Ferrostatin-1 (Fer-1)   中文名称: 非罗司他丁-1

Ferroptosis (EC50 = 60 nM)

Ferrostatin-1 抑制由erastin 产生的脂质和胞质ROS 的积累。在器官型大鼠脑切片中，ferrostatin-1 可抑制谷氨酸引起的神经毒性 [1]。 Ferrostatin-1（2 μM；24 小时）可保护大鼠器官型海马切片培养物 (OHSC) 免受谷氨酸 (5 mM) 诱导的神经毒性 [2]。 Ferrostatin-1 抑制脂质过氧化，但不抑制溶酶体膜的通透性或线粒体中活性氧的产生 [2]。在肾衰竭、脑室周围白质软化 (PVL) 和亨廷顿病 (HD) 的细胞模型中，ferrostatin-1 可减少细胞死亡 [2]。在 HT-1080 细胞中，ferrostatin-1（1 μM；6 小时）可防止不饱和脂肪酸氧化降解，从而增加健康中型多棘神经元 (MSN) 的数量 [3]。

在横纹肌溶解症小鼠中，Ferrostatin-1（5 mg/kg；腹膜内注射；单剂量，注射甘油前 30 分钟给药）可改善肾功能；然而，这种益处在缺乏泛半胱天冬酶抑制剂 zVAD 或 RIPK3 的小鼠中并未显示出来。 Frostatin-1（0.8 mg/kg；尾静脉注射）可以有效治疗 LPS 引起的急性肺损伤（ALI）[4]。 Ferrostatin-1（5 mg/kg；腹腔注射；C57BL/6J 小鼠）可改善横纹肌溶解症小鼠的肾功能 [5]。

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研究结果显示，Ferrostatin-1（Fer-1）显著减轻了由缺氧和缺血引发的脑损伤。Fer-1显著增加SLC3A2、SLC7A11、ACSL3、GSS和GPX4的表达（P<0.05），显著降低GFAP、ACSL4、TFRC、FHC、FLC、4-HNE、HIF-1α和ROS的表达（P<0.05）。 结论：Fer-1通过潜在靶向GPX4/ACL3/ACSL4轴抑制铁下垂并缓解HIBD；然而，其具体机制值得进一步探索。[6]

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绝经后口干症的机制尚未完全阐明。本研究旨在探讨绝经后动物模型中口干症的机制以及铁蛋白抑制剂去铁胺（DFO）和Ferrostatin-1（FER）对唾液腺功能障碍的影响。24只雌性Sprague-Dawley大鼠随机分为四组：SHAM组（n=6，假手术大鼠）、OVX组（n=5，去卵巢大鼠），FER组（n=7，切除卵巢大鼠腹腔注射FER）和DFO组（n=8，切除卵巢的大鼠腹腔内注射DFO）。分析GPX4活性、铁积累、脂质过氧化、炎症、纤维化和唾液腺功能。DFO组GPX4活性恢复，铁积累和细胞质MDA+HAE减少。此外，与OVX组相比，DFO组的I型胶原、III型胶原、TGF-β、IL-6、TNF-α和TGF-β水平降低。FER组还观察到GPX4活性和线粒体形态的恢复，以及细胞质MDA+HAE的减少。此外，在DFO和FER组中观察到炎性细胞因子和纤维化标志物的表达降低，AQP5的表达增加。绝经后唾液腺功能障碍与铁下垂有关，DFO和FER可能逆转绝经后的唾液腺功能障碍。因此，DFO和FER被认为是治疗绝经后口干症的有前景的方法。[7]

