在现代集约化养殖产业背景下，畜禽面临着复杂的生长环境，如环境应激、病原体感染以及营养失衡等，这些因素均可诱发神经细胞损伤。神经损伤在临床上主要表现为显著的行为学异常，如攻击性行为增强、刻板运动增多以及探究行为反射减弱等。更为重要的是，神经损伤导致动物摄食和生殖机能紊乱，对动物健康产生负面影响，进而影响畜牧生产的经济效益。近年来，研究发现铁死亡与神经元损伤及神经系统疾病密切相关。铁死亡是一种铁依赖性脂质过氧化物积累的程序性细胞死亡方式。本研究利用铁死亡诱导剂IKE诱导HT-22神经细胞铁死亡，随后通过高通量质谱蛋白质组学技术，筛选铁死亡的差异表达蛋白并系统性分析其功能，最终确定胱硫醚 γ-裂合酶（Cystathionine gamma-lyase，CTH）为IKE诱导HT-22细胞铁死亡的关键蛋白。目前，CTH与神经细胞铁死亡之间的关系尚不清楚。因此，本研究旨在深入探究CTH对IKE诱导神经细胞铁死亡的作用及其分子调控机制。

IKE诱导HT-22细胞铁死亡模型的建立及CTH的筛选与验证

本研究利用IKE构建HT-22细胞铁死亡模型，并通过实验室前期的TMT蛋白组学分析，筛选出HT-22细胞铁死亡的关键靶蛋白，并对其表达量进行验证。首先本研究采用不同浓度的IKE处理HT-22细胞，并设置不同的处理时间，结果显示，IKE（5 μM）处理HT-22细胞24 h，与对照组相比，细胞活力下降50%左右，且检测到细胞内ROS、lipid ROS、Fe²⁺、MDA含量增加以及PTGS2基因和蛋白表达升高，GSH含量和GPX4基因和蛋白表达下降，证明铁死亡造模成功。在蛋白组学数据分析过程中，设定P<0.05且Ratio>1.5作为筛选参数，对铁死亡组与对照组的蛋白进行差异分析。经筛选，共鉴定出105个差异蛋白，其中41个蛋白表达上调，63个蛋白表达下调。在蛋白组学GO-BP展示的前20个高度富集的差异通路中，“羧酸代谢过程”富集的差异蛋白数量最多。基于该通路中各蛋白的差异变化倍数，筛选出呈表达差异变化较大的CTH作为靶标蛋白。通过Western blot和RT-qPCR进行验证IKE处理可显著上调CTH基因和蛋白表达，与蛋白组学分析结果趋势一致。并检测到细胞内硫化氢（H₂S）和半胱氨酸（Cysteine，Cys）含量下降，确定CTH的生物功能增强。由此得出结论：5 μM IKE处理24 h可以导致HT-22细胞发生铁死亡，并且上调HT-22细胞中CTH表达。

CTH调控IKE诱导HT-22细胞铁死亡

为探究CTH在IKE诱导HT-22细胞铁死亡中的功能，本研究构建CTH的siRNA和过表达质粒，对CTH进行功能验证。首先，转染siCth，借助RT-qPCR、Western blot以及免疫荧光等技术手段，并且检测细胞内H₂S和Cys含量变化，从基因、蛋白表达和CTH功能层面，发现CTH表达和功能下降，证明siCth转染成功。同时发现抑制CTH缓解IKE导致的细胞活力下降、细胞内ROS、Fe²⁺、MDA及GSH含量升高，由此证明抑制CTH可以显著缓解IKE导致的HT-22细胞铁死亡。随后转染CTH过表达质粒，借助RT-qPCR、Western blot以及免疫荧光结果显示，CTH基因及蛋白表达升高，检测到细胞内H₂S和Cys含量升高，进一步从CTH功能层面证明CTH过表达质粒构建成功。并发现过表达CTH导致细胞活力下降、细胞内ROS、Fe²⁺、MDA及GSH含量升高。得出结论：IKE上调CTH表达导致HT-22细胞发生铁死亡。

CTH通过NRF2/HO-1通路促进HT-22细胞铁死亡

为深入探究CTH如何参与IKE诱导的HT-22细胞铁死亡，本部分研究结合数据库分析、通过对通路蛋白干扰和过表达实验，旨在确定CTH在铁死亡过程中的调控机制。本试验中首先使用铁死亡抑制剂（Fer-1）、自噬抑制剂（3-Ma）和ROS清除剂（NAC），与OE Cth共处理HT-22细胞，发现NAC能极显著缓解OE Cth导致的细胞活力下降。基于此，联合Pathcards数据库收集的氧化应激反应蛋白与CTH进行联合分析，结果显示CTH与NRF2和HO1等氧化应激蛋白存在调控关系。其次借助Western blot和RT-qPCR，检测调控CTH对NRF2/HO1表达的影响，结果显示，siCth可以逆转IKE导致的NRF2、HO1蛋白和基因水平的表达升高；而OE Cth则上调两者的表达，与预测结果一致。有研究表明NRF2作为一种核转录因子，HO1是其下游靶蛋白，NRF2在细胞内的定位对HO1表达至关重要。因此，本研究通过提取细胞浆和细胞核蛋白，结果显示IKE处理与过表达CTH均能促使NRF2向细胞核内转移。而当转染siCth时，可有效逆转IKE所诱导的NRF2核转位。为进一步明确NRF2/HO1在CTH促进HT-22细胞铁死亡过程中的作用机制，本研究将siNrf2、siHo1分别与OE Cth共转染，发现阻断NRF2/HO1表达可以缓解OE Cth导致的细胞活力下降，细胞内ROS和Fe²⁺含量升高，证明抑制NRF2和HO1可以缓解OE Cth导致的细胞铁死亡。为验证NRF2、HO1与抑制CTH缓解IKE诱导的HT-22铁死亡之间的关系，在IKE处理HT-22细胞下，OE Nrf2和OE Ho1分别与siCth的共转染实验，发现OE Nrf2、OE Ho1分别可以逆转siCth缓解的IKE诱导的HT-22铁死亡。由此确定，CTH激活NRF2/HO1促进IKE诱导HT-22细胞铁死亡发生。

