铁过载及铁死亡在瘢痕疙瘩成纤维细胞中的作用及机制

The role and mechanism of iron overload and ferroptosis in keloid fibroblasts

目的了解瘢痕疙瘩与正常皮肤组织中铁含量及转铁蛋白受体1(TfR1)表达水平,在体外构建Erastin诱导的瘢痕疙瘩成纤维细胞(KFB)铁死亡模型,检测Erastin和铁抑制素-1(Fer-1)对细胞活力、亚铁离子(Fe^(2+))含量及脂质过氧化物、铁死亡和纤维化相关调节因子的影响。方法收集2022年3至6月新疆医科大学第一附属医院6例瘢痕疙瘩组织与6例包皮组织,采用组织铁含量试剂盒测定2种组织真皮层中铁含量,Western blotting法检测2种组织中TfR1蛋白表达情况。采用组织块培养法获取原代KFB和正常皮肤成纤维细胞(NFB),使用Erastin诱导KFB铁死亡模型并用CCK-8法检测不同浓度的Erastin和Fer-1对细胞活性的影响,筛选合适的药物浓度。后续实验分为5组:NFB组、control组、Erastin(0.6μmol/L)组、Fer-1(1μmol/L)组、Erastin(0.6μmol/L)+Fer-1(1μmol/L)组,其中后4组采用KFB作为实验对象;采用划痕实验检测细胞迁移能力,荧光探针法和试剂盒检测各组细胞中丙二醛(MDA)、活性氧(ROS)和Fe^(2+)含量;Western blotting法检测各组细胞中TfR1、谷胱甘肽过氧化物酶4(GPx4)、溶质载体家族7成员11(SLC7A11)、α-平滑肌肌动蛋白(α-SMA)、Ⅰ型胶原蛋白(COL-1)表达水平,免疫荧光检测KFB中TfR1、Gpx4蛋白表达及定位情况。采用GraphPad Prism 9.0统计软件,计量资料以±s表示,2组间比较采用独立样本t检验,多组间比较采用单因素方差分析,组间两两比较采用LSD-t检验。P<0.05表示差异具有统计学意义。结果与正常皮肤组织比较,瘢痕疙瘩中的铁含量及TfR1蛋白表达均明显较高(P<0.01)。不同浓度Erastin处理的KFB增殖率逐渐下降,IC50为0.61μmol/L,Fer-1在0.1~20μmol/L对KFB无明显毒性。划痕实验显示,control组迁移率显著高于NFB组(P<0.01);与control组相比,Erastin干预后KFB迁移率明显下降(P<0.01);与Erastin组相比,Erastin+Fer-1组KFB迁移明显加快(P<0.01)。control组ROS、MDA水平显著高于NFB组(P<0.01);与control组相比,Erastin组ROS、MDA水平及Fe^(2+)含量显著升高(P<0.01),而Fer-1组ROS、MDA水平和Fe^(2+)含量显著降低(P<0.05);与Erastin组相比,Erastin+Fer-1组MDA、ROS水平及Fe^(2+)含量均显著降低(P<0.01)。Western blotting显示,与NFB组相比,control组铁死亡指标SLC7A11、GPx4蛋白表达明显减少(P<0.01),TfR1蛋白表达增加(P<0.01),纤维化指标α-SMA、COL-1蛋白表达显著增加(P<0.01);与control组相比,Erastin组SLC7A11表达减少(P<0.01),TfR1、COL-1表达增加(P<0.01),而Fer-1组SLC7A11、GPx4表达增加(P<0.01),TfR1、α-SMA、COL-1表达显著减少(P<0.01);与Erastin组相比,Erastin+Fer-1组GPx4、SLC7A11表达增加(P<0.01),TfR1、α-SMA、COL-1表达显著减少(P<0.01),提示Fer-1能够逆转Erastin诱导的KFB铁死亡和促纤维化作用。免疫荧光显示,GPx4在细胞核及细胞质中均有表达,与control组相比,Fer-1增加了KFB中GPx4的荧光强度(P<0.01);与Erastin组相比,Erastin+Fer-1组中GPx4的荧光强度显著增加(P<0.01);TfR1主要在细胞质中表达,与control组相比,Erastin增加了KFB中TfR1的荧光强度(P<0.05),而Fer-1组中TfR1荧光强度显著降低(P<0.01);与Erastin组相比,Erastin+Fer-1组TfR1荧光强度显著降低(P<0.01)。结论瘢痕疙瘩中铁过载且游离铁增多,Erastin可诱导KFB铁死亡并加重瘢痕疙瘩纤维化,Fer-1可逆转Erastin诱导形成的氧化损伤及铁蓄积,有效抑制KFB发生铁死亡和瘢痕疙瘩纤维化。

