类探针不会和铁蛋白或其他物质中的络合铁发生反应, 并可以在Fe³⁺或其他二价金属存在的情况, 自身荧光强度不发生变化, 且具有高渗透性和低细胞毒性的优点, 因此, 它们常被应用于铁死亡细胞Fe²⁺的成像^{[60, 61]}。另外, 一类以FIP-1为代表的基于内过氧化物反应的荧光共振能量转移(fluorescence resonance energy transfer, FRET) 探针, 也被开发用于特异性监测铁死亡过程中的铁离子变化^[62]。FIP-1探针通过过氧桥键连接两个荧光基团(供体荧光为荧光素, 受体荧光为Cy3), Fe²⁺能够诱导过氧桥键发生断裂, 从而降低了供体基团与受体基团之间的FRET效应, 使得供体基团的荧光显著提高^[63] (图 3C)。此外, Gao等^[64]也基于荧光共振能量转移技术设计了一种能用于监测铁死亡细胞中不稳定Fe³⁺变化的荧光探针DRhFe。总之, 这些特异性探针在监测铁死亡过程铁离子的变化规律中发挥了重要的作用。

5 谷胱甘肽代谢通路相关检测方法

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谷胱甘肽(glutathione, GSH) 耗竭是细胞执行铁死亡的重要原因之一^[65], 检测GSH消耗和胱氨酸摄取等指标可用于监测铁死亡的过程。

5.1 GSH的检测

GSH的检测方法主要包括试剂盒和LC-MS法, 其中试剂盒检测法操作简便、灵敏, 是目前用于检测GSH水平最为常用的一种方法。它的检测原理为GSH能够与二硫代二硝基苯甲酸反应, 产生一种在波长为412 nm处有最大光吸收的黄色产物, 通过比色法可对GSH的水平进行定量测定。Jang等^[38]采用该方法检测了细胞及线粒体的GSH含量, 帮助阐明了线粒体GSH在心肌细胞铁死亡的作用。一些荧光探针如Thiol Fluorescent Probe IV (激发波长和发射波长分别为400和465 nm) 借助荧光酶标仪等仪器也可以用于GSH的含量测定, 实现细胞内GSH的可视化分析^[66], 但是这种探针对GSH的专一性不高。相比传统的GSH检测方法, LC-MS法则具有高灵敏度度, 低检测限和定量准等优点, 目前, LC-MS可实现对铁死亡过程中的GSH及氧化型GSH (glutathione disulfide, GSSG) 的定量检测^{[67, 68]}, 而它的局限性在于测试成本较高、实验条件要求较高、分析方法较复杂, 以及需要配备特殊设备和专业操作人员。

5.2 胱氨酸摄取活性的检测

谷氨酸/胱氨酸转运体系统(system X_c^-) 及其相关调控蛋白在调控铁死亡中发挥重要作用^[8], 因此, 胱氨酸摄取活性作为表征system X_c^-活性的检测指标被广泛应用于铁死亡的相关研究。同位素标记法是目前用于检测胱氨酸摄取活性最为常用的方法。如Zhang等^[69]采用放射性同位素¹⁴C标记胱氨酸, 发现了SLC7A11介导的胱氨酸摄取促进了GPX4的蛋白合成。Jiang等^[70]采用¹⁴C标记胱氨酸进行胱氨酸摄取实验, 辅助证明了p53通过抑制SLC7A11的表达进而抑制胱氨酸的摄取, 增强了细胞对铁死亡的敏感性。这种方法能够对胱氨酸进行示踪, 缺点是需要配备专门的检测仪器及对实验条件的要求相对苛刻。另外, 一些荧光探针也可用于检测胱氨酸的摄取活性, 如Mukhopadhyay等^[71]采用BioTracker Cystine-FITC活细胞荧光探针对胱氨酸的摄取活性进行检测, 辅助证明了自噬参与调控SLC7A11的活性与功能。此外, 一些研究人员也设计开发了用于检测胱氨酸摄取活性更为简便的方法, 如Shimomura等^[72]开发了一种基于硒代半胱氨酸和荧光素O, O'-二丙烯酸酯的反应来测量X_c^-活性的方法, 能够实现对各种细胞中的胱氨酸摄取活性和抑制性X_c^-抑制剂的有效性进行评估。

