Ferroptosis occurs through an osmotic mechanism and propagates independently of cell rupture.
铁死亡通过渗透机制发生，并且独立于细胞破裂而传播。

The proper regulation of cell death is important for normal organismal development and the maintenance of tissue homeostasis in adulthood. It was once thought that programmed cell death occurred exclusively through apoptosis, whereas necrotic death resulted only from acute cell stress or injury. However, numerous new cell death modalities have recently been discovered, including programmed forms of necrosis that are regulated by specific and distinct cellular machineries¹¹. One form of regulated necrosis called ferroptosis involves the iron-dependent accumulation of lipid peroxide species in cell membranes^{1, 2}. Under physiological conditions, ferroptosis is prevented by antioxidant enzymes that limit the buildup of oxidized lipids, including GPX4, which uses glutathione as a cofactor to detoxify peroxidation products¹². Cell death can be triggered by GPX4 inactivation, either through direct inhibition or depletion of cellular glutathione, thereby allowing the accumulation of phospholipid peroxides and cell damage. Recent work has uncovered an additional ferroptosis-preventing mechanism controlled by Ferroptosis Suppressor Protein 1 (FSP1), which catalyzes the reduction of the lipophilic antioxidant coenzyme Q10 (CoQ) ^{13, 14}.
细胞死亡的适当调节对于正常的有机体发育和成年期组织稳态的维持非常重要。人们曾经认为程序性细胞死亡仅通过细胞凋亡发生，而坏死性死亡仅由急性细胞应激或损伤引起。然而，最近发现了许多新的细胞死亡方式，包括由特定和独特的细胞机制调节的程序性坏死形式¹¹ 。一种称为铁死亡的受调节坏死形式涉及细胞膜中铁依赖性脂质过氧化物的积累^{1 , 2} 。在生理条件下，抗氧化酶可以限制氧化脂质的积累，从而防止铁死亡，其中包括 GPX4，它使用谷胱甘肽作为辅助因子来解毒过氧化产物¹² 。 GPX4 失活可以通过直接抑制或消耗细胞谷胱甘肽来触发细胞死亡，从而导致磷脂过氧化物的积累和细胞损伤。最近的工作发现了另一种由铁死亡抑制蛋白 1 (FSP1) 控制的铁死亡预防机制，该机制可催化亲脂性抗氧化剂辅酶 Q10 (CoQ) 的还原^{13 , 14} 。

Ferroptosis was previously shown to spread through cell populations, resulting in spatiotemporal patterns of cell death with a wave-like appearance not previously observed in other forms of cell death^{7, 8}. It is unknown what mechanism underlies this phenomenon and whether death propagation between neighboring cells is a consistent feature of ferroptosis or occurs only under certain conditions. Given emerging links between ferroptosis and degenerative diseases that often involve large, continuous areas of tissue damage, the propagative nature of ferroptosis is important to understand¹⁵. Furthermore, while factors that affect the accumulation of lipid peroxides and thereby modulate ferroptosis have been elucidated^{1, 16}, little is known about how lipid peroxidation leads to plasma membrane permeabilization. Whether cell lysis is involved in the intercellular propagation of ferroptosis is also unknown¹⁷. Here we investigated the wave-like nature of ferroptosis, the mechanism of ferroptotic cell rupture, and the link between the two processes.
