电离辐射对皮肤细胞铁死亡的影响及其抑制剂Ferrostatin-1的防护作用研究

目的 探讨电离辐射对皮肤细胞铁死亡的影响以及铁死亡抑制剂Ferrostatin-1（Fer-1）对受照射皮肤细胞的保护作用及机制。方法 为检测Fer-1对人永生化角质形成细胞（HaCaT）辐照后的影响,按照射与给药方式分为对照组、Fer-1组、单纯照射组、照射+Fer-1组。采用CCK-8法和乳酸脱氢酶（LDH）释放检测X射线照射以及Fer-1处理对HaCaT细胞存活和细胞死亡的影响。采用流式细胞术检测X射线照射以及Fer-1处理后对HaCaT细胞脂质过氧化水平的影响。利用结晶紫染色法检测X射线照射以及Fer-1处理对HaCaT细胞克隆形成能力的影响。Western blot检测X射线照射以及Fer-1处理对铁死亡相关蛋白ACSL4和GPX4表达的影响。结果 单纯照射组经不同剂量的X射线照射后细胞存活率明显降低（t=5.63、8.74,P<0.05）,LDH的释放明显增加（t=3.98、5.08、9.27,P<0.05）,Fer-1能够提高受照射皮肤细胞存活率（t=5.79,P<0.05）,降低LDH的释放量（t=12.36、11.96、18.13、9.96,P<0.05）。单纯照射组经10 Gy的X射线照射后细胞的脂质过氧化水平明显升高（t=9.59,P<0.05）,克隆存活能力明显减弱（t=4.26,P<0.05）,而Fer-1能够降低X射线照射引起的脂质过氧化水平的升高（t=6.48、17.04,P<0.05）,增加受照射皮肤细胞的克隆存活能力（t=3.96,P<0.05）。10 Gy的X射线照射会导致ACSL4表达水平的升高和GPX4的表达水平的降低,而Fer-1的治疗能促进ACSL4和GPX4的表达水平恢复正常（t=5.23、7.16、4.78、8.29、6.43,P<0.05）。结论 铁死亡抑制剂Fer-1在细胞水平上能够通过抑制电离辐射后铁死亡的发生而保护皮肤细胞,为放射性皮肤损伤的防护提供了一种新的策略。     

Comprehensive Analysis of the Expression and Clinical Significance of a Ferroptosis-Related Genome in Ovarian Serous Cystadenocarcinoma: A Study Based on TCGA Data

Background: Epithelial ovarian cancer (EOC) is the deadliest malignancy among the gynecologic tumors, and ovarian serous cystadenocarcinoma (OV) is the dominant histological type. Ferroptosis is a novel iron-dependent, programmed form of cell death, and agents that trigger ferroptosis may constitute potential anti-cancer therapies. Materials and Methods: We herein extracted the genes that participate in the process of ferroptosis from the online FerrDb database to create a ferroptosis-related genome (FRG), and then comprehensively analyzed the relationship between the mRNA expression of each gene and the clinicopathologic features of The Cancer Genome Atlas (TCGA)-OV cohort. Results: We found that most of the FRG genes were differently expressed between OV and normal ovarian tissue and were significantly related to the prognosis of OV. In addition, gene ontology (GO) analysis revealed that the candidate genes of the FRG were primarily associated with responses to nutrient levels, oxidative stress, oxygen levels, and neuronal death. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated that candidate genes of the FRG were primarily associated with ferroptosis, hepatitis B, mitophagy, prostate cancer, and bladder cancer. The protein–protein interaction (PPI) network exhibited 132 nodes, 565 edges, an average node degree of 9.94, and an average local clustering coefficient of 0.539, indicating that the proteins were closely interrelated with one another and constitute a biological cluster. A comprehensive analysis of the top 5 genes involved in promoting and preventing ferroptosis revealed that these genes were differently expressed between OV and normal ovarian tissue, were significantly related to the prognosis of OV, reflected excellent diagnostic value, and were significantly correlated with immune cell infiltration in the tumor microenvironment (TME), polarization of macrophages, and with immune checkpoints. The protein levels of top 5 genes involved in promoting ferroptosis were differentially expressed between OV and normal ovarian tissue, and the top 5 genes involved in preventing ferroptosis were constitutively expressed in most of the normal tissues and tumors. Conclusions: We herein ascertained that the majority of the FRG was differentially expressed between OV and normal ovarian tissue, that the genes were significantly related to prognosis, and that the dysregulation of ferroptosis appeared to influence the development and progression of OV. The FRG showed an excellent diagnostic value for OV, and we postulate that it is significantly correlated with immune cell infiltration and immune checkpoints in the TME. We posit that targeting ferroptosis might comprise a novel anti-cancer therapy in OV.     