6 铁死亡相关诱导和调控分子表达的检测

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铁死亡的发生及调控过程涉及铁代谢、脂质过氧化反应、谷胱甘肽代谢等多条信号通路的交叉网络, 因此, 采用Western blot、免疫荧光、免疫组织化学、实时荧光定量PCR技术等方法测定铁死亡诱导和调控分子的表达水平可为细胞铁死亡检测提供依据。

6.1 铁死亡相关诱导和调控分子蛋白表达水平的检测

免疫检测法包括Western blot、免疫荧光和免疫组织化学等, 可通过检测调控铁死亡过程多个通路中的关键分子如SLC7A11、GPX4、TfR1等的表达水平来表征铁死亡的发生。如Jiang等^[70]运用Western blot技术研究了p53调控SLC7A11在驱动铁死亡发生中的作用。Friedmann等^[73]运用该技术检测GPX4的蛋白表达水平, 发现了GPX4在急性肾损伤中的作用。其他研究人员也采用该技术检测了铁死亡通路中的其他调控蛋白如TfR1^[46]、ALOX12^[74]、iPLA₂β^[75]、Keap1-NRF2^[76]、ACSL4^[13]、ALOX15^[77]的表达, 来研究铁死亡的病理过程和调控机制。该方法的优点主要为检测灵敏, 检测专一性较强, 不足之处是步骤较为繁多, 实验过程中容易产生错误或主观的结果。并且, 由于铁死亡过程涉及的蛋白分子较多, 有时候需要同时检测多个铁死亡关键蛋白, 并结合其他的技术手段来共同表征铁死亡。

6.2 铁死亡相关诱导和调控分子基因表达水平的检测

荧光定量PCR方法可通过检测铁死亡关键分子的基因表达水平来表征铁死亡的发生。除了那些编码铁死亡关键调控蛋白的基因, 编码前列腺素内过氧化物合成酶2的基因PTGS2和谷胱甘肽特异性γ-谷氨酰基环转移酶1的基因CHAC1的表达水平也常常作为铁死亡的生物标记来使用, 并广泛应用在铁死亡相关的体内和体外研究中^{[40, 78-80]}。虽然PTGS2和CHAC1的基因表达水平在多种铁死亡模型中均表达上调, 但它们作为铁死亡生物标志物仍然具有一定的局限性, 如PTGS2和CHAC1并不是只在铁死亡诱导的模型中才能高表达, 在其他一些模型中也能够发生显著上调^{[81, 82]}。

7 总结与展望

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铁死亡涉及复杂的诱导和调控机制, 多种信号通路与代谢途径参与, 因此针对铁死亡的研究方法和策略具有多样性(表 3)。如细胞死亡相关检测方法包括LDH法和PI、SYTOX Green等虽然不具有铁死亡检测的特异性, 但这些方法结合铁死亡工具药的使用, 能够实现对铁死亡进行早期筛选及检识。基于透射电子显微镜的形态学检测法则是鉴别铁死亡最为直观的方法, 但由于其主观性较强, 操作复杂, 通常作为其他检测方法的基础。针对铁死亡过程中脂质过氧化的检测, 荧光探针、化学发光、免疫分析等方法虽然在定性和定量分析生物样品中复杂的脂质氧化产物上存在较大的局限性, 但它们为实验室提供了检测脂质过氧化常规且重要的技术手段。基于LC-MS的氧化脂质组学能够在常规检测方法使用的基础上, 实现快速和准确定量分析铁死亡细胞中不同类型的脂质过氧化物, 并且能够实现对脂质氧化产物修饰蛋白质及修饰位点的分析。针对铁离子含量的检测, ICP-MS能够实现对铁离子含量的准确测定, 但其对设备要求高, 针对不同类型铁离子检测的特异性不强。荧光探针法则是目前实验室用于检测铁离子最为常用的方法, 可实现对铁离子含量的特异性定量分析及定位检测。针对谷胱甘肽代谢途径的检测, 试剂盒检测法是目前用于检测GSH最为常用的方法, LC-MS法由于具有准确性高、特异性强、灵敏度高的优势, 能够实现GSH及GSSG的精确定量。而放射性同位素虽然对仪器和实验的要求较高, 但它能够对GSH合成过程的关键氨基酸胱氨酸的摄取活性进行动态监测。针对铁死亡调控分子的检测, 免疫分析法及实时荧光定量PCR法可以实现对铁死亡关键调控蛋白的表达水平进行定量分析。以上这些仅是铁死亡研究过程中常用的检测方法, 均有各自的优势和不足之处。在实际应用过程中应针对实验目的来选择不同的实验方法, 并注意针对铁死亡的不同过程, 结合使用多种检测手段, 这样才能扬长避短, 以更系统准确地对铁死亡进行检识、确认与机制研究。随着技术的不断发展及研究人员对铁死亡探索的不断深入, 相信未来会有更多特异、准确、简便的检测方法被开发运用, 帮助研究者更好地阐明铁死亡的调控机制及其在治疗疾病中的作用。