综上所述，本研究揭示CTH/NRF2/HO-1通路在铁死亡中的调控机制，为神经损伤和神经退行性疾病等铁死亡相关病理过程提供潜在治疗靶点，为开发新型神经保护策略提供科学依据

Domesticated animals within intensive livestock production systems face a barrage of environmental stressors, including ambient conditions, pathogen exposure, and dietary imbalances. This multifactorial stress burden can lead to neurophysiological disruptions via neuronal cytotoxic mechanisms. These factors can collectively contribute to potential nerve cell damage. Nerve damage is clinically manifested by significant behavioral abnormalities, such as increased aggressive behavior, increased stereotyped movements, and weakened reflexes for exploratory behavior. More importantly, nerve damage leads to disorders in feeding and reproductive functions, which negatively affects animal health and consequently the economic benefits of livestock production. In recent years, it has been found that iron death is closely related to neuronal damage and neurological diseases. Iron death is a programmed cell death mode of iron-dependent lipid peroxide accumulation. In this study, ferroptosis of HT-22 nerve cells was induced by the ferroptosis inducer IKE, and then screened the differentially expressed proteins of iron death and systematically analyzed their functions by high-throughput mass spectrometry proteomics, and finally identified cystathionine gamma-lyase (CTH) as the IKE-induced iron death in HT-22 cells. key protein. Currently, the relationship between CTH and iron death in neuronal cells is unclear. Therefore, the present study aimed to deeply investigate the role of CTH on IKE-induced iron death in neuronal cells and its molecular regulatory mechanism.

1. Establishment of IKE-induced iron death model of HT-22 cells and screening and validation of CTH

In this study, we utilized the IKE-induced iron death model of HT-22 cells and screened the key target proteins of iron death in HT-22 cells and validated their expression by pre-laboratory TMT proteomics analysis. Firstly, in this study, HT-22 cells were treated with different concentrations of IKE and set different treatment times. The results showed that IKE (5 μM) treatment of HT-22 cells for 24 h resulted in a decrease in cell viability of about 50% compared with the control group, and an increase in intracellular ROS, lipid ROS, Fe²⁺, and MDA content as well as an elevation of PTGS2 gene and protein expression, and a decrease in GSH content and GPX4 gene and protein expression were detected, which proved that iron death modeling was successful. During the proteomics data analysis, P < 0.05 and Ratio > 1.5 was set as a screening parameter to analyze the proteins of the iron death group and the control group differently. After screening, a total of 105 differential proteins were identified, of which 41 proteins were up-regulated and 63 proteins were down-regulated in expression. Among the top 20 highly enriched differential pathways demonstrated by proteomics GO-BP, “carboxylic acid metabolism” had the highest number of enriched differential proteins. Based on the multiplicity of differential changes of the proteins in this pathway, CTH, which showed large differential changes in expression, was selected as a target protein. It was verified by Western blot and RT-qPCR that IKE treatment significantly up-regulated CTH gene and protein expression, which was consistent with the trend of proteomic analysis. A decrease in intracellular hydrogen sulfide (H₂S) and cysteine (Cys) content was also detected, confirming the enhanced biological function of CTH. It was concluded that 5 μM IKE treatment for 24 h could lead to iron death in HT-22 cells and up-regulate CTH expression in HT-22 cells.

2. CTH regulates IKE-induced iron death in HT-22 cells

To investigate the function of CTH in IKE-induced iron death in HT-22 cells, siRNA and overexpression plasmid of CTH were constructed in this study to functionally verify CTH. Firstly, siCth was transfected, with the help of RT-qPCR, Western blot and immunofluorescence, and the changes of intracellular H₂S and Cys content were detected, and the decrease of CTH expression and function was found from the level of genes, protein expression and CTH function, which proved the success of siCth transfection. At the same time, it was found that inhibition of CTH alleviated the decrease in cell viability and the increase in intracellular ROS, Fe²⁺, MDA, and GSH content caused by IKE, thus proving that inhibition of CTH could significantly alleviate iron death in HT-22 cells caused by IKE. Subsequently, the CTH overexpression plasmid was transfected, and with the help of RT-qPCR, Western blot and immunofluorescence results, the expression of CTH gene and protein was elevated, and the contents of intracellular H₂S and Cys were detected to be increased, which further proved that the construction of CTH overexpression plasmid was successful from the functional level of CTH. It was also found that overexpression of CTH resulted in decreased cell viability and increased intracellular ROS, Fe²⁺, MDA, and GSH content. It was concluded that IKE up-regulation of CTH expression led to iron death in HT-22 cells.