柳氮磺吡啶通过铁死亡途径增强结直肠癌细胞放射敏感性研究

目的 探究低剂量柳氮磺吡啶（SAS）对结直肠癌细胞的放疗增敏作用。方法 CCK‐8检测不同浓度SAS对结直肠癌细胞的增殖抑制作用,采用凋亡、自噬等抑制剂联合SAS处理结直肠癌细胞,CCK‐8法比较细胞活力。采用锥虫蓝染色、平板克隆形成、细胞生长曲线等实验评价低浓度SAS联合放疗对结直肠癌细胞的体外放疗增敏作用。通过检测细胞内脂质过氧化物、铁死亡蛋白标志物（GPX4、PTGS2）等,探究低浓度SAS联合放疗对细胞铁死亡的促进作用。利用小鼠皮下移植瘤模型,明确SAS的体内放疗增敏效应。结果 高浓度SAS可诱导结直肠癌细胞凋亡与铁死亡。低浓度SAS增强结直肠癌细胞放疗敏感性,该效应可被铁死亡抑制剂（Fer‐1）阻断。低浓度SAS联合放疗显著增加细胞内脂质过氧化物含量与PTGS2表达,降低GPX4表达。皮下移植瘤模型中SAS亦显示显著的放疗增敏效应。结论 低浓度SAS通过铁死亡途径增强结直肠癌细胞放疗敏感性。     

Ferroptosis and kidney disease

Cell death is a finely regulated process occurring through different pathways. Regulated cell death, either through apoptosis or regulated necrosis offers the possibility of therapeutic intervention. Necroptosis and ferroptosis are among the best studied forms of regulated necrosis in the context of kidney disease. We now review the current evidence supporting a role for ferroptosis in kidney disease and the implications of this knowledge for the design of novel therapeutic strategies. Ferroptosis is defined functionally, as a cell modality characterized by peroxidation of certain lipids, constitutively suppressed by GPX4 and inhibited by iron chelators and lipophilic antioxidants. There is functional evidence of the involvement of ferroptosis in diverse forms of kidneys disease. In a well characterized nephrotoxic acute kidney injury model, ferroptosis caused an initial wave of death, triggering an inflammatory response that in turn promoted necroptotic cell death that perpetuated kidney dysfunction. This suggests that ferroptosis inhibitors may be explored as prophylactic agents in clinical nephrotoxicity or ischemia–reperfusion injury such as during kidney transplantation. Transplantation offers the unique opportunity of using anti-ferroptosis agent ex vivo, thus avoiding bioavailability and in vivo pharmacokinetics and pharmacodynamics issues.     