蛋白质印迹[4]
在我们的研究中，使用放射免疫沉淀分析裂解缓冲液裂解细胞样品，并使用Pierce BCA蛋白质分析试剂盒检测不同组的总蛋白质浓度。在我们的研究中，细胞裂解物（20 μg/泳道）用10%SDS-PAGE凝胶分离，然后转移到硝化纤维膜上。用在PBS中稀释的5%脱脂奶粉封闭膜，并进一步与一级抗体在4 °C。在此，使用的不同一级抗体是：抗SLC7A11（1:3000；细胞信号，类别号：12691）、抗GPX4（1:1000）、抗FTH（1:2000）和抗GAPDH（1:3000）。所用的第二抗体是：抗小鼠IgG（HRP缀合；1:5000）和抗兔IgG（HRP-缀合；1:10000）。最后，使用SuperSignal West Femto Maximum Sensitivity Substrate和ChemiDoc Images对每条泳道中的蛋白质带进行可视化。最后使用ImageJ1.x软件对结果进行量化。整个论文中图像的所有未切割的原始印迹如补充图所示。1。[4]
丙二醛（MDA）、4-羟基壬烯醛（4-HNE）和铁水平的评估[4]
在我们的研究中，为了评估不同组的脱铁水平，检测各组的MDA、4-HNE和铁水平。细胞裂解物中的MDA浓度、4-HNE浓度和铁浓度根据制造商的说明使用脂质过氧化（MDA）测定试剂盒、脂质过氧化测定试剂盒和铁测定试剂盒进行评估。

细胞活力测定[4]
为了评估细胞活力，我们的研究中使用CCK-8方法作为参考。简而言之，将BEAS-2B细胞以5的浓度接种到96孔板中 × 104个细胞/孔。细胞培养24小时 h、 然后用不同浓度的LPS和Fer-1处理16 h，然后添加20 μl CCK-8溶液直接加入培养基（200 μl/孔）并在37 °C 4 h.在450时检测不同组的吸光度（Abs） nm（n= 3.在空白组中，孔仅包含培养基，未经任何处理的细胞用作对照组。这里，细胞活力 = （实验组Abs空白组Abs）/（对照组Abs × 100%.

Animal/Disease Models: Male C57BL/6 mice (LPS-induced ALI)[4]
Doses: 0.8 mg/kg
Route of Administration: Tail vein injection
Experimental Results: Exerted therapeutic action against LPS-induced ALI.

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In our study, the male C57BL/6 mice were divided randomly into 4 groups (n = 4 per group, 8–10 weeks old, weight = 23–25 g): the control group receiving 0.9% NaCl (containing 0.1% DMSO), the LPS group receiving LPS plus 0.9% NaCl (containing 0.1% DMSO), the Fer-1 group receiving Fer-1 only, and the LPS + Fer-1 group receiving both Fer-1 and LPS. The LPS-induced ALI model was induced by instilling intratracheally 50 μl of LPS solution (0.2 g/L), then Fer-1 (0.8 mg/kg) was administered after LPS challenge via tail vein injection. The Fer-1 was dissolved in DMSO first, and diluted with 0.9% NaCl. The final concentration of Fer-1 and DMSO was 0.2 mg/ml and 0.1% respectively. After the treatments for 16 h, the mice in each group were euthanized and bronchoalveolar lavage (BAL) fluid was collected via lung lavage. To analyze the differential BAL cell counts, the cells were concentrated using a Cytospin 4. Cell staining was performed using the Shandon Kwik-Diff kit. Additionally, the total protein concentration and the levels of IL-6 and TNF-α in each sample were detected with the Pierce BCA Protein Assay Kit, IL-6 ELISA Kit ELISA kit and TNF-α ELISA Kit according to the manufacturer’s instructions. Lung tissues in different groups were collected for qPCR and western blot detection, and part of lung tissues was fixed using 10% buffered formalin, then the tissues were embedded in paraffin for histological analyses as the references. Herein, a scoring system of 0–4 was used for the evaluation of lung injury as the reference.

[1]. Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell. 2012;149(5):1060-1072.

[2]. Ferrostatins inhibit oxidative lipid damage and cell death in diverse disease models. J Am Chem Soc. 2014;136(12):4551-4556.

[3]. Antifungal Activity of the Lipophilic Antioxidant Ferrostatin-1. Chembiochem. 2017;18(20):2069-2078.

[4]. Ferrostatin-1 alleviates lipopolysaccharide-induced acute lung injury via inhibiting ferroptosis. Cell Mol Biol Lett. 2020;25:10. Published 2020 Feb 27.