3. CTH Promotes Iron Death in HT-22 Cells via the NRF2/HO-1 Pathway

In order to deeply investigate how CTH is involved in IKE-induced iron death in HT-22 cells, this part of the study, combined with database analysis, and through interference with pathway proteins and overexpression experiments, aimed to determine the regulatory mechanism of CTH in the iron death process. In this experiment, we firstly used iron death inhibitor (Fer-1), autophagy inhibitor (3-Ma) and ROS scavenger (NAC) to co-treat HT-22 cells with OE Cth, and found that NAC could extremely significantly alleviate the decrease in cell viability caused by OE Cth. Based on this, joint analysis of oxidative stress response proteins collected from the Pathcards database in conjunction with CTH showed that CTH has a regulatory relationship with oxidative stress proteins such as NRF2 and HO1. Next, with the help of Western blot and RT-qPCR, the effect of regulating CTH on NRF2/HO1 expression was examined, and the results showed that siCth could reverse the elevated expression of NRF2 and HO1 protein and gene levels caused by IKE; while OE Cth upregulated the expression of both, which was in line with the predicted results. It has been shown that NRF2 acts as a nuclear transcription factor and HO1 is its downstream target protein, and the localization of NRF2 in the cell is critical for HO1 expression. Therefore, in this study, by extracting cytoplasmic and nuclear proteins, the results showed that both IKE treatment and overexpression of CTH could promote the translocation of NRF2 to the nucleus. And when transfected with siCth, the NRF2 entry into the nucleus induced by IKE could be effectively reversed. To further clarify the mechanism of NRF2/HO1 in the process of CTH-promoted iron death in HT-22 cells, in this study, siNrf2 and siHo1 were cotransfected with OE Cth, respectively, and it was found that blocking the expression of NRF2/HO1 alleviated the decrease in cell viability and the elevation of intracellular ROS and Fe²⁺ content caused by OE Cth, demonstrating that that inhibition of NRF2 and HO1 could alleviate cellular iron death caused by OE Cth. To verify the relationship between NRF2, HO1, and inhibition of CTH to alleviate IKE-induced iron death in HT-22, cotransfection experiments of OE Nrf2 and OE Ho1 with siCth under IKE-treated HT-22 cells revealed that OE Nrf2 and OE Ho1, respectively, could reverse IKE-induced iron death in HT-22 alleviated by siCth. It was thus determined that CTH activation of NRF2/HO1 promotes the occurrence of IKE-induced iron death in HT-22 cells.

In summary, the present study reveals the regulatory mechanism of the CTH/NRF2/HO-1 pathway in iron death, provides potential therapeutic targets for iron death-related pathological processes such as nerve injury and neurodegenerative diseases, and provides a scientific basis for the development of novel neuroprotective strategies.

[1] AbeK,KimuraH. The possible role of hydrogen sulfide as an endogenous neuromodulator[J]. Journal of Neuroscience, 1996, 16(3):1066-1071.

[2] Abou-Hamdan A, Guedouari-Bounihi H, Lenoir V, et al. Oxidation of H2S in mammalian cells and mitochondria[J]. Methods Enzymol, 2015, 554:201-228.

[3] Aggarwal S, Talukdar N C, Yadav A K. Advances in higher order multiplexing techniques in proteomics[J]. Journal of Proteome Research, 2019, 18(6):2360-2369.

[4] Alam J, Stewart D, Touchard C, et al. Nrf2, a cap'n'collar transcription factor, regulates induction of the Heme Oxygenase-1 gene[J]. Journal of Biological Chemistry, 1999, 274(37):26071-26078.

[5] Araki S, Takata T, Ono K, et al. Cystathionine γ-lyase self-inactivates by polysulfidation during cystine metabolism[J]. International Journal of Molecular Sciences, 2023, 24(12): 24(12):9982.

[6] Bannai S. Exchange of cystine and glutamate across plasma membrane of human fibroblasts[J]. Journal of Biological Chemistry, 1986, 261(5):2256-2263.

[7] Bao W D, Pang P, Zhou X T, et al. Loss of ferroportin induces memory impairment by promoting ferroptosis in alzheimer's disease[J]. Cell Death and Differentiation, 2021, 28(5):1548-1562.

[8] Bauermeister A, Mannochio-Russo H, Costa-Lotufo L V, et al. Mass spectrometry-based metabolomics in microbiome investigations[J]. Nature Reviews Microbiology, 2022, 20(3):143-160.

[9] Bedoui S, Herold M J, Strasser A. Emerging connectivity of programmed cell death pathways and its physiological implications[J]. Nature Reviews Molecular Cell Biology, 2020, 21(11):678-695.

[10] Bibli S I, Hu J, Sigala F, et al. Cystathionine γ lyase sulfhydrates the RNA binding protein human antigen r to preserve endothelial cell function and delay atherogenesis[J]. Circulation, 2019, 139(1):101-114.

[12] Bogdan A R, Miyazawa M, Hashimoto K, et al. Regulators of iron homeostasis: new players in metabolism, cell death, and disease[J]. Trends in Biochemical Sciences, 2016, 41(3):274-286.

[13] Cano-Galiano A, Oudin A, Fack F, et al. Cystathionine-γ-lyase drives antioxidant defense in cysteine-restricted idh1-mutant astrocytomas[J]. Neuro-Oncology Advances, 2021, 3(1): vdab057.

[14] Cao X, Ding L, Xie Z Z, et al. A review of hydrogen sulfide synthesis, metabolism, and measurement: is modulation of hydrogen sulfide a novel therapeutic for cancer? [J]. Antioxidants & Redox Signaling, 2019, 31(1):1-38.

[15] Chen N C, Yang F, Capecci L M, et al. Regulation of homocysteine metabolism and methylation in human and mouse tissues[J]. Faseb Journal, 2010, 24(8):2804-2817.

[16] Chen T, Majerníková N, Marmolejo-Garza A, et al. Mitochondrial transplantation rescues neuronal cells from ferroptosis[J]. Free Radical Biology and Medicine, 2023, 208:62-72.

[17] Chen X, Kang R, Kroemer G, et al. Organelle-specific regulation of ferroptosis[J]. Cell Death and Differentiation, 2021a, 28(10):2843-2856.

[18] Chen X, Li J, Kang R, et al. Ferroptosis: machinery and regulation[J]. Autophagy, 2021b, 17(9):2054-2081.

[19] Costa I, Barbosa D J, Benfeito S, et al. Molecular mechanisms of ferroptosis and their involvement in brain diseases[J]. Pharmacology & Therapeutics, 2023, 244:108373.

[20] Dai Y, Chen Y, Mo D, et al. Inhibition of Acsl4 ameliorates tubular ferroptotic cell death and protects against fibrotic kidney disease[J]. Communications Biology, 2023, 6(1):907.

[21] Dang R, Wang M, Li X, et al. Edaravone ameliorates depressive and anxiety-like behaviors via Sirt1/Nrf2/Ho-1/Gpx4 pathway[J]. Journal of Neuroinflammation, 2022, 19(1):41.