先前显示铁死亡在细胞群中扩散，导致细胞死亡的时空模式，具有波状外观，此前在其他形式的细胞死亡中未观察到^{7 , 8} 。目前尚不清楚这种现象背后的机制是什么，以及相邻细胞之间的死亡传播是否是铁死亡的一致特征，还是仅在某些条件下发生。鉴于铁死亡和退行性疾病之间新出现的联系通常涉及大面积、连续的组织损伤，了解铁死亡的繁殖性质非常重要¹⁵ 。此外，虽然影响脂质过氧化物积累并从而调节铁死亡的因素已被阐明^{1 , 16} ，但人们对脂质过氧化如何导致质膜透化知之甚少。细胞裂解是否参与铁死亡的细胞间传播也是未知的¹⁷ 。在这里，我们研究了铁死亡的波状性质、铁死亡细胞破裂的机制以及两个过程之间的联系。

We previously observed wave-like spreading of ferroptosis when cells were treated with ferroptosis-inducing nanoparticles called C’ dots (Fig. 1a,b and Supplementary Video 1)⁸, and a similar phenomenon was reported in mouse renal tubules treated with the ferroptosis-inducing agent erastin⁷. However, the spatiotemporal patterns of ferroptosis have not been systematically investigated¹⁵. To quantitatively study propagation, we performed live cell imaging of several cell lines (MCF10A mammary epithelium, MCF7 breast cancer, U937 promonocytic leukemia, HAP1 chronic myelogenous leukemia, and B16F10 melanoma) in the presence of the cell death indicator SYTOX Green and different ferroptosis-inducing agents (C’ dots⁸, erastin², the GPX4 inhibitor ML162^{10, 18}, or a combination of ferric ammonium citrate (FAC) and buthionine sulfoxamine (BSO), Extended Data Fig. 1a,b). We then used a bootstrapping approach to quantify potential non-random patterns of cell death. For each movie, we calculated the mean time difference between neighboring cell deaths, μ_expΔt, and compared this experimental value to a distribution of means derived from computationally generated permutations representing random orders of death (Fig. 1c,d). Consistent with wave-like propagation, ferroptosis occurred with non-random spatiotemporal patterns when it was induced by erastin, C’ dots, or FAC and BSO, as determined by comparing μ_expΔt to the 95^th percentile of the random distribution, μ_perm95Δt (Fig. 1b, d, g; Extended Data Fig. 1c). Interestingly, when ferroptosis was induced by inhibition of GPX4 through treatment with ML162, μ_expΔt was more similar to the 95^th percentile of the random permutations (Fig. 1g and Extended Data Fig. 1d, e).
我们之前观察到，当细胞用称为 C' 点的铁死亡诱导纳米颗粒处理时，铁死亡呈波状扩散（ Fig. 1a , b 和 Supplementary Video 1 ) ⁸ ，并且在用铁死亡诱导剂erastin ⁷治疗的小鼠肾小管中也报道了类似的现象。然而，铁死亡的时空模式尚未得到系统研究¹⁵ 。为了定量研究增殖，我们在存在细胞死亡指示剂 SYTOX Green 和不同铁死亡的情况下，对几种细胞系（MCF10A 乳腺上皮、MCF7 乳腺癌、U937 早单核细胞白血病、HAP1 慢性粒细胞白血病和 B16F10 黑色素瘤）进行了活细胞成像。诱导剂（C'dots ⁸ 、erastin ² 、GPX4 抑制剂ML162 ^{10 、 18}或柠檬酸铁铵(FAC)和丁硫氨酸亚磺胺(BSO)的组合， Extended Data Fig. 1a , b ）。然后，我们使用引导方法来量化细胞死亡的潜在非随机模式。对于每部电影，我们计算了相邻细胞死亡之间的平均时间差 μ _expΔt ，并将该实验值与从代表随机死亡顺序的计算生成的排列得出的均值分布进行比较（ Fig. 1c , d ）。与波状传播一致，当由erastin、C'点或FAC和BSO诱导时，铁死亡以非随机时空模式发生，通过将μ _expΔt与随机分布的^第95个百分位数μ _perm95Δt进行比较来确定（ Fig. 1b , d , g ; Extended Data Fig. 1c ）。 有趣的是，当通过 ML162 处理抑制 GPX4 诱导铁死亡时，μ _expΔt更类似于随机排列的^第95 个百分位（ Fig. 1g 和 Extended Data Fig. 1d , e ）。

To measure intercellular death propagation in different forms of cell death, we induced necrosis by treatment with hydrogen peroxide (H₂O₂), and apoptosis using TNF-related apoptosis-inducing ligand (TRAIL). While H₂O₂-induced necrosis and TRAIL-induced apoptosis displayed no visually obvious wave-like spreading of death (Fig. 1e, f, Supplementary Video 2), they did result in death patterns with non-random spatiotemporal features (Fig. 1g). In order to better compare the propagative features of these different forms of cell death, we devised a measure termed the spatial propagation index (SPI). When propagation is not the major determinant of the spatiotemporal distribution of cell death across a population, we expect μ_expΔt to have similar or larger values than μ_perm95Δt, as death occurs independently of neighboring cell deaths in the vicinity. However, when propagation does play a major role, i.e. cells are affected by the death of their neighbors, we expect μ_expΔt to be much smaller than μ_perm95Δt due to the non-random spatial order of death. Thus we defined the 𝑆⁢𝑃⁢𝐼 =𝜇𝑝⁢𝑒⁢𝑟⁢𝑚⁢95⁢Δ⁢𝑡 − 𝜇𝑒⁢𝑥⁢𝑝⁢Δ⁢𝑡𝜇𝑝⁢𝑒⁢𝑟⁢𝑚⁢95⁢Δ⁢𝑡 , the spatial contribution to the observed death patterns as a fraction of the neighboring death times expected if death order were spatially random. The SPI indicates that cell death propagation plays a dominant role when ferroptosis is induced by erastin, C’ dots, or FAC and BSO, but not when induced by ML162 (Fig. 1h). Similarly, H₂O₂-induced necrosis and TRAIL-induced apoptosis also do not exhibit propagative features (Fig. 1h). These results demonstrate that the ability to spread in wave-like patterns by propagating between neighboring cells is a feature of particular forms of ferroptosis.
