Hemin-induced platelet activation and ferroptosis is mediated through ROS-driven proteasomal activity and inflammasome activation: Protection by Melatonin

Reactive oxygen species (ROS) are capable of inducing cell death or apoptosis. Recently, we demonstrated that lipid-ROS can mediate ferroptosis and activation of human platelets. Ferroptosis is an intracellular iron-mediated cell death, distinct from classical apoptosis and necrosis, which is mediated through the accumulation of ROS, lipid peroxides and depletion of cellular GSH. Lately, we demonstrated that hemoglobin degradation product hemin induces ferroptosis in platelets via ROS and lipid peroxidation. In this study, we demonstrate that hemin-induced ferroptosis in platelets is mediated through ROS-driven proteasome activity and inflammasome activation, which were mitigated by Melatonin (MLT). Although inflammasome activation is linked with pyroptosis, it is still not clear whether ferroptosis is associated with inflammasome activation. Our study for the first time demonstrates an association of platelet activation/ferroptosis with proteasome activity and inflammasome activation. Although, high-throughput screening has recognized ferrostatin-1 (Fer-1) and liproxstatin-1 (Lip-1) as potent ferroptosis inhibitors, having an endogenous antioxidant such as MLT as ferroptosis inhibitor is of high interest. MLT is a well-known chronobiotic hormone that regulates the circadian rhythms in vertebrates. It also exhibits potent antioxidant and ROS quenching capabilities. MLT can regulate fundamental cellular functions by exhibiting cytoprotective, oncostatic, antiaging, anti-venom, and immunomodulatory activities. The ROS scavenging capacity of MLT is key for its cytoprotective and anti-apoptotic properties. Considering the anti-ferroptotic and anti-apoptotic potentials of MLT, it could be a promising clinical application to treat hemolytic, thrombotic and thrombocytopenic conditions. Therefore, we propose MLT as a pharmacological and therapeutic agent to inhibit ferroptosis and platelet activation.     

老年缺血性心力衰竭的心脏DNA甲基化编码重编程与心肌细胞焦亡、铁死亡的关联

摘要 目的：探讨老年缺血性心力衰竭的心脏DNA甲基化编码重编程与心肌细胞焦亡、铁死亡的关联性。方法：2019年12月到2021月2月,选择在本院诊治的老年缺血性心力衰竭115例作为心衰组,同期选择在本院体检的非心血管疾病老年人群115例作为对照组。检测心脏DNA甲基化编码重编程、心肌细胞焦亡、铁死亡指标表达情况并进行相关性分析。结果：心衰组的心脏DNA甲基化编码重编程指标-miR-92a、miR-130a相对表达水平高于对照组（P<0.05）。心衰组的Caspase-1蛋白、Caspase-4蛋白相对表达水平高于对照组（P<0.05）。心衰组的铁调素含量高于对照组（P<0.05）。在两组230例入选者中,Spearsman相关分析显示：缺血性心力衰竭与miR-92a、miR-130a、半胱氨酸蛋白酶1（Caspase-1）、半胱氨酸蛋白酶4（Caspase-4）、铁调素存在正向相关性（P<0.05）。Logistic回归分析显示：miR-92a、miR-130a、Caspase-1、Caspase-4、铁调素为导致缺血性心力衰竭发生的重要因素（P<0.05）。结论：老年缺血性心力衰竭患者多伴随有心脏DNA甲基化编码重编程与心肌细胞焦亡、铁死亡,后三者与缺血性心力衰竭的发生存在关联性,也是导致缺血性心力衰竭发生的重要因素。     

Understanding the mechanistic regulation of ferroptosis in cancer: the gene matters