[22] David S, Jhelum P, Ryan F, et al. Dysregulation of iron homeostasis in the central nervous system and the role of ferroptosis in neurodegenerative disorders[J]. Antioxidants & Redox Signaling, 2022, 37(1-3):150-170.

[23] De Cicco P, Panza E, Ercolano G, et al. Atb-346, A novel hydrogen sulfide-releasing anti-inflammatory drug, induces apoptosis of human melanoma cells and inhibits melanoma development in vivo[J]. Pharmacological Research, 2016, 114:67-73.

[24] di Masi A, Ascenzi P. H2S: a "double face" molecule in health and disease[J]. Biofactors, 2013, 39(2):186-196.

[25] Dixon S J, Lemberg K M, Lamprecht M R, et al. Ferroptosis: an iron-dependent form of Nonapoptotic Cell Death[J]. Cell, 2012, 149(5):1060-1072.

[26] Dixon S J, Olzmann J A. The cell biology of ferroptosis[J]. Nature Reviews Molecular Cell Biology, 2024, 25(6):424-442.

[27] Dixon S J, Patel D N, Welsch M, et al. Pharmacological inhibition of cystine-glutamate exchange induces endoplasmic reticulum stress and ferroptosis[J]. eLife, 2014, 3: e02523.

[28] Doll S, Freitas F P, Shah R, et al. Fsp1 is a glutathione-independent ferroptosis suppressor[J]. Nature, 2019, 575(7784):693-698.

[29] Doll S, Proneth B, Tyurina Y Y, et al. Acsl4 dictates ferroptosis sensitivity by shaping cellular lipid composition[J]. Nature Chemical Biology, 2017, 13(1):91-98.

[30] Dolma S, Lessnick S L, Hahn W C, et al. Identification of genotype-selective antitumor agents using synthetic lethal chemical screening in engineered human tumor cells[J]. Cancer Cell, 2003, 3(3):285-296.

[31] Fan D, Tang H, Yang X, et al. Improving statins production: from non-genetic strategies to genetic strategies[J]. Biotechnology Journal, 2023, 18(12): e2300229.

[32] Fão L, Mota S I, Rego A C. Shaping the Nrf2-are-related pathways in alzheimer’s and parkinson’s diseases[J]. Aging Research Reviews, 2019, 54:100942.

[33] Fu M, Zhang W, Wu L, et al. Hydrogen sulfide (H2S) metabolism in mitochondria and its regulatory role in energy production[J]. Proceedings of the National Academy of Sciences of the United States of America, 2012, 109(8):2943-2948.

[34] Gao W, Wang X, Zhou Y, et al. Autophagy, ferroptosis, pyroptosis, and necroptosis in tumor immunotherapy[J]. Signal Transduction and Targeted Therapy, 2022, 7(1):196.

[35] Giannakopoulou E, Konstantinou F, Ragia G, et al. Epigenetics-by-sex interaction for coronary artery disease risk conferred by the cystathionine γ-lyase gene promoter methylation[J]. Omics, 2017, 21(12):741-748.

[36] Giovinazzo D, Bursac B, Sbodio J I, et al. Hydrogen sulfide is neuroprotective in alzheimer's disease by sulfhydrating Gsk3β and inhibiting tau hyperphosphorylation[J]. PNAS, 2021, 118(4)：e2017225118.

[37] Gout P W, Buckley A R, Simms C R, et al. Sulfasalazine, a potent suppressor of lymphoma growth by inhibition of the xc− cystine transporter: a new action for an old drug[J]. Leukemia, 2001, 15(10):1633-1640.

[38] Guo S, Zhang D, Dong Y, et al. Sulfiredoxin-1 accelerates erastin-induced ferroptosis in HT-22 hippocampal neurons by driving heme oxygenase-1 activation[J]. Free Radical Biology and Medicine, 2024, 223:430-442.

[39] Guo S, Zhong A, Zhang D, et al. Atp2b3 Inhibition Alleviates erastin-induced ferroptosis in HT-22 cells through the P62-Keap1-Nrf2-Ho-1 pathway[J]. The International Journal of Molecular Sciences, 2023, 24(11):9199.

[40] Guo X, Qi X, Li H, et al. Deferoxamine alleviates iron overload and brain injury in a rat model of brainstem hemorrhage[J]. World Neurosurg, 2019, 128: e895-e904.

[41] Han C, Liu Y, Dai R, et al. Ferroptosis and its potential role in human diseases[J]. Frontiers in Pharmacology, 2020, 11:239.

[42] Hao M, Jiang Y, Zhang Y, et al. Ferroptosis regulation by methylation in cancer[J]. Biochimica et Biophysica Acta (BBA) - Reviews on Cancer, 2023, 1878(6):188972.

[43] Harding H P, Zhang Y, Zeng H, et al. An integrated stress response regulates amino acid metabolism and resistance to oxidative stress[J]. Molecular Cell, 2003, 11(3):619-633.

[44] Hassan M I, Boosen M, Schaefer L, et al. Platelet-derived growth factor-bb induces cystathionine γ-lyase expression in rat mesangial cells via a redox-dependent mechanism[J]. The British Journal of Pharmacology, 2012, 166(8):2231-2242.

[45] Hassannia B, Vandenabeele P, Vanden Berghe T. Targeting ferroptosis to iron out cancer[J]. Cancer Cell, 2019, 35(6):830-849.

[46] Hayano M, Yang W S, Corn C K, et al. Loss of cysteinyl-trna synthetase (cars) induces the transsulfuration pathway and inhibits ferroptosis induced by cystine deprivation[J]. Cell Death and Differentiation, 2016, 23(2):270-278.

[47] Henning Y, Blind U S, Larafa S, et al. Hypoxia aggravates ferroptosis in RPE cells by promoting the fenton reaction[J]. Cell Death and Disease, 2022, 13(7):662.

[48] Hirschhorn T, Stockwell B R. The development of the concept of ferroptosis[J]. Free Radical Biology and Medicine, 2019, 133:130-143.