为了测量不同形式的细胞死亡中细胞间死亡传播，我们通过过氧化氢 (H2O) 处理诱导坏死，并使用 TNF 相关凋亡诱导配体 (TRAIL) 诱导细胞凋亡。虽然 H2O 诱导的坏死和 TRAIL 诱导的细胞凋亡没有表现出视觉上明显的波状死亡扩散 (, , )，但它们确实导致了具有非随机时空特征的死亡模式 ()。为了更好地比较这些不同形式的细胞死亡的增殖特征，我们设计了一种称为（SPI）的测量方法。当传播不是群体中细胞死亡时空分布的主要决定因素时，我们预计 μ 具有相似或大于 μ 的值，因为死亡的发生独立于附近的相邻细胞死亡。然而，当传播确实发挥主要作用时，即细胞受到其邻居死亡的影响时，由于死亡的非随机空间顺序，我们预计μ远小于μ。因此我们定义了𝑆 ⁢ 𝑃 ⁢ 𝐼 = 𝜇 𝑝 ⁢ 𝑒 ⁢ 𝑟 ⁢ 𝑚 ⁢ 9 5 ⁢ Δ ⁢ 𝑡 - 𝜇 𝑒 ⁢ 𝑥 ⁢ 𝑝 ⁢Δ ⁢ 𝑡 𝜇 𝑝 ⁢ 𝑒 ⁢ 𝑟 ⁢ 𝑚 ⁢ 9 5 ⁢ Δ ⁢ 𝑡 ，如果死亡顺序是空间随机的，则对观察到的死亡模式的空间贡献作为预期的邻近死亡时间的一部分。 SPI 表明，当由erastin、C' 点或FAC 和BSO 诱导铁死亡时，细胞死亡传播起主导作用，但由ML162 诱导时则不然。同样，H2O 诱导的坏死和 TRAIL 诱导的细胞凋亡也不表现出增殖特征 ()。 这些结果表明，通过在相邻细胞之间传播以波状模式传播的能力是特定形式的铁死亡的特征。

To examine the mechanism of ferroptotic propagation, we first asked whether iron and lipid peroxidation, two known drivers of ferroptosis, are required for propagation. The addition of the lipid peroxidation inhibitor liproxstatin-1 or the iron chelator deferoxamine (DFO) to cell cultures after the initiation of ferroptosis stopped death from spreading (Fig. 2a–d, Supplementary Video 3), demonstrating that iron and lipid peroxidation are both required for continuous ferroptosis propagation. Because iron and lipid peroxidation are also necessary for ferroptosis to occur in individual cells, these results suggested that the full execution of ferroptosis, including cell lysis, could be required for the spreading of death between cells.