Ferroptosis has emerged as a crucial regulated cell death involved in a variety of physiological processes or pathological diseases, such as tumor suppression. Though initially being found from anticancer drug screening and considered not essential as apoptosis for growth and development, numerous studies have demonstrated that ferroptosis is tightly regulated by key genetic pathways and/or genes, including several tumor suppressors and oncogenes. In this review, we introduce the basic concepts of ferroptosis, characterized by the features of non-apoptotic, iron-dependent, and overwhelmed accumulation of lipid peroxides, and the underlying regulated circuits are considered to be pro-ferroptotic pathways. Then, we discuss several established lipid peroxidation defending systems within cells, including SLC7A11/GPX4, FSP1/CoQ, GCH1/BH4, and mitochondria DHODH/CoQ, all of which serve as anti-ferroptotic pathways to prevent ferroptosis. Moreover, we provide a comprehensive summary of the genetic regulation of ferroptosis via targeting the above-mentioned pro-ferroptotic or anti-ferroptotic pathways. The regulation of pro- and anti-ferroptotic pathways gives rise to more specific responses to the tumor cells in a context-dependent manner, highlighting the unceasing study and deeper understanding of mechanistic regulation of ferroptosis for the purpose of applying ferroptosis induction in cancer therapy.     

mTOR in programmed cell death and its therapeutic implications

Mechanistic target of rapamycin (mTOR), a highly conserved serine/threonine kinase, is involved in cellular metabolism, protein synthesis, and cell death. Programmed cell death (PCD) assists in eliminating aging, damaged, or neoplastic cells, and is indispensable for sustaining normal growth, fighting pathogenic microorganisms, and maintaining body homeostasis. mTOR has crucial functions in the intricate signaling pathway network of multiple forms of PCD. mTOR can inhibit autophagy, which is part of PCD regulation. Cell survival is affected by mTOR through autophagy to control reactive oxygen species production and the degradation of pertinent proteins. Additionally, mTOR can regulate PCD in an autophagy-independent manner by affecting the expression levels of related genes and phosphorylating proteins. Therefore, mTOR acts through both autophagy-dependent and -independent pathways to regulate PCD. It is conceivable that mTOR exerts bidirectional regulation of PCD, such as ferroptosis, according to the complexity of signaling pathway networks, but the underlying mechanisms have not been fully explained. This review summarizes the recent advances in understanding mTOR-mediated regulatory mechanisms in PCD. Rigorous investigations into PCD-related signaling pathways have provided prospective therapeutic targets that may be clinically beneficial for treating various diseases.     

miR-20b-5p靶向MAPK1调控脑出血过程中脑微血管内皮细胞铁死亡

摘要 目的：探讨miR-20b-5p对氧糖剥夺（OGD）/Hemin处理的脑微血管内皮细胞（BMVEC）功能的影响及机制。方法：将BMVEC分为Control组、agomir-NC组、agomir-miR-20b-5p组、antagomir-NC组和antagomir-miR-20b-5p组。使用Lipofectamine 2000试剂对细胞进行相应的转染处理。BMVEC转染后，将BMVEC再分为Control组、OGD/Hemin组（O/H组）、OGD/Hemin+agomir-NC组（O/H+agomir-NC组）、OGD/Hemin+agomir-miR-20b-5p组（O/H+agomir-miR-20b-5p组）、OGD/Hemin+antagomir-NC组（O/H+antagomir-NC组）和OGD/Hemin+antagomir-miR-20b-5p组（O/H+antagomir-miR-20b-5p组）。Control组BMVEC正常培养，其他组BMVEC进行OGD/Hemin处理。MTT法检测BMVEC增殖，TUNEL染色检测BMVEC凋亡，Transwell检测BMVEC迁移。使用试剂盒检测超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GSH-Px）和丙二醛（MDA）水平。使用Iron Assay试剂盒检测Fe²⁺含量。通过qRT-PCR检测miR-20b-5p和MAPK1 mRNA水平。通过Western blot检测MAPK1、Bax、Bcl-2、谷胱甘肽过氧化物酶4（GPX4）和前列腺素内过氧化物合酶2（PTGS2）蛋白表达水平。通过免疫荧光染色检测MAPK1的荧光强度水平。结果：与Control组和agomir-NC组比较，agomir-miR-20b-5p组BMVEC中的miR-20b-5p水平升高（P<0.05）。与Control组和antagomir-NC组比较，antagomir-miR-20b-5p组BMVEC中的miR-20b-5p水平降低（P<0.05）。与Control组比较，O/H组BMVEC中的miR-20b-5p水平降低，细胞活力降低，TUNEL阳性率和Bax蛋白表达水平升高，Bcl-2蛋白表达水平降低，迁移数量降低，SOD和GSH-Px活性降低，MDA含量升高，Fe²⁺含量和PTGS2的蛋白表达水平升高，GPX4的蛋白表达水平降低，MAPK1的mRNA和蛋白表达水平以及相对荧光强度升高（P<0.05）。与O/H组和O/H+agomir-NC组比较，O/H+agomir-miR-20b-5p组BMVEC中的miR-20b-5p水平升高，细胞活力升高，TUNEL阳性率和Bax蛋白表达水平降低，Bcl-2蛋白表达水平升高，迁移数量升高，SOD和GSH-Px活性升高，MDA含量降低，Fe²⁺含量和PTGS2的蛋白表达水平降低，GPX4的蛋白表达水平升高，MAPK1的mRNA和蛋白表达水平以及相对荧光强度降低（P<0.05）。与O/H组和O/H+antagomir-NC组比较，O/H+antagomir-miR-20b-5p组BMVEC中的miR-20b-5p水平降低，细胞活力降低，TUNEL阳性率和Bax蛋白表达水平升高，Bcl-2蛋白表达水平降低，迁移数量降低，SOD和GSH-Px活性降低，MDA含量升高，Fe²⁺含量和PTGS2的蛋白表达水平升高，GPX4的蛋白表达水平降低，MAPK1的mRNA和蛋白表达水平以及相对荧光强度升高（P<0.05）。结论：本研究表明上调miR-20b-5p通过抑制OGD/Hemin处理的BMVEC中MAPK1的表达从而抑制了铁死亡途径。     