[49] Hostettler I C, Seiffge D J, Werring D J. Intracerebral hemorrhage: an update on diagnosis and treatment[J]. Expert Review of Neurotherapeutics, 2019, 19(7):679-694.

[50] Hu B, Zhang X X, Zhang T, et al. Dissecting molecular mechanisms underlying ferroptosis in human umbilical cord mesenchymal stem cells: role of cystathionine γ-lyase/hydrogen sulfide pathway[J]. World Journal of Stem Cells, 2023, 15(11):1017-1034.

[51] Jia B, Li J, Song Y, et al. Acsl4-mediated ferroptosis and its potential role in central nervous system diseases and injuries[J]. The International Journal of Molecular Sciences, 2023, 24(12):10021.

[52] Jiang Y, Glandorff C, Sun M. GSH and ferroptosis: side-by-side partners in the fight against tumors[J]. Antioxidants (Basel), 2024, 13(6):697.

[53] Jiang Z, Yang H, Ni W, et al. Attenuation of neuronal ferroptosis in intracerebral hemorrhage by inhibiting Hdac1/2: microglial heterogenization via the Nrf2/Ho1 pathway[J]. CNS Neuroscience & Therapeutics, 2024, 30(3): e14646.

[54] Jin R, Yang R, Cui C, et al. Ferroptosis due to cystathionine γ lyase/hydrogen sulfide downregulation under high hydrostatic pressure exacerbates vsmc dysfunction[J]. Frontiers in Cell and Developmental Biology, 2022, 10:829316.

[55] Jurkowska H, Kaczor-Kamińska M, Bronowicka-Adamska P, et al. [Cystathionine γ-Lyase] [J]. Postepy Higieny I Medycyny Doswiadczalnej, 2014a, 68:1-9.

[56] Jurkowska H, Roman H B, Hirschberger L L, et al. Primary hepatocytes from mice lacking cysteine dioxygenase show increased cysteine concentrations and higher rates of metabolism of cysteine to hydrogen sulfide and thiosulfate[J]. Amino Acids, 2014b, 46(5):1353-1365.

[57] Kadir F H, al-Massad F K, Moore G R. Haem binding to horse spleen ferritin and its effect on the rate of iron release[J]. Biochemical Journal, 1992, 282 (Pt 3):867-870.

[58] Kandil S, Brennan L, McBean G J. Glutathione depletion causes a Jnk and P38mapk-mediated increase in expression of cystathionine-gamma-lyase and upregulation of the transsulfuration pathway in c6 glioma cells[J]. Neurochemistry International, 2010, 56(4):611-619.

[59] Kansanen E, Kuosmanen S M, Leinonen H, et al. The Keap1-Nrf2 pathway: mechanisms of activation and dysregulation in cancer[J]. Redox Biology, 2013, 1(1):45-49.

[60] Kimura H. Hydrogen sulfide induces cyclic amp and modulates the NMDA receptor[J]. Biochemical and Biophysical Research Communications, 2000, 267(1):129-133.

[61] Koppula P, Zhuang L, Gan B. Cystine transporter Slc7a11/Xct in cancer: ferroptosis, nutrient dependency, and cancer therapy[J]. Protein Cell, 2021, 12(8):599-620.

[62] Kuang F, Liu J, Tang D, et al. Oxidative damage and antioxidant defense in ferroptosis[J]. Frontiers in Cell and Developmental Biology, 2020, 8:586578.

[63] Kwon M Y, Park E, Lee S J, et al. Heme oxygenase-1 accelerates erastin-induced ferroptotic cell death[J]. Oncotarget, 2015, 6(27):24393-24403.

[64] Latunde-Dada G O. Ferroptosis: role of lipid peroxidation, iron and ferritinophagy[J]. Biochimica Et Biophysica Acta-general Subjects, 2017, 1861(8):1893-1900.

[65] Li F J, Long H Z, Zhou Z W, et al. System x(c) (-)/Gsh/Gpx4 axis: an important antioxidant system for the ferroptosis in drug-resistant solid tumor therapy[J]. Frontiers in Pharmacology 2022a, 13:910292.

[66] Li J, Jia Y C, Ding Y X, et al. The crosstalk between ferroptosis and mitochondrial dynamic regulatory networks[J]. Biochimica Et Biophysica Acta-general Subjects, 2023, 19(9):2756-2771.

[67] Li L, Hsu A, Moore P K. Actions and interactions of nitric oxide, carbon monoxide and hydrogen sulphide in the cardiovascular system and in inflammation-a tale of three gases! [J]. Pharmacology & Therapeutics, 2009, 123(3):386-400.

[68] Li X, Zhang W, Xing Z, et al. Targeting Sirt3 sensitizes glioblastoma to ferroptosis by promoting mitophagy and inhibiting slc7a11[J]. Cell Death and Disease, 2024, 15(2):168.

[69] Li Y, Xu B, Ren X, et al. Inhibition of Cisd2 promotes ferroptosis through ferritinophagy-mediated ferritin turnover and regulation of P62-Keap1-Nrf2 pathway[J]. Cellular & Molecular Biology Letters, 2022b, 27(1):81.

[70] Liang C, Zhang X, Yang M, et al. Recent progress in ferroptosis inducers for cancer therapy[J]. Advanced Materials, 2019, 31(51): e1904197.

[71] Lieu P T, Heiskala M, Peterson P A, et al. The roles of iron in health and disease[J]. Molecular Aspects of Medicine, 2001, 22(1):1-87.

[72] Lima S F, Pires S, Rupert A, et al. The gut microbiome regulates the clinical efficacy of sulfasalazine therapy for ibd-associated spondyloarthritis[J]. Cell Reports Medicine, 2024, 5(3):101431.

[73] Lin W, Wang C, Liu G, et al. Slc7a11/Xct in cancer: biological functions and therapeutic implications[J]. American Journal of Cancer Research, 2020, 10(10):3106-3126.