为了研究铁死亡繁殖的机制，我们首先询问铁死亡的两个已知驱动因素铁和脂质过氧化是否是繁殖所必需的。在铁死亡开始后，向细胞培养物中添加脂质过氧化抑制剂 liproxstatin-1 或铁螯合剂去铁胺 (DFO) 可阻止死亡蔓延。 Fig. 2a – d , Supplementary Video 3 ），证明铁和脂质过氧化都是持续铁死亡繁殖所必需的。由于铁和脂质过氧化对于单个细胞中发生铁死亡也是必要的，因此这些结果表明铁死亡的完全执行（包括细胞裂解）可能是细胞之间死亡传播所必需的。

How ferroptosis is executed downstream of lipid peroxidation is not clearly defined. We noted from time-lapse imaging that ferroptotic cells appeared to round and swell prior to cell death (Fig. 2e). Like cell death, swelling also appeared to spread through cell populations in a manner that was blocked by treatment with liproxstatin-1 or DFO (Supplementary Video 3). Expression of an mKate-tagged version of the zebrafish cPLA2 enzyme, which localizes to the nuclear envelope upon osmotic swelling in HeLa cells¹⁹, confirmed that ferroptotic cells indeed swell prior to undergoing rupture (Fig. 2f, Supplementary Video 4). Cell swelling is also known to occur during pyroptosis and necroptosis, both of which involve the formation of pores in the plasma membrane, leading to the influx of extracellular ions and water molecules^{3, 4}. Pore-mediated cell rupture can be inhibited by incubating cells with large carbohydrates known as osmoprotectants⁴. Osmoprotectants with a diameter larger than the pores protect cells from lysis by osmotically balancing large intracellular molecules that cannot diffuse freely across the perforated membrane, while smaller osmoprotectants do not. Thus, while osmoprotectants of sufficient size do not block plasma membrane permeabilization, pore-mediated ion exchange, or cell death, they prevent osmotic cell lysis caused by pore formation. Indeed, cell rupture resulting from FAC and BSO-induced ferroptosis, as measured by the release of lactate dehydrogenase (LDH), was inhibited by the addition of polyethylene glycols (PEGs) with molecular weights of 1450 and 3350 Da, but not by the smaller osmoprotectants sucrose and raffinose (Fig. 3a). The translocation of cPLA2-mKate to the nuclear envelope was also reduced by PEG1450 and PEG3350 (Fig. 3b), suggesting that ferroptotic swelling and rupture may be caused by the opening of nano-scale pores in the plasma membrane. Induction of ferroptosis with erastin or the GPX4 inhibitors RSL3 and ML162 likewise resulted in cell rupture that was inhibited by treatment with PEG 1450 or 3350 (Fig. 3d,e). LDH release caused by H₂O₂-induced death, on the other hand, was not affected by osmoprotectants (Fig. 3c).
脂质过氧化下游如何执行铁死亡尚不清楚。我们从延时成像中注意到，铁死亡细胞在细胞死亡之前似乎变圆并膨胀（ Fig. 2e ）。与细胞死亡一样，肿胀似乎也在细胞群中扩散，但用 liproxstatin-1 或 DFO 治疗可阻断这种方式。 Supplementary Video 3 ）。斑马鱼 cPLA2 酶的 mKate 标记版本的表达（该酶在 HeLa 细胞中渗透性膨胀时定位于核膜¹⁹ ）证实了铁死亡细胞在破裂之前确实会膨胀（ Fig. 2f , Supplementary Video 4 ）。众所周知，细胞肿胀也发生在细胞焦亡和坏死性凋亡过程中，这两种情况都涉及质膜中孔的形成，导致细胞外离子和水分子的流入^{3 , 4} 。将细胞与被称为渗透保护剂的大碳水化合物一起孵育可以抑制孔介导的细胞破裂⁴ 。直径大于孔的渗透保护剂通过渗透平衡不能自由扩散穿过穿孔膜的大细胞内分子来保护细胞免于裂解，而较小的渗透保护剂则不能。因此，虽然足够大小的渗透保护剂不会阻止质膜透化、孔介导的离子交换或细胞死亡，但它们可以防止由孔形成引起的渗透细胞裂解。事实上，通过添加分子量为 1450 和 3350 Da 的聚乙二醇 (PEG) 可以抑制由 FAC 和 BSO 诱导的铁死亡引起的细胞破裂（通过乳酸脱氢酶 (LDH) 的释放来测量），但较小的聚乙二醇 (PEG) 则不会抑制细胞破裂。渗透保护剂蔗糖和棉子糖（ Fig. 3a ）。 PEG1450 和 PEG3350 也减少了 cPLA2-mKate 向核膜的易位（ Fig. 3b ），表明铁死亡膨胀和破裂可能是由质膜中纳米级孔的打开引起的。用erastin或GPX4抑制剂RSL3和ML162诱导铁死亡同样会导致细胞破裂，而用PEG 1450或3350处理可抑制细胞破裂（ Fig. 3d , e ）。另一方面，H ₂ O ₂诱导的死亡引起的 LDH 释放不受渗透保护剂的影响（ Fig. 3c ）。