[5]. FP282 FERROPTOSIS-MEDIATED CELL DEATH IS DECREASED BY CURCUMIN IN RENAL DAMAGE ASSOCIATED TO RHABDOMYOLYSIS, Nephrology Dialysis Transplantation, Volume 34, Issue Supplement_1, June 2019, gfz106.FP282.

[6]. Ferrostatin-1 attenuates hypoxic-ischemic brain damage in neonatal rats by inhibiting ferroptosis. Transl Pediatr. 2023 Nov 28;12(11):1944-1970.

[7]. Effect of deferoxamine and ferrostatin-1 on salivary gland dysfunction in ovariectomized rats. Aging (Albany NY). 2023 Apr 6;15(7):2418-2432.

Ferrostatin-1 is an ethyl ester resulting from the formal condensation of the carboxy group of 3-amino-4-(cyclohexylamino)benzoic acid with ethanol. It is a potent inhibitor of ferroptosis, a distinct non-apoptotic form of cell death caused by lipid peroxidation. It is also a radical-trapping antioxidant and has the ability to reduce the accumulation of lipid peroxides and chain-carrying peroxyl radicals. It has a role as a ferroptosis inhibitor, a radiation protective agent, an antioxidant, a radical scavenger, an antifungal agent and a neuroprotective agent. It is a substituted aniline, an ethyl ester and a primary arylamine.

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Background: Ferroptosis is a newly recognized type of cell death, which is different from traditional necrosis, apoptosis or autophagic cell death. However, the position of ferroptosis in lipopolysaccharide (LPS)-induced acute lung injury (ALI) has not been explored intensively so far. In this study, we mainly analyzed the relationship between ferroptosis and LPS-induced ALI. Methods: In this study, a human bronchial epithelial cell line, BEAS-2B, was treated with LPS and ferrostatin-1 (Fer-1, ferroptosis inhibitor). The cell viability was measured using CCK-8. Additionally, the levels of malondialdehyde (MDA), 4-hydroxynonenal (4-HNE), and iron, as well as the protein level of SLC7A11 and GPX4, were measured in different groups. To further confirm the in vitro results, an ALI model was induced by LPS in mice, and the therapeutic action of Fer-1 and ferroptosis level in lung tissues were evaluated. Results: The cell viability of BEAS-2B was down-regulated by LPS treatment, together with the ferroptosis markers SLC7A11 and GPX4, while the levels of MDA, 4-HNE and total iron were increased by LPS treatment in a dose-dependent manner, which could be rescued by Fer-1. The results of the in vivo experiment also indicated that Fer-1 exerted therapeutic action against LPS-induced ALI, and down-regulated the ferroptosis level in lung tissues. Conclusions: Our study indicated that ferroptosis has an important role in the progression of LPS-induced ALI, and ferroptosis may become a novel target in the treatment of ALI patients.[4]

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Background: Hypoxic-ischemic brain damage (HIBD) is a type of brain damage that is caused by perinatal asphyxia and serious damages the central nervous system. At present, there is no effective drug for the treatment of this disease. Besides, the pathogenesis of HIBD remains elusive. While studies have shown that ferroptosis plays an important role in HIBD, its role and mechanism in HIBD are yet to be fully understood. Methods: The HIBD model of neonatal rats was established using the Rice-Vannucci method. A complete medium of PC12 cells was adjusted to a low-sugar medium, and the oxygen-glucose deprivation model was established after continuous hypoxia for 12 h. Laser Doppler blood flow imaging was used to detect the blood flow intensity after modeling. 2,3,5-triphenyl tetrazolium chloride staining was employed to detect ischemic cerebral infarction in rat brain tissue, and hematoxylin and eosin staining and transmission electron microscopy were used to observe brain injury and mitochondrial damage. Immunofluorescence was applied to monitor the expression of GFAP. Real-time quantitative polymerase chain reaction, western blot, and immunofluorescence were utilized to detect the expression of messenger RNA and protein. The level of reactive oxygen species (ROS) in cells was detected using the ROS detection kit. Results: The results showed that ferrostatin-1 (Fer-1) significantly alleviated the brain injury caused by hypoxia and ischemia. Fer-1 significantly increased the expression of SLC3A2, SLC7A11, ACSL3, GSS, and GPX4 (P<0.05) and dramatically decreased the expressions of GFAP, ACSL4, TFRC, FHC, FLC, 4-HNE, HIF-1α, and ROS (P<0.05). Conclusions: Fer-1 inhibits ferroptosis and alleviates HIBD by potentially targeting the GPX4/ACSL3/ACSL4 axis; however, its specific mechanism warrants further exploration.[6]