[74] Liu A, Wu Q, Guo J, et al. Statins: adverse reactions, oxidative stress and metabolic interactions[J]. Pharmacology & Therapeutics, 2019, 195:54-84.

[75] Liu M, Zeng C, Zhang Y, et al. Protective role of hydrogen sulfide against diabetic cardiomyopathy by inhibiting pyroptosis and myocardial fibrosis[J]. Biomed Pharmacother, 2024, 175:116613.

[76] Loboda A, Damulewicz M, Pyza E, et al. Role of Nrf2/Ho-1 system in development, oxidative stress response and diseases: an evolutionarily conserved mechanism[J]. Cellular and Molecular Life Sciences, 2016, 73(17):3221-3247.

[77] Lönnerdal B. Calcium and iron absorption-mechanisms and public health relevance[J]. International Journal for Vitamin and Nutrition Research, 2010, 80(4-5):293-299.

[78] Lossi L, Alasia S, Salio C, et al. Cell death and proliferation in acute slices and organotypic cultures of mammalian CNS[J]. Progress in Neurobiology, 2009, 88(4):221-245.

[79] Lu B, Chen X B, Ying M D, et al. The role of ferroptosis in cancer development and treatment response[J]. Frontiers in Pharmacology 2017, 8:992.

[80] Lu J, Tang Z, Xu M, et al. Skeletal muscle cystathionine γ-lyase deficiency promotes obesity and insulin resistance and results in hyperglycemia and skeletal muscle injury upon hfd in mice[J]. Redox Report, 2024, 29(1):2347139.

[81] Ma W, Hu N, Xu W, et al. Ferroptosis inducers: a new frontier in cancer therapy[J]. Bioorganic Chemistry, 2024, 146:107331.

[82] Mao C, Liu X, Zhang Y, et al. Dhodh-mediated ferroptosis defence is a targetable vulnerability in cancer[J]. Nature, 2021, 593(7860):586-590.

[83] Masters C L, Bateman R, Blennow K, et al. Alzheimer's disease[J]. Naturereviews Disease Primers, 2015, 1:15056.

[84] Meijer H A, Smith E M, Bushell M. Regulation of miRNA strand selection: follow the leader? [J]. Biochemical Society Transactions, 2014, 42(4):1135-1140.

[85] Miao Y, Zhang S, Liang Z, et al. Hydrogen sulfide ameliorates endothelial dysfunction in aging arteries by regulating ferroptosis[J]. Nitric Oxide, 2023, 140-141:77-90.

[86] Mitsuishi Y, Motohashi H, Yamamoto M. The Keap1-Nrf2 system in cancers: stress response and anabolic metabolism[J]. Frontiers In Oncology, 2012, 2:200.

[87] Morrison B, 3rd, Pringle A K, McManus T, et al. L-arginyl-3,4-spermidine is neuroprotective in several in vitro models of neurodegeneration and in vivo ischaemia without suppressing synaptic transmission[J]. The British Journal of Pharmacology, 2002, 137(8):1255-1268.

[88] Nobuta H, Yang N, Ng Y H, et al. Oligodendrocyte death in pelizaeus-merzbacher disease is rescued by iron chelation[J]. Cell Stem Cell, 2019, 25(4):531-541.e536.

[89] Offermanns S. Hydroxy-carboxylic acid receptor actions in metabolism[J]. Trends in Endocrinology and Metabolism, 2017, 28(3):227-236.

[90] Papapetropoulos A, Pyriochou A, Altaany Z, et al. Hydrogen sulfide is an endogenous stimulator of angiogenesis[J]. PNAS, 2009, 106(51):21972-21977.

[91] Park M W, Cha H W, Kim J, et al. Nox4 promotes ferroptosis of astrocytes by oxidative stress-induced lipid peroxidation via the impairment of mitochondrial metabolism in alzheimer's diseases[J]. Redox Biology, 2021, 41:101947.

[92] Paul B D. Cysteine metabolism and hydrogen sulfide signaling in huntington's disease[J]. Free Radical Biology and Medicine, 2022, 186:93-98.

[93] Paul B D, Sbodio J I, Snyder S H. Cysteine metabolism in neuronal redox homeostasis[J]. Trends in Pharmacological Sciences, 2018, 39(5):513-524.

[94] Paul B D, Sbodio J I, Xu R, et al. Cystathionine γ-lyase deficiency mediates neurodegeneration in huntington's disease[J]. Nature, 2014, 509(7498):96-100.

[95] Paul B D, Snyder S H, Kashfi K. Effects of hydrogen sulfide on mitochondrial function and cellular bioenergetics[J]. Redox Biology, 2021, 38:101772.

[96] Pedrera L, Espiritu R A, Ros U, et al. Ferroptotic pores induce Ca2+ fluxes and escrt-iii activation to modulate cell death kinetics[J]. Cell Death & Differentiation, 2021, 28(5):1644-1657.

[97] Pelizzoni I, Macco R, Zacchetti D, et al. Iron and calcium in the central nervous system: a close relationship in health and sickness[J]. Biochemical Society Transactions, 2008, 36(6):1309-1312.

[98] Polhemus D J, Calvert J W, Butler J, et al. The cardioprotective actions of hydrogen sulfide in acute myocardial infarction and heart failure[J]. Scientifica (Cairo), 2014, 2014:768607.

[99] Reichert C O, de Freitas F A, Sampaio-Silva J, et al. Ferroptosis mechanisms involved in neurodegenerative diseases[J]. The International Journal of Molecular Sciences, 2020, 21(22):8765.

[100] Renga B, Cipriani S, Carino A, et al. Reversal of endothelial dysfunction by gpbar1 agonism in portal hypertension involves a Akt/Foxoa1 dependent regulation of H2S generation and endothelin-1[J]. PLoS One, 2015, 10(11): e0141082.