Objective To investigate the iron content and transferrin receptor 1(TfR1)expression levels in keloid and normal skin tissues.Erastin induced ferroptosis model of keloid fibroblasts(KFB)is constructed in vitro,and the effects of Erastin and Ferrostatin-1(Fer-1)on cell viability,ferrous ion(Fe^(2+))content and lipid peroxidation,ferroptosis and fibrosis-related regulatory factors are examined.Methods Six keloid tissues and six prepuces were collected from the First Affiliated Hospital of Xinjiang Medical University from March to June 2022.The tissue iron content kit was used to determine the iron content in the dermis,and TfR1 protein expression level was detected by Western blotting.Primary KFB and normal skin fibroblasts(NFB)were obtained by tissue cultivation,Erastin-induced KFB ferroptosis model and CCK-8 assay were used to detect the effects of different concentrations of Erastin and Fer-1 on cell viability,and to screen the appropriate drug concentration.The subsequent experiments were divided into five groups:NFB group,control group,Erastin(0.6μmol/L)group,Fer-1(1μmol/L)group,and Erastin(0.6μmol/L)+Fer-1(1μmol/L)group.KFB was used in the last 4 groups.Cell migration ability was detected by scratch assay.The contents of malondialdehyde(MDA),reactive oxygen species(ROS)and Fe^(2+)were detected by fluorescence probe and kits;the protein expression levels of TfR1,glutathione peroxidase 4(GPx4),solute carrier family 7 member 11(SLC7A11),α-smooth muscle actin(α-SMA)and type I collagen(COL-1)in each group of cells were detected by Western blotting;the protein expression and localization of TfR1 and Gpx4 in KFB were detected by immunofluorescence staining.GraphPad Prism 9.0 statistical software was used in the statistical analyses,and the measurement data were expressed as Mean±SD.Independent samples t-test was used for comparison between 2 groups,and one-way ANOVA was used for comparison between multiple groups,LSD-t test was used for pairwise comparison between groups.P<0.05 indicated statistical significance.Results The iron content and TfR1 protein expression level were significantly higher in keloids compared with normal skin tissue(P<0.01).The proliferation rate of KFB decreased as the Erastin concentration increased,the IC50 was 0.61μmol/L,and Fer-1 had no obvious toxicity to KFB in the range of 0.1-20μmol/L.Scratch test showed that the migration rate of control group was significantly higher than that of NFB group(P<0.01);compared with the control group,KFB migration rate decreased significantly after Erastin intervention(P<0.01);compared with the Erastin group,KFB migration was significantly accelerated in the Erastin+Fer-1 group(P<0.01).Compared with the NFB group,ROS,MDA levels were significantly increased in the control group(P<0.01);compared with the control group,ROS,MDA levels and Fe^(2+)content were significantly higher in the Erastin group(P<0.01),while ROS,MDA levels and Fe^(2+)content were significantly lower in the Fer-1 group(P<0.05);compared with the Erastin group,MDA,ROS levels and Fe^(2+)content in the Erastin+Fer-1 group were significantly decreased(P<0.01).Western blotting results showed that,compared with the NFB group,ferroptosis indexes of SLC7A11 and GPx4 protein expression levels were significantly reduced(P<0.01),TfR1 protein expression was increased(P<0.01),and protein expression of fibrosis indexes,α-SMA and COL-1 were significantly increased(P<0.01)in the control group;compared with the control group,Erastin group had reduced SLC7A11 expression(P<0.01)and increased TfR1,COL-1 expression(P<0.01),while SLC7A11,GPx4 expression increased(P<0.01)and TfR1,α-SMA,COL-1 decreased(P<0.01)in the Fer-1 group;compared with the Erastin group,the GPx4 and SLC7A11 expression levels were increased(P<0.01)and TfR1,α-SMA,COL-1 expression levels were significantly decreased(P<0.01)in the Erastin+Fer-1 group,suggesting that Fer-1 was able to reverse the Erastin-induced ferroptosis and pro-fibrotic effects in KFB.Immunofluorescence staining showed that GPx4 was expressed in both the nucleus and the cytoplasm.Compared with the control group,Fer-1 increased the fluorescence intensity of GPx4 in KFB(P<0.01).Compared with the Erastin group,the fluorescence intensity of GPx4 in Erastin+Fer-1 group was significantly increased(P<0.01).TfR1 was mainly expressed in the cytoplasm.Compared with the control group,Erastin increased the fluorescence intensity of TfR1 in KFB(P<0.05),while Fer-1 group significantly decreased it(P<0.01).Compared with Erastin group,the fluorescence intensity of TfR1 in Erastin+Fer-1 group was significantly reduced(P<0.01).Conclusion Iron overload is present in keloids and the free iron level is increased.Erastin is able to induce ferroptosis in KFB and aggravate keloid fibrosis.Fer-1 can reverse the oxidative damage and iron accumulation induced by Erastin,and is able to effectively inhibit ferroptosis and keloid fibrosis in KFB.