长链非编码RNA PRMT5-AS1调控电离辐射诱导肝癌细胞铁死亡机制的研究

目的 探讨长链非编码RNA PRMT5-AS1对电离辐射诱导的肝癌细胞铁死亡的影响。方法 在MHCC-97H细胞中构建PRMT5-AS1过表达模型,在HepG2细胞中构建PRMT5-AS1敲低模型。使用X射线照射,吸收剂量为10 Gy,剂量率为3 Gy/min。采用Western blot和qRT-PCR实验检测基因表达水平。采用台盼蓝染色流式细胞术检测PRMT5-AS1表达对受照肝癌细胞脂质过氧化以及铁死亡的影响。采用CCK-8实验检测PRMT5-AS1表达水平对电离辐射照射后肝癌细胞死亡的影响。双荧光素酶报告实验检测let-7c-5p与PRMT5-AS1和SLC7A11之间结合作用。结果 MHCC-97H细胞中过表达PRMT5-AS1能够显著降低电离辐射引起的细胞死亡(对照组vs. PRMT5-AS1过表达组:27.57% vs.18.30%,t=14.94,P<0.05)。HepG2细胞中敲低PRMT5-AS1可显著增加电离辐射引起的细胞死亡(对照组vs. PRMT5-AS1敲低组:17.26% vs.28.26%,t=13.63,P<0.05)。过表达PRMT5-AS1能够明显抑制由电离辐射诱导的细胞内脂质活性氧(ROS)水平增加(对照组vs. PRMT5-AS1过表达组:17.01% vs.12.52%,t=12.80,P<0.05),敲低PRMT5-AS1可显著增加电离辐射诱导的脂质ROS水平增加(对照组vs. PRMT5-AS1敲低组:14.54% vs.17.72%,t=5.93,P<0.05)。CCK-8实验结果表明,过表达PRMT5-AS1能够显著抑制Erastin诱导的细胞活性降低(对照组vs. PRMT5-AS1过表达组:87.92% vs.109.06%,t=2.87,P<0.05),敲低PRMT5-AS1则促进Erastin抑制细胞活性(对照组vs. PRMT5-AS1敲低组:82.56%vs.60.58%,t=38.35,P<0.05)。Western blot和荧光定量PCR结果表明,过表达PRMT5-AS1能够明显提高SLC7A11的蛋白和mRNA水平(t=26.24,P<0.05),敲低PRMT5-AS1后SLC7A11的蛋白和mRNA水平均显著降低(t=5.60,P<0.05)。荧光素酶报告基因实验表明PRMT5-AS1与let-7c-5p之间存在相互作用(t=9.74,P<0.05)。PRMT5-AS1可以与let-7c-5p形成ceRNA网络,靶向调节SLC7A11。let-7c-5p能够逆转由过表达PRMT5-AS1引起的SLC7A11表达水平增加、脂质ROS水平和细胞死亡减少(t=3.01、4.11,P<0.05),而敲低SLC7A11能够逆转PRMT5-AS1引起的脂质ROS抑制和细胞死亡减少(t=21.35、7.15,P<0.05)。结论 长链非编码RNA PRMT5-AS1通过PRMT5-AS1/let-7c-5p/SLC7A11轴抑制电离辐射诱导肝癌细胞铁死亡的发生。     