[101] Renga B, Mencarelli A, Migliorati M, et al. Bile-acid-activated farnesoid x receptor regulates hydrogen sulfide production and hepatic microcirculation[J]. World Journal of Gastroenterology, 2009, 15(17):2097-2108.

[102] Rochette L, Dogon G, Rigal E, et al. Lipid peroxidation and iron metabolism: two corner stones in the homeostasis control of ferroptosis[J]. The International Journal of Molecular Sciences, 2022, 24(1):449.

[103] Ruwolt M, Schnirch L, Borges Lima D, et al. Optimized tmt-based quantitative cross-linking mass spectrometry strategy for large-scale interactomic studies[J]. Analytical chemistry, 2022, 94(13):5265-5272.

[104] Sahebkar A, Foroutan Z, Katsiki N, et al. Ferroptosis, a new pathogenetic mechanism in cardiometabolic diseases and cancer: is there a role for statin therapy? [J]. Metabolism, 2023, 146:155659.

[105] Sbodio J I, Snyder S H, Paul B D. Regulators of the transsulfuration pathway[J]. The British Journal of Pharmacology, 2019, 176(4):583-593.

[106] Sen N, Paul B D, Gadalla M M, et al. Hydrogen sulfide-linked sulfhydration of Nf-κb mediates its antiapoptotic actions[J]. Molecular Cell, 2012, 45(1):13-24.

[107] Sharma K B, Aggarwal S, Yadav A K, et al. Studying autophagy using a tmt-based quantitative proteomics approach[J]. Methods in Molecular Biology, 2022, 2445:183-203.

[108] Shefa U, Kim M S, Jeong N Y, et al. Antioxidant and cell-signaling functions of hydrogen sulfide in the central nervous system[J]. Oxidative Medicine and Cellular Longevity, 2018, 2018:1873962.

[109] Singh S, Padovani D, Leslie R A, et al. Relative contributions of cystathionine beta-synthase and gamma-cystathionase to H2S biogenesis via alternative trans-sulfuration reactions[J]. Journal of Biological Chemistry, 2009, 284(33):22457-22466.

[110] Song, C, Li, D, Huang, L, et al. Role of ferroptosis regulation by Nrf2/Nqo1 pathway in alcohol-induced cardiotoxicity in vitro and in vivo[J]. Chemical Research in Toxicology, 2024, 37(6):1044-1052.

[112] Song X, Long D. Nrf2 and ferroptosis: a new research direction for neurodegenerative diseases[J]. Frontiers in Neuroscience, 2020, 14:267.

[113] Stockwell B R, Friedmann Angeli J P, Bayir H, et al. Ferroptosis: a regulated cell death nexus linking metabolism, redox biology, and disease[J]. Cell, 2017, 171(2):273-285.

[114] Subramaniam S, Snyder S H. Huntington’s disease is a disorder of the corpus striatum: focus on rhes (ras homologue enriched in the striatum) [J]. Neuropharmacology, 2011, 60(7):1187-1192.

[115] Sui X, Zhang R, Liu S, et al. RSL3 drives ferroptosis through GPX4 inactivation and ROS production in colorectal cancer[J]. Frontiers in Pharmacology 2018, 9:1371.

[116] Sun Y-Y, Zhu H-J, Zhao R-Y, et al. Remote ischemic conditioning attenuates oxidative stress and inflammation via the Nrf2/Ho-1 pathway in MCAO mice[J]. Redox Biology, 2023a, 66:102852.

[117] Sundstrom L, Morrison B 3rd, Bradley M, et al. Organotypic cultures as tools for functional screening in the CNS[J]. Drug Discovery Today, 2005, 10(14):993-1000.

[118] Szabo C, Papapetropoulos A. International union of basic and clinical pharmacology. Cii: pharmacological modulation of H2S levels: H2S donors and H2S biosynthesis inhibitors[J]. Pharmacological Reviews 2017, 69(4):497-564.

[119] Tang D, Chen X, Kang R, et al. Ferroptosis: molecular mechanisms and health implications[J]. Cell Research, 2021, 31(2):107-125.

[120] Tang D, Kang R, Berghe T V, et al. The molecular machinery of regulated cell death[J]. Cell Research, 2019, 29(5):347-364.

[121] Traber G M, Yu A M. Rnai-based therapeutics and novel RNA bioengineering technologies[J]. Journal of Pharmacology and Experimental Therapeutics, 2023, 384(1):133-154.

[122] Viswanathan V S, Ryan M J, Dhruv H D, et al. Dependency of a therapy-resistant state of cancer cells on a lipid peroxidase pathway[J]. Nature, 2017, 547(7664):453-457.

[123] Vitvitsky V, Thomas M, Ghorpade A, et al. A functional transsulfuration pathway in the brain links to glutathione homeostasis[J]. Journal of Biological Chemistry, 2006, 281(47):35785-35793.

[124] Wang Y, Gao L, Chen J, et al. Pharmacological modulation of Nrf2/ho-1 signaling pathway as a therapeutic target of parkinson's disease[J]. Frontiers in Pharmacology 2021, 12:757161.

[125] Wang Y H, Huang J T, Chen W L, et al. Dysregulation of cystathionine γ-lyase promotes prostate cancer progression and metastasis[J]. Embo Reports, 2019, 20(10): e45986.

[126] Wang Y Q, Chang S Y, Wu Q, et al. The protective role of mitochondrial ferritin on erastin-induced ferroptosis[J]. Frontiers in Aging Neuroscience, 2016, 8:308.

[127] Wang Y Y, Shi G W, Shi S F, et al. Everolimus accelerates erastin and rsl3-induced ferroptosis in renal cell carcinoma[J]. Gene, 2022, 809:145992.

[128] Wang Z, Ding Y, Wang X, et al. Pseudolaric acid b triggers ferroptosis in glioma cells via activation of nox4 and inhibition of xct[J]. Cancer Letters, 2018, 428:21-33.