死亡相关的LncRNAs在甲状腺癌中的预后价值及预后风险模型构建

目的:探讨铁死亡相关的长链非编码RNAs(LncRNAs)在甲状腺癌中的预后价值并构建预后风险模型。方法:从癌症基因组图谱(TCGA)数据库下载甲状腺癌的转录本数据和临床数据,铁死亡相关的基因数据集是从铁死亡数据库(http://www.zhounan.org/ferrdb/)下载的259个基因集。得到铁死亡相关LncRNAs,与患者临床信息合并后,通过单因素Cox回归分析和Kaplan-Meier生存分析两种方法得到与甲状腺癌预后相关的铁死亡LncRNAs,通过R的survival包构建COX模型以此来建立最佳预后风险模型并予以验证。结果:获得30个铁死亡相关的LncRNAs,多因素cox分析得到10个与甲状腺癌预后相关的铁死亡LncRNAs,包括AL136366.1、AL162231.2、CRNDE、AC004918.3、LINC02471、AC092279.1、AC046143.1、LINC02454、DOCK9-DT、AC008063.1。Kaplan-Meier生存分析表明高风险组预后较差。单因素和多因素Cox分析表明风险评分可以作为独立预后因子。KEGG通路富集分析发现,差异基因可能与嘧啶代谢、核苷酸切除修复、NOTCH_信号通路等通路有关。结论:通过生物信息学方法筛选出10个与甲状腺癌预后的铁死亡相关LncRNAs,并成功构建预后风险模型。     

有氧运动对高脂膳食小鼠心肌损伤中Nrf2/GPX4/Ferroptosis通路的作用*

目的: 探讨核因子E2相关因子2(Nrf 2)激活谷胱甘肽过氧化物酶4(GPX4)抑制铁死亡(Ferroptosis)的通路在有氧运动预防高脂膳食小鼠心肌损伤中的保护作用。方法: 40只5周龄SPF 级C57BL/6雄性小鼠随机分为安静对照组(NC)、运动组(NE)、高脂组(HC)和高脂+运动组(HE,高脂与跑台运动同时开始),每组10只。高脂膳食采用60% Kcal SPF级高脂模型饲料喂养,自由进食。有氧运动采用递增负荷跑台运动,每周5 d,60 min/d,速度从13 m/min开始,每两周速度递增1 m/min。14周后取心肌和血液。HE染色观察心肌组织结构变化。Western blot 检测心肌Nrf2/GPX4/ Ferroptosis相关蛋白表达。分光光度法测定心肌过氧化物浓度和抗氧化酶活性。ELISA法检测心肌线粒体8-OHdG和血清胰岛素水平。结果: 与对照组相比,高脂组的心肌纤维间隙脂质集聚增加,FBG和FINS显著增加,而ISI显著下降(P＜0.01);与高脂组相比,高脂运动组的心肌纤维间隙脂质集聚减少, T-AOC、T-SOD、GSH活性显著增强,心肌线粒体8-OHdG和心肌铁含量降低(P＜0.01),FPN1、FTH1、GPX4、GLUT1和细胞核内Nrf2显著升高(P＜0.01)。结论: 有氧运动可促进小鼠心肌Nrf2转位入核增强GPX4表达,抑制心肌Ferroptosis发生,同时促进心肌抗氧化酶活性,抑制心肌线粒体过氧化损伤。     