As ferroptotic cell rupture could be inhibited using osmoprotectants, we sought to examine whether cell lysis is required for ferroptosis propagation. When HAP1 cells were treated with FAC and BSO in the presence of the osmoprotectant PEG1450, we observed waves of cell rounding that spread through cell colonies and appeared similar to waves of cell death (Supplementary Video 5). However, SYTOX uptake was reduced, consistent with the inhibition of cell rupture. To quantify these waves, we expressed a fluorescent sensor of nuclear calcium (GCaMP6-NLS) in HAP1 cells, reasoning that pore formation might lead to a spike in intracellular calcium levels that could be used as a readout of cell permeabilization. Live imaging of ferroptotic cells demonstrated that GCaMP fluorescence indeed increased prior to the uptake of SYTOX, and that GCaMP signals spread through cell populations in a similar manner to SYTOX and cell rounding (Fig. 4a, Supplementary Video 6). We compared the relative timing of GCaMP and SYTOX fluorescence for individual cells and found a high degree of correlation, indicating that GCaMP signals could be used instead of SYTOX uptake to assess propagation (Fig. 4b). When cells were treated with PEG1450 to inhibit rupture, wave-like spreading of GCaMP fluorescence still occurred (Fig. 4c, Supplementary Video 7). We quantitatively examined the spatiotemporal GCaMP patterns, and found that their non-random nature was similar to SYTOX death waves in both the presence and absence of osmoprotectants (Fig. 4d,e), demonstrating that propagation occurs in the absence of cell rupture.
由于使用渗透保护剂可以抑制铁死亡细胞破裂，因此我们试图检查铁死亡传播是否需要细胞裂解。当 HAP1 细胞在渗透保护剂 PEG1450 存在的情况下用 FAC 和 BSO 处理时，我们观察到细胞变圆波在细胞集落中扩散，看起来与细胞死亡波类似。 Supplementary Video 5 ）。然而，SYTOX 的摄取减少，与细胞破裂的抑制一致。为了量化这些波，我们在 HAP1 细胞中表达了核钙荧光传感器 (GCaMP6-NLS)，推断孔的形成可能会导致细胞内钙水平的峰值，这可以用作细胞透化的读数。铁死亡细胞的实时成像表明，GCaMP 荧光确实在吸收 SYTOX 之前增加，并且 GCaMP 信号以与 SYTOX 和细胞圆整类似的方式在细胞群中传播。 Fig. 4a , Supplementary Video 6 ）。我们比较了单个细胞的 GCaMP 和 SYTOX 荧光的相对时间，发现了高度的相关性，表明可以使用 GCaMP 信号代替 SYTOX 摄取来评估增殖（ Fig. 4b ）。当细胞用 PEG1450 处理以抑制破裂时，GCaMP 荧光的波状扩散仍然发生（ Fig. 4c , Supplementary Video 7 ）。我们定量检查了时空 GCaMP 模式，发现在存在和​​不存在渗透保护剂的情况下，它们的非随机性质与 SYTOX 死亡波相似（ Fig. 4d , e ），证明增殖是在没有细胞破裂的情况下发生的。

While treatment with osmoprotectants did not prevent propagation, we wondered if it might affect wave speed. To test this we used U937 cells, which exhibit long-lived, unidirectional waves of ferroptosis that can be imaged by differential interference contrast (DIC) microscopy even in the absence of SYTOX staining (Fig. 5a). Treatment of U937 cells with PEG3350 inhibited cell lysis (Fig. 5b) yet had no effect on the induction of cell death waves (Supplementary Video 8), consistent with the HAP1 data. However, when we measured the speed of these ferroptosis waves, we found them to be slightly but significantly slower in the presence of PEG3350 (1.66 vs. 1.37 μm/min, see Fig. 5c), demonstrating that ferroptosis propagation is faster when cells are able to fully lyse.