[129] Wei R, Zhao Y, Wang J, et al. Tagitinin c induces ferroptosis through Perk-Nrf2-Ho-1 signaling pathway in colorectal cancer cells[J]. Biochimica et Biophysica Acta-general Subjects, 2021, 17(11):2703-2717.

[130] Wei Y, Song X, Gao Y, et al. Iron toxicity in intracerebral hemorrhage: physiopathological and therapeutic implications[J]. Brain Research Bulletin, 2022, 178:144-154.

[131] Wu C-T, Deng J-S, Huang W-C, et al. Salvianolic acid c against acetaminophen-induced acute liver injury by attenuating inflammation, oxidative stress, and apoptosis through inhibition of the Keap1/Nrf2/Ho-1 signaling[J]. Oxidative Medicine and Cellular Longevity, 2019, 2019(1):9056845.

[132] Wu D, Tan B, Sun Y, et al. Cystathionine γ lyase s-sulfhydrates drp1 to ameliorate heart dysfunction[J]. Redox Biology, 2022, 58:102519.

[133] Wu X, Li Y, Zhang S, et al. Ferroptosis as a novel therapeutic target for cardiovascular disease[J]. Theranostics, 2021, 11(7):3052-3059.

[134] Xia H, Wu Y, Zhao J, et al. N6-methyladenosine-modified circsav1 triggers ferroptosis in copd through recruiting ythdf1 to facilitate thetranslation of ireb2[J]. Cell Death and Differentiation, 2023, 30(5):1293-1304.

[135] Yagoda N, von Rechenberg M, Zaganjor E, et al. Ras-Raf-Mek-Dependent oxidative cell death involving voltage-dependent anion channels[J]. Nature, 2007, 447(7146):864-868.

[136] Yang R, Gao W, Wang Z, et al. Polyphyllin i induced ferroptosis to suppress the progression of hepatocellular carcinoma through activation of the mitochondrial dysfunction via Nrf2/Ho-1/Gpx4 axis[J]. Phytomedicine, 2024a, 122:155135.

[137] Yang S, Wu Y, Zhong W, et al. GSH/PH dual activatable cross-linked and fluorinated pei for cancer gene therapy through endogenous iron de-hijacking and in situ ROS amplification[J]. Advanced Materials, 2024b, 36(2): e2304098.

[138] Yang S, Xie Z, Pei T, et al. Salidroside attenuates neuronal ferroptosis by activating the Nrf2/Ho1 signaling pathway in aβ (1-42)-induced alzheimer's disease mice and glutamate-injured HT-22 cells[J]. Chinese Medical Journal, 2022, 17(1):82.

[139] Yang W S, SriRamaratnam R, Welsch M E, et al. Regulation of ferroptotic cancer cell death by Gpx4[J]. Cell, 2014, 156(1-2):317-331.

[140] Yang W S, Stockwell B R. Synthetic lethal screening identifies compounds activating iron-dependent, nonapoptotic cell death in oncogenic-ras-harboring cancer cells[J]. Chemistry Biology, 2008, 15(3):234-245.

[141] Yang Z, Su W, Wei X, et al. Hif-1α drives resistance to ferroptosis in solid tumors by promoting lactate production and activating slc1a1[J]. Cell Reports, 2023, 42(8):112945.

[142] Ye L, Wen X, Qin J, et al. Metabolism-regulated ferroptosis in cancer progression and therapy[J]. Cell Death and Disease, 2024, 15(3):196.

[143] Yuan H, Pratte J, Giardina C. Ferroptosis and its potential as a therapeutic target[J]. Biochemical Pharmacology, 2021, 186:114486.

[144] Zeng F, Nijiati S, Tang L, et al. Ferroptosis detection: from approaches to applications[J]. Angewandte Chemie-International Edition, 2023, 62(35): e202300379.

[145] Zhang H, Pan J, Huang S, et al. Hydrogen sulfide protects cardiomyocytes from doxorubicin-induced ferroptosis through the Slc7a11/Gsh/Gpx4 pathway by Keap1 s-sulfhydration and Nrf2 activation[J]. Redox Biology, 2024a, 70:103066.

[146] Zhang H, Zhou J, Dong L, et al. Unveiling the impact of glutathione (GSH) and p53 gene deletion on tumor cell metabolism by amino acid and proteomics analysis[J]. Journal of Gastrointestinal Oncology, 2024b, 15(3):1002-1019.

[147] Zhang W, Liu Y, Liao Y, et al. Gpx4, ferroptosis, and diseases[J]. Biomed Pharmacother, 2024c, 174:116512.

[148] Zhang Y, Tan H, Daniels J D, et al. Imidazole ketone erastin induces ferroptosis and slows tumor growth in a mouse lymphoma model[J]. Cell Chemical Biology, 2019, 26(5):623-633.e629.

[149] Zhang Z, Ji Y, Hu N, et al. Ferroptosis-induced anticancer effect of resveratrol with a biomimetic nano-delivery system in colorectal cancer treatment[J]. Asian Journal of Pharmaceutical Sciences, 2022, 17(5):751-766.

[150] Zhao Y, Li Y, Zhang R, et al. The role of erastin in ferroptosis and its prospects in cancer therapy[J]. Oncotargets and Therapy, 2020, 13:5429-5441.

[151] Zheng H H, Fu P F, Chen H Y, et al. Pseudorabies virus: from pathogenesis to prevention strategies[J]. Viruses, 2022, 14(8):1638.

[152] Zheng M, Zhou M, Lu T, et al. Tmt and prm based quantitative proteomics to explore the protective role and mechanism of iristectorin b in stroke[J]. The International Journal of Molecular Sciences, 2023, 24(20):15195.

[153] Zhou J, Jin Y, Lei Y, et al. Ferroptosis is regulated by mitochondria in neurodegenerative diseases[J]. Neurodegener Disease, 2020, 20(1):20-34.

[154] Zhu J, Berisa M, Schwörer S, et al. Transsulfuration activity can support cell growth upon extracellular cysteine limitation[J]. Cell Metabolism, 2019, 30(5):865-876.e865.