作者贡献: 何蓉蓉为文章提供综述的思路设计及总指导; 欧阳淑桦完成文献的查阅、整理、撰写综述; 吴燕萍、孙万阳和闫昌誉负责完成综述部分内容; 李怡芳和栗原博提供了修改建议并完善相关内容。

利益冲突: 所有作者均声明不存在利益冲突。

基金

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国家自然科学基金项目(82125038)
国家自然科学基金项目(81873209)
国家自然科学基金项目(82004012)
珠江学者计划项目(GDUPS2019)
广东省基础与应用基础研究基金项目(2021B1515120023)
广东省基础与应用基础研究基金项目(2020A1515110596)
珠江创新团队项目(2017BT01Y036)
广州市科技计划-基础与应用基础研究资助项目(201903010062)

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2022年第57卷第6期

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doi: 10.16438/j.0513-4870.2022-0276

接收时间：2022-03-01
首发时间：2025-12-23
出版时间：2022-06-12

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收稿日期：2022-03-01
修回日期：2022-04-15

基金

国家自然科学基金项目(82125038)

国家自然科学基金项目(81873209)

国家自然科学基金项目(82004012)

珠江学者计划项目(GDUPS2019)

广东省基础与应用基础研究基金项目(2021B1515120023)

广东省基础与应用基础研究基金项目(2020A1515110596)

珠江创新团队项目(2017BT01Y036)

广州市科技计划-基础与应用基础研究资助项目(201903010062)

作者信息

1.暨南大学, 广东省疾病易感性及中医药研发工程技术研究中心, 广东广州 510632

2.暨南大学, 中药及天然药物研究所, 广东广州 510632

3.暨南大学, 广东省中药药效物质基础及创新药物研究重点实验室, 广东广州 510632

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*何蓉蓉, Tel / Fax: 86-20-85221559, E-mail: rongronghe@jnu.edu.cn

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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TxT

	Small molecule	Structure	Mechanism
Inducer	RSL3		Inhibit GPX4 activity
ML162		Inhibit GPX4 activity
ML210		Inhibit GPX4 activity
FIN56		Decrease GPX4 levels
FINO₂		Induce lipid peroxidation
Erastin		Inhibit system X_c^-
Sorafenib		Inhibit system X_c^-
BSO		Deplete GSH
FeCl₂	–	Iron overload
(NH₄)₂Fe(SO₄)₂	–	Iron overload
Inhibitor	Deferoxamine (DFO)		Chelate iron
Vitamin E		Inhibit propagation of lipid peroxidation
Ferrostain-1		Inhibit lipid peroxidation
Liproxstatin-1		Inhibit lipid peroxidation
CoQ₁₀		Inhibit lipid peroxidation
Monounsaturated fatty acid		Inhibit lipid peroxidation

Small molecule

Structure

Mechanism

Inducer

RSL3

Inhibit GPX4 activity

ML162

Inhibit GPX4 activity

ML210

Inhibit GPX4 activity

FIN56

Decrease GPX4 levels

FINO₂

Induce lipid peroxidation

Erastin

Inhibit system X_c^-

Sorafenib

Inhibit system X_c^-

BSO

Deplete GSH

FeCl₂

–

Iron overload

(NH₄)₂Fe(SO₄)₂

–

Iron overload

Inhibitor

Deferoxamine (DFO)

Chelate iron

Vitamin E

Inhibit propagation of lipid peroxidation

Ferrostain-1

Inhibit lipid peroxidation

Liproxstatin-1

Inhibit lipid peroxidation

CoQ₁₀

Inhibit lipid peroxidation

Monounsaturated fatty acid

Inhibit lipid peroxidation

Type of cell death	Typical morphological feature
Ferroptosis	Increased mitochondrial membrane densities Small size of mitochondria Mitochondrial crista vanished
Apoptosis	Membrane blebbing Pseudopod retraction Cell shrinkage Chromatin condensation and margination
Necrosis	Membrane rupture Cytoplasmic and organelle swelling
Necroptosis	Dilation of the perinuclear space Cell rounding and swelling Membrane rupture
Pyroptosis	Cell swelling Membrane rupture Deformation of organelles Chromatin condensation
Autophagy	Formation of double membrane structures