虽然渗透保护剂的处理并不能阻止传播，但我们想知道它是否会影响波速。为了测试这一点，我们使用了 U937 细胞，该细胞表现出长寿命、单向的铁死亡波，即使在没有 SYTOX 染色的情况下，也可以通过微分干涉对比 (DIC) 显微镜对其进行成像。 Fig. 5a ）。用 PEG3350 处理 U937 细胞可抑制细胞裂解（ Fig. 5b ）但对细胞死亡波的诱导没有影响（ Supplementary Video 8 ），与HAP1数据一致。然而，当我们测量这些铁死亡波的速度时，我们发现它们在 PEG3350 存在的情况下稍微但显着变慢（1.66 与 1.37 μm/min，参见 Fig. 5c ），证明当细胞能够完全裂解时，铁死亡传播速度更快。

Together these data indicate that wave-like spreading is a feature of specific forms of ferroptosis that requires the continuous presence of iron and lipid peroxidation, and involves a signal that propagates upstream of cell rupture. While ferroptosis propagation has been previously observed^{7, 8}, here we quantitatively establish the existence of non-random spatiotemporal patterns of ferroptosis in multiple contexts. Our method allowed us to distinguish two types of ferroptosis: cell-autonomous or “single-cell ferroptosis” observed in response to GPX4 inhibition, and propagative or “multicellular ferroptosis” that is induced by treatments that inhibit the generation of glutathione (erastin, BSO) and/or increase cellular iron concentrations (FAC, C’ dots). Why direct GPX4 inhibition does not induce propagative ferroptosis is important to examine in future studies, and may relate to activities of iron or functions of glutathione that do not directly involve GPX4²⁰.
这些数据共同表明，波状扩散是铁死亡特定形式的一个特征，需要铁和脂质过氧化的持续存在，并且涉及在细胞破裂上游传播的信号。虽然之前已经观察到铁死亡的传播^{7 , 8} ，但在这里我们定量地确定了多种情况下铁死亡的非随机时空模式的存在。我们的方法使我们能够区分两种类型的铁死亡：响应 GPX4 抑制而观察到的细胞自主性或“单细胞铁死亡”，以及通过抑制谷胱甘肽生成（erastin、BSO）的治疗诱导的增殖性或“多细胞铁死亡”。 ）和/或增加细胞铁浓度（FAC、C'点）。为什么直接 GPX4 抑制不会诱导增殖性铁死亡对于未来的研究很重要，并且可能与不直接涉及 GPX4 的铁活性或谷胱甘肽功能有关²⁰ 。

Non-autonomous cell death effects have been described elsewhere, most notably in the radiation-induced bystander effect (RIBE), where damage and death rates are increased in cells adjacent to those exposed to radiation²¹. While RIBE may increase death frequencies, such phenotypes appear distinct from the wave-like death observed during ferroptosis that, in many cases, leads to the near-complete elimination of a cell population^{7, 8, 15}. Further discovery of the underlying molecular mechanisms is required to determine whether death propagation in these different systems involves similar signaling mechanisms. Numerous factors are proposed to mediate RIBE, including gap junctions, TGFβ²², p53, and cyclooxygenase-2 (COX-2) signaling²¹. While our U937 data suggest that gap junctions are not involved in ferroptosis propagation, since these cells do not form cell junctions (Supplementary Video 8), whether other RIBE signals could play a role in ferroptosis spreading is not yet known. Our finding that the presence of an osmoprotectant slows propagation could suggest that the release of a spreadable factor is enhanced by cell rupture, although further experiments are needed to test this.
非自主细胞死亡效应已在其他地方描述过，最引人注目的是辐射诱导的旁观者效应 (RIBE)，其中与暴露于辐射的细胞相邻的细胞的损伤和死亡率增加²¹ 。虽然 RIBE 可能会增加死亡频率，但这种表型似乎与铁死亡期间观察到的波状死亡不同，铁死亡期间观察到的波状死亡在许多情况下导致细胞群几乎完全消除^{7 , 8 , 15} 。需要进一步发现潜在的分子机制，以确定这些不同系统中的死亡传播是否涉及相似的信号机制。许多因素被认为可以介导 RIBE，包括间隙连接、TGFβ ²² 、p53 和环氧合酶-2 (COX-2) 信号传导²¹ 。虽然我们的 U937 数据表明间隙连接不参与铁死亡传播，因为这些细胞不形成细胞连接（ Supplementary Video 8 ），其他 RIBE 信号是否在铁死亡扩散中发挥作用尚不清楚。我们发现渗透保护剂的存在会减缓增殖，这可能表明细胞破裂会增强可扩散因子的释放，尽管需要进一步的实验来测试这一点。