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The mechanism underlying xerostomia after menopause has not yet been fully elucidated. This study aimed to investigate the mechanism of xerostomia and the effect of the ferroptosis inhibitors deferoxamine (DFO) and ferrostatin-1 (FER) on salivary gland dysfunction in a postmenopausal animal model. Twenty-four female Sprague-Dawley rats were randomly divided into four groups: a SHAM group (n = 6, sham-operated rats), an OVX group (n = 6, ovariectomized rats), an FER group (n = 6, ovariectomized rats injected intraperitoneally with FER), and a DFO group (n = 6, ovariectomized rats injected intraperitoneally with DFO). GPX4 activity, iron accumulation, lipid peroxidation, inflammation, fibrosis, and salivary gland function were analyzed. Recovery of GPX4 activity and a decrease in iron accumulation and cytosolic MDA + HAE were observed in the DFO group. In addition, collagen I, collagen III, TGF-β, IL-6, TNF-α, and TGF-β levels were decreased in the DFO group compared to the OVX group. Recovery of GPX4 activity and the morphology of mitochondria, and reduction of cytosolic MDA + HAE were also observed in the FER group. In addition, decreased expression of inflammatory cytokines and fibrosis markers and increased expression of AQP5 were observed in both the DFO and FER groups. Postmenopausal salivary gland dysfunction is associated with ferroptosis, and DFO and FER may reverse the postmenopausal salivary gland dysfunction after menopause. DFO and FER are hence considered promising treatments for postmenopausal xerostomia.[7]

C15H22N2O2

262.35

262.168

C, 68.67; H, 8.45; N, 10.68; O, 12.20

347174-05-4

4068248

Gray to gray purple solid

1.1±0.1 g/cm3

437.3±35.0 °C at 760 mmHg

218.3±25.9 °C

0.0±1.1 mmHg at 25°C

1.595

3.9

64.35

2

4

5

19

290

0

O(C([H])([H])C([H])([H])[H])C(C1C([H])=C([H])C(=C(C=1[H])N([H])[H])N([H])C1([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C1([H])[H])=O

UJHBVMHOBZBWMX-UHFFFAOYSA-N

InChI=1S/C15H22N2O2/c1-2-19-15(18)11-8-9-14(13(16)10-11)17-12-6-4-3-5-7-12/h8-10,12,17H,2-7,16H2,1H3

3-amino-4-(cyclohexylamino)-benzoic acid, ethyl ester

Frer-1; 3-amino-4-(cyclohexylamino)-benzoic acid, ethyl ester; Ferrostatin-1; 347174-05-4; Ethyl 3-amino-4-(cyclohexylamino)benzoate; Fer-1; Ferrostatin-1 (Fer-1); Ferrostatin 1; ferrrostatin 1; MFCD08072959;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

配方 1 中的溶解度: ≥ 2.5 mg/mL (9.53 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: 2.5 mg/mL (9.53 mM) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 悬浊液; 超声助溶。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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配方 3 中的溶解度: ≥ 2.08 mg/mL (7.93 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 20.8 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 4 中的溶解度: 0.2 mg/mL (0.76 mM) in 10% DMSO + 90% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液; 超声助溶.
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 5 中的溶解度: 2% DMSO+50% PEG 300+5% Tween 80+ddH2O: 5mg/mL

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
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7、 以上所有助溶剂都可在 Invivochem.cn网站购买。