Type of cell death

Typical morphological feature

Ferroptosis

Increased mitochondrial membrane densities
Small size of mitochondria
Mitochondrial crista vanished

Apoptosis

Membrane blebbing
Pseudopod retraction
Cell shrinkage
Chromatin condensation and margination

Necrosis

Membrane rupture
Cytoplasmic and organelle swelling

Necroptosis

Dilation of the perinuclear space
Cell rounding and swelling
Membrane rupture

Pyroptosis

Cell swelling
Membrane rupture
Deformation of organelles
Chromatin condensation

Autophagy

Formation of double membrane structures

Assay and tool	Method	Advantage	Disadvantage
LDH assay	Detects cell death based on chemiluminescence reaction	Easy to operate and low equipment requirements	Not specific for detecting ferroptosis
PI	A fluorescent probe for detecting cell death	Easy to operate and achieve quantitative and visual analysis of cell death	Not specific for detecting ferroptosis
SYTOX Green	A fluorescent probe for detecting cell death	Easier to operate and achieve quantitative and visual analysis of cell death than PI	Not specific for detecting ferroptosis
TEM	Detects morphological feature of ferroptosis	Directly observe the morphological features of ferroptosis	Subjectivity, complex to operate
C11-BODIPY^581/591	Lipid sensor for detecting lipid ROS in living cells	Highly lipophilic, easy for membrane entry, low toxicity	Not specific for detecting lipid peroxidation
Click-iT LAA	Detects lipid peroxidation-derived protein modifications in fixed cells	Localizes and quantifies lipid peroxidation in cell membranes	Linoleic acid dependence, not specific for detecting lipid peroxidation
Liperfluo	A fluorescent probe for relatively specific detecting lipid peroxidation in living cells	Specifically detects and quantifies lipid peroxidation in the membrane of living cells	Unable to distinguish different types of lipid peroxides
TBARS assay	Detects MDA product of lipid peroxidation	Easy to operate and low equipment requirements	Less specific for detecting lipid peroxidation, prone to false positives
DNPH assay	Detects 4-HNE product of lipid peroxidation	Easy to operate and low equipment requirements	Less specific for detecting lipid peroxidation, unable to distinguish different types of aldehydes
Immunoassays for lipid peroxidation products	Detects MDA- and 4-HNE-protein adduct	Easy to operate, strong specific for the detection of MDA- and 4-HNE-protein adduct	Unable to distinguish the specific modification of MDA- and 4-HNE-protein adduct
Oxidative lipidomic based on LC-MS	Identification and structural characterization of oxidatively modified lipids	High specificity, sensitivity, accuracy for detecting ferroptosis related lipid peroxidation	Complex to operate, require special equipment and technician
ICP-MS	Detects total iron level	Accurate determination of iron levels	Only for the total iron level detection
Prussian blue staining	Detects the distribution and amount of iron deposits in tissues	Good stability	Not suitable for the detection of free iron levels in living cells
Calcein-AM	A fluorescent probe detects iron level based on iron chelation	Sensitive	Not specific
RhoNox-1	A fluorescent probe detects Fe²⁺ level in living cells	Sensitive, high specificity for Fe²⁺	Not suitable for fixed cell and paraffin slices
FerrOrrange	A fluorescent probe detects Fe²⁺ level in living cells	Sensitive, high specificity for Fe²⁺, low toxicity	Not suitable for fixed cell and paraffin slices
Mito-FerroGreen	A fluorescent probe detects Fe²⁺ level in mitochondria	Sensitive, high specificity for Fe²⁺, low toxicity	Not suitable for fixed cell and paraffin slices
FRET iron probe 1	Detects Fe²⁺ level based on FRET	High specificity for Fe²⁺	Not suitable for fixed cell and paraffin slices
GSH assay kit	Detects GSH content	Sensitive, easy to operate and low equipment requirements	Unable to visualize the content of GSH
LC-MS	Detects GSH/GSSG content	High specificity, sensitivity, accuracy for detecting GSH/GSSG	Complex to operate, require special equipment and technician
BioTracker cystine-FITC	Detects cystine uptake	Achieve quantitative and visual analysis of cystine uptake	Prone to false positives
Radioisotope labeled cystine	Detects cystine uptake	High specificity, achieve tracking the cystine	Complex to operate, require special equipment
Immunoassays for key regulators of ferroptosis	Detects key regulators of ferroptosis	Sensitive, strong specific for detecting key regulators of ferroptosis	Subjectivity, prone to false positives
PCR analysis for key regulators of ferroptosis	Detects key regulators of ferroptosis	Sensitive, easy to operate, specific for detecting key regulators of ferroptosis	Relative expensive