铁死亡在重症急性胰腺炎引起的急性肾损伤中的作用及其机制

目的探讨铁死亡在重症急性胰腺炎(SAP)诱发急性肾损伤(AKI)的作用及其机制。方法胰腺炎建模后24 h,将36只SD大鼠(购自青岛大学医学部实验室动物中心)采用完全随机分组法分为3组(n=12):假手术组(SO组)、重症急性胰腺炎组(SAP组)、铁死亡抑制剂Ferrostatin-1干预组(SAP+Fer组)。建立SAP模型24 h后,检测血清淀粉酶(AMY)、肌酐(Cr)和血尿素氮(BUN)水平;检测肾组织中铁含量、丙二醛(MDA)、活性氧(ROS)、谷胱甘肽(GSH)变化及谷胱甘肽过氧化物酶4(GPX4)活性;苏木精-伊红(HE)染色观察胰腺和肾脏组织学改变;透射电镜(TEM)观察铁死亡的形态学特征;蛋白质印迹法(Western blot)、反转录-聚合酶链反应(RT-PCR)和免疫荧光染色等方法分析长链脂酰CoA合成酶4(ACSL4)、谷胱甘肽过氧化物酶4(GPX4)和铁蛋白重链1(FTH1)等铁死亡相关蛋白和基因的表达。组间比较采用方差分析,进一步两两比较采用LSD-t检验。结果SAP组大鼠血清AMY[(6276.5±562.2)比(1086.6±192.3)U/L]、Cr[(98.0±16.2)比(19.5±5.2)μmol/L]、BUN[(17.1±2.8)比(7.2±1.7)mmol/L]水平明显高于SO组(t=30.314、15.927、10.375,P值均<0.01),差异均有统计学意义;肾组织铁[(0.65±0.13)比(0.44±0.12)mg/g]、MDA[(7.25±0.81)比(1.84±0.24)μmol/g]、ROS[(68.54±7.04)比(15.67±5.21)×104/mg]高于SO组(t=3.855、22.167、11.496,P值均<0.01),GSH[(358.38±41.59)比(518.64±55.72)nmol/g]和GPX4[(7.75±1.09)比(14.72±2.56)U/mg]明显低于SO组(t=7.984、8.674,P值均<0.01),差异有统计学意义;胰腺和肾组织病理损伤程度增加;线粒体出现明显萎缩;肾脏ACSL4蛋白(0.84±0.03比0.34±0.02)表达高于SO组(t=7.876,P<0.01),GPX4、FTH1蛋白(0.69±0.03比1.04±0.06、0.26±0.02比0.83±0.04)表达低于SO组(t=5.651、8.134,P值均<0.01),差异有统计学意义;ACSL4、铁响应元件结合蛋白2(IREB2)mRNA(0.87±0.06比0.24±0.03、1.23±0.05比0.32±0.02)表达高于SO组(t=12.864、15.163,P均<0.01),差异有统计学意义。SAP+Fer组大鼠血清AMY[(5124.3±483.5)比(6276.5±562.2)U/L]、Cr[(55.2±13.7)比(98.0±16.2)μmol/L]、BUN[(9.8±2.1)比(17.1±2.8)mmol/L]水平显著低于SAP组(t=5.287、6.989、7.359,P值均<0.01),差异有统计学意义;肾组织铁[(0.54±0.07)比(0.65±0.13)mg/g]、MDA[(4.67±0.73)比(7.25±0.81)μmol/g]、ROS[(28.39±6.36)比(68.54±7.04)×104/mg]低于SAP组(t=2.639、7.176、8.247,P值均<0.05),GSH[(448.21±45.51)比(358.38±41.59)nmol/g]和GPX4[(11.31±1.89)比(7.75±1.09)U/mg]高于SAP组(t=5.048、5.639,P值均<0.01),差异有统计学意义;胰腺和肾组织病理损伤程度减轻;线粒体轻度萎缩;肾脏ACSL4蛋白(0.49±0.02比0.84±0.03)表达低于SAP组(t=2.781,P<0.05),GPX4、FTH1蛋白(0.69±0.03比1.04±0.06、0.26±0.02比0.83±0.04)表达高于SAP组(t=2.427、2.876,P值均<0.05),差异有统计学意义;ACSL4、IREB2 mRNA(0.52±0.04比0.87±0.06、0.74±0.04比1.23±0.05)表达低于SAP组(t=5.843、6.781,P值均<0.01),差异均有统计学意义。结论铁死亡参与SAP引起的AKI,抑制铁死亡能改善肾功能,减轻肾组织脂质过氧化和病理损伤。     

铁死亡在急性肺损伤中的研究进展

铁死亡是一种以细胞内脂质过氧化物堆积为特征的新型细胞程序性死亡方式,其在多种疾病中发挥了重要作用。急性肺损伤是由多种原因引起的危重疾病,患者死亡率很高。随着对急性肺损伤机制研究的不断深入,更多证据表明铁死亡在急性肺损伤的发病机制中发挥了重要作用。本文通过归纳既往铁死亡相关研究成果,发现缺血再灌注、脓毒血症、放射线、淹溺、油酸诱发的急性肺损伤均有铁死亡的参与,并总结了铁死亡的作用机制及在急性肺损伤发病机制中的作用研究,以期能够给未来诊疗急性肺损伤提出有希望的治疗策略提供理论依据。     