Our data also indicate that ferroptosis is an osmotic process, as it involves cell swelling (Fig. 5d), and can be blocked by the addition of large osmoprotectants. The ability of osmoprotectants to block lysis following induction of necroptosis or pyroptosis both in culture and in vivo, and the observed size-dependence of the protective effects of different osmoprotectants (Fig. 3), have been interpreted previously as evidence for the existence of pore-like structures that trigger lysis in these forms of necrosis^{3, 4}. This is indeed known to occur during pyroptosis, in which the caspase-dependent cleavage of Gasdermin D (GSDMD) triggers its oligomerization in the plasma membrane^{23, 24}. Similarly, necroptosis may involve plasma membrane permeabilization mediated by the pseudokinase MLKL^{25, 26}. Our data thus suggest that ferroptotic rupture is mediated by the formation of plasma membrane pores of a few nanometers in diameter and that cell permeabilization during ferroptosis could be a regulated process. Intriguingly, lipid peroxidation has been proposed to lead to conformational changes in lipid domains and plasma membrane regions^{27, 28}, raising the possibility that pore formation could occur through a lipid-based mechanism rather than by activation of a pore-forming protein. Ferroptotic pore formation could regulate not only cell death execution but also the potential release of pro-inflammatory cytokines or DAMPs, which is known to occur during pyroptosis²⁹. We previously showed that ferroptosis induction in mouse xenografts leads to tumor regression and a concomitant immune response, implying that ferroptosis-inducing agents may be promising cancer therapies^{8, 15}. Ferroptosis is also implicated in cell death resulting from ischemia reperfusion injury during stroke or myocardial infarction, as well as in acute kidney injury, all of which result in the formation of large zones of necrotic tissue, possibly indicating a role for ferroptosis propagation in these diseases^{1, 15}. Intriguingly, the paper by Katikaneni et al. published in this issue shows large waves of cellular deformation occurring in intact zebrafish larvae following microperfusion of arachidonic acid (AA). As AA is a known driver of ferroptosis, this finding suggests that wave-like propagation of ferroptosis may also occur in vivo, causing widespread tissue damage³⁰. Uncovering the molecular mechanisms that regulate ferroptosis execution and propagation through cell populations will ultimately further our understanding of how modulators of ferroptosis may be leveraged for therapeutic benefit.
我们的数据还表明铁死亡是一个渗透过程，因为它涉及细胞肿胀（ Fig. 5d ），并且可以通过添加大量渗透保护剂来阻断。渗透保护剂在培养物和体内诱导坏死性凋亡或焦亡后阻止裂解的能力，以及观察到的不同渗透保护剂的保护作用的大小依赖性（ Fig. 3 ），之前已被解释为存在类孔结构的证据，这些结构会引发这些形式的坏死^{3 、 4 的}溶解。这确实已知发生在焦亡期间，其中 Gasdermin D (GSDMD) 的半胱天冬酶依赖性裂解触发其在质膜中的寡聚化^{23 、 24} 。类似地，坏死性凋亡可能涉及由假激酶 MLKL ^25、26介导的质膜透化。因此，我们的数据表明，铁死亡破裂是由直径为几纳米的质膜孔的形成介导的，并且铁死亡期间的细胞透化可能是一个受调节的过程。有趣的是，脂质过氧化被认为会导致脂质结构域和质膜区域^27、28的构象变化，从而提高了通过基于脂质的机制而不是通过成孔蛋白的激活来发生孔形成的可能性。铁死亡孔的形成不仅可以调节细胞死亡的执行，还可以调节促炎细胞因子或 DAMP 的潜在释放，已知这发生在细胞焦亡期间²⁹ 。 我们之前表明，小鼠异种移植物中的铁死亡诱导导致肿瘤消退和伴随的免疫反应，这意味着铁死亡诱导剂可能是有前途的癌症疗法^{8 , 15} 。铁死亡还与中风或心肌梗塞期间的缺血再灌注损伤以及急性肾损伤引起的细胞死亡有关，所有这些都会导致大片坏死组织的形成，可能表明铁死亡在这些疾病中传播的作用^{1 , 15} .有趣的是，Katikaneni等人的论文。本期发表的研究表明，在微灌注花生四烯酸 (AA) 后，完整的斑马鱼幼虫中发生了大波的细胞变形。由于 AA 是铁死亡的已知驱动因素，这一发现表明铁死亡的波状传播也可能在体内发生，导致广泛的组织损伤³⁰ 。揭示调节铁死亡执行和通过细胞群传播的分子机制最终将进一步加深我们对如何利用铁死亡调节剂获得治疗益处的理解。