Assay and tool

Method

Advantage

Disadvantage

LDH assay

Detects cell death based on chemiluminescence reaction

Easy to operate and low equipment requirements

Not specific for detecting ferroptosis

A fluorescent probe for detecting cell death

Easy to operate and achieve quantitative and visual analysis of cell death

Not specific for detecting ferroptosis

SYTOX Green

A fluorescent probe for detecting cell death

Easier to operate and achieve quantitative and visual analysis of cell death than PI

Not specific for detecting ferroptosis

TEM

Detects morphological feature of ferroptosis

Directly observe the morphological features of ferroptosis

Subjectivity, complex to operate

C11-BODIPY^581/591

Lipid sensor for detecting lipid ROS in living cells

Highly lipophilic, easy for membrane entry, low toxicity

Not specific for detecting lipid peroxidation

Click-iT LAA

Detects lipid peroxidation-derived protein modifications in fixed cells

Localizes and quantifies lipid peroxidation in cell membranes

Linoleic acid dependence, not specific for detecting lipid peroxidation

Liperfluo

A fluorescent probe for relatively specific detecting lipid peroxidation in living cells

Specifically detects and quantifies lipid peroxidation in the membrane of living cells

Unable to distinguish different types of lipid peroxides

TBARS assay

Detects MDA product of lipid peroxidation

Easy to operate and low equipment requirements

Less specific for detecting lipid peroxidation, prone to false positives

DNPH assay

Detects 4-HNE product of lipid peroxidation

Easy to operate and low equipment requirements

Less specific for detecting lipid peroxidation, unable to distinguish different types of aldehydes

Immunoassays for lipid peroxidation
products

Detects MDA- and 4-HNE-protein adduct

Easy to operate, strong specific for the detection of MDA- and 4-HNE-protein adduct

Unable to distinguish the specific modification of MDA- and 4-HNE-protein adduct

Oxidative lipidomic based on LC-MS

Identification and structural characterization of oxidatively modified lipids

High specificity, sensitivity, accuracy for detecting ferroptosis related lipid peroxidation

Complex to operate, require special equipment and technician

ICP-MS

Detects total iron level

Accurate determination of iron levels

Only for the total iron level detection

Prussian blue staining

Detects the distribution and amount of iron deposits in tissues

Good stability

Not suitable for the detection of free iron levels in living cells

Calcein-AM

A fluorescent probe detects iron level based on iron chelation

Sensitive

Not specific

RhoNox-1

A fluorescent probe detects Fe²⁺ level in living cells

Sensitive, high specificity for Fe²⁺

Not suitable for fixed cell and paraffin slices

FerrOrrange

A fluorescent probe detects Fe²⁺ level in living cells

Sensitive, high specificity for Fe²⁺, low toxicity

Not suitable for fixed cell and paraffin slices

Mito-FerroGreen

A fluorescent probe detects Fe²⁺ level in mitochondria

Sensitive, high specificity for Fe²⁺, low toxicity

Not suitable for fixed cell and paraffin slices

FRET iron probe 1

Detects Fe²⁺ level based on FRET

High specificity for Fe²⁺

Not suitable for fixed cell and paraffin slices

GSH assay kit

Detects GSH content

Sensitive, easy to operate and low equipment requirements

Unable to visualize the content of GSH

LC-MS

Detects GSH/GSSG content

High specificity, sensitivity, accuracy for detecting GSH/GSSG

Complex to operate, require special equipment and technician

BioTracker cystine-FITC

Detects cystine uptake

Achieve quantitative and visual analysis of cystine uptake

Prone to false positives

Radioisotope labeled cystine

Detects cystine uptake

High specificity, achieve tracking the cystine

Complex to operate, require special equipment

Immunoassays for key regulators of ferroptosis

Detects key regulators of ferroptosis

Sensitive, strong specific for detecting key regulators of ferroptosis

Subjectivity, prone to false positives

PCR analysis for key regulators of ferroptosis

Detects key regulators of ferroptosis

Sensitive, easy to operate, specific for detecting key regulators of ferroptosis

Relative expensive