基于单细胞数据揭示铁死亡相关预后基因IL-33在HCC发育早期阶段内皮细胞中的作用

目的：基于单细胞测序数据对HCC铁死亡相关预后基因的表达状态进行分析,为HCC的精准治疗提供有效靶点。方法：利用TCGA数据库和铁死亡相关基因数据库,获取HCC患者和正常对照组的铁死亡相关差异基因,构建风险预后评估模型;利用单细胞测序数据,通过tSNE聚类降维和Monocle软件包对预后风险基因的在不同发育阶段细胞中的表达情况进行分析,并进一步通过CellChat软件包对预后风险基因在HCC细胞间的相互作用进行分析;结果：共发现25个铁死亡相关差异基因与HCC患者的预后相关。在此基础上,筛选出NT5DC2、G6PD、STMN1和IL-33共4个基因作为预后基因。其中NT5DC2主要在上皮细胞和内皮细胞中表达,IL-33主要在内皮细胞中表达,G6PD和STMN1在细胞中广泛表达;IL-33主要在发育早期阶段的内皮细胞中表达,NT5DC2、G6PD、STMN在不同的发育阶段均有表达;此外IL-33只在发育早期阶段的内皮细胞中与受体存在广泛交互。结论：IL-33作为良性预后基因,在HCC患者发育早期阶段的内皮细胞中特异性表达并发挥作用,提示维持血管内皮细胞的这种原始状态或促进IL-33介导的原始状态内皮细胞铁死亡可能是有效的HCC治疗靶点。     

铁死亡在胰腺癌中的作用研究及进展

胰腺癌是一种恶性程度极高的消化系统肿瘤,侵袭性强,早期诊断困难,大多数患者因确诊时已处于晚期而无法接受根治性手术治疗,亦无其他有效治疗手段,故预后极差。铁死亡是一种铁依赖性新型细胞程序性死亡方式,以细胞内铁过载、脂质过氧化物增多和活性氧异常蓄积为特征。近年研究发现铁死亡在抑制胰腺癌细胞生长、增殖方面具有重要作用,并能够提高化疗药物疗效,有望成为胰腺癌治疗的潜在靶点。笔者对铁死亡在胰腺癌发生发展及治疗中的作用研究进展作一综述。     

CircEXOC5 promotes ferroptosis by enhancing ACSL4 mRNA stability via binding to PTBP1 in sepsis-induced acute lung injury

BackgroundSepsis causes severe acute lung injury (ALI). Circular RNA is involved in the regulation of sepsis-related ALI progression. The regulation mechanism of circEXOC5 in sepsis-induced ALI is still unclear. Whether circEXOC5 is involved in the regulation of ferroptosis remains to be explored.MethodsWe constructed a mouse model of sepsis through cecal ligation and puncture (CLP). LPS induced mouse lung microvascular endothelial cells (MPVECs) to construct a sepsis cell model. The expression of circEXOC5 in the sepsis model was detected by qPCR. The extent of lung injury in mice was analyzed by HE staining. The contents of GSH/GSSG, iron, MDA and 4HNE in mice lung tissues and cells were detected by the kit. And further the ROS content was detected in the cells. Finally, the binding relationship between circEXOC5 and PTBP1 was detected by RIP and RNA pulldown.ResultsOur results showed that the circEXOC5 expression was significantly increased in the in vivo and in vitro models of sepsis. And after inhibiting circEXOC5, it improved the lung injury of septic mice. It was confirmed in cell models that ROS levels and ferroptosis in cells were reduced after knocking down circEXOC5. In addition, the expressions of ACSL4 and Gpx4 proteins were regulated by the level of circEXOC5. Finally, we also found that circEXOC5 had a direct binding relationship with PTBP1.ConclusionOur study found that the expression of cell ferroptosis and circEXOC5 increased in ALI induced by sepsis, and circEXOC5 aggravated ferroptosis in septic cells by regulating the PTBP1/ACSL4 axis.