肾透明细胞癌患者铁死亡相关基因的预后作用

探讨铁死亡相关基因在肾透明细胞癌患者中的表达及其预后价值。通过TCGA数据库下载KIRC的相关测序数据与检索到的铁死亡相关基因取交集,进行铁死亡相关基因的差异分析。之后利用单变量和多变量Cox回归分析,筛选具有预后价值的基因,构建预测患者生存情况的风险评分模型,并对模型进行验证。对高低风险组进行GO与KEGG通路富集,探讨风险差异的可能原因;通过ssGSEA分析,评估高低风险组间的免疫浸润情况。在KIRC患者的肿瘤组织和正常组织中,共得到21个差异的铁死亡相关基因;通过单因素Cox回归分析,获得 28 个与KIRC预后相关的基因;之后进行Lasso回归与多因素Cox回归分析,结果显示有10个基因被纳入模型,计算公式为:风险值(Risk score)=(0.024 5)×ALOX5表达值+(0.126 0)×CBS表达值+(0.199 5)×CD44表达值+(0.218 3)×CHAC1表达值+(-0.295 9)×HMGCR表达值+(0.036 7)×MT1G表达值+(0.061 4)×SLC7A11表达值+(-0.080 7)×FDFT1表达值+(0.160 3)×PEBP1表达值+(-0.220 5)×GOT1表达值。生存状态图表明,高风险组死亡病例数多于低风险组;ROC曲线表明风险评分模型具备一定预测能力;K-M生存分析显示,高风险组总体生存率低于低风险组(P=5.73×10^-13)。GO与KEGG富集分析提示,高低风险组间免疫情况及IL-17信号通路存在显著差异;进一步的ssGSEA富集显示,高低风险组间大部分免疫细胞的评分存在显著差异。基于铁死亡相关基因的预后风险评分模型可用于KIRC的预后预测,针对铁死亡相关基因设计靶点可能是治疗KIRC的一种新选择。     

Role of ferroptosis in cisplatin-induced acute nephrotoxicity in mice

BackgroundCisplatin is widely used as an antitumor drug for the treatment of solid tumors. However, its use has been limited owing to nephrotoxicity, a major side effect. The mechanism of cisplatin-induced nephrotoxicity (CIN) has long been investigated in order to develop preventive/therapeutic drugs. Ferroptosis is a newly identified form of non-apoptotic regulated cell death induced by iron-mediated lipid peroxidation and is involved in the pathophysiology of various diseases. In this study, we examined the role of ferroptosis in CIN.MethodsWe evaluated the role of ferroptosis in CIN by in vivo experiments in a mouse model.ResultsCisplatin increased the protein expressions of transferrin receptor-1 and ferritin, and iron content in the kidney of mice. In addition, treatment with cisplatin augmented renal ferrous iron and hydroxyl radical levels with co-localization. Mice administered cisplatin demonstrated kidney injury, with renal dysfunction and increased inflammatory cytokine expression; these changes were ameliorated by Ferrostatin-1 (Fer-1), an inhibitor of ferroptosis. The expression of the ferroptosis markers, COX2 and 4-hydroxynonenal (4-HNE), increased with cisplatin administration, and decreased with the administration of Fer-1. By contrast, cisplatin-induced apoptosis and necroptosis were inhibited by treatment with Fer-1. Moreover, deferoxamine, an iron chelator, also inhibited CIN, with a decrease in the expression of COX-2 and 4-HNE.ConclusionFerroptosis is involved in the pathogenesis of CIN and might be used as a new preventive target for CIN.