摘要
目的探讨在骨髓增生异常综合征(myelodysplastic syndrome,MDS)中SMAD家族成员7(SMAD family member 7,SMAD7)信号通路异常与铁死亡关系,并探讨积雪草苷(asiaticoside,AC)提高MDS细胞系SMAD7的表达对铁死亡的影响。方法通过基因表达综合(Gene Expression Omnibus,GEO)数据库筛选MDS相关数据集,比较MDS患者与健康对照人群的差异表达基因,再与铁死亡数据库(Ferroptosis Database,FerrDb)取交集,以寻找MDS铁死亡潜在靶点。收集2022年10月至2024年11月在解放军总医院第一医学中心血液科初次确诊的MDS患者骨髓血18例(MDS组)和同期16例健康供者骨髓血(Con组),提取骨髓单个核细胞(bone marrow mononuclear cells,BMMNCs),RT-qPCR检测SMAD7及铁死亡相关基因表达水平。免疫磁珠分选髓系标志CD33^(+)细胞,RT-qPCR与Western blot检测SMAD7及铁死亡相关基因与蛋白表达水平。同时使用人正常骨髓细胞系(HS-5)与MDS细胞系(MUTZ-1、SKM-1),测定SMAD7及铁死亡相关指标的水平。采用不同浓度的SMAD7激动剂AC(AC组)和铁抑素-1(Ferrostatin-1,Fer-1)处理MDS细胞,探究其对MDS细胞铁死亡的影响。同时构建SMAD7过表达质粒(SMAD7过表达组),进一步研究SMAD7在铁死亡调控中的作用。Western blot与RT-qPCR实验检测细胞处理前后SMAD7与铁死亡相关基因的表达;同时检测谷胱甘肽(glutathione,GSH)、丙二醛(malondialdehyde,MDA)和超氧歧化酶(superoxide dismutase,SOD)水平,流式细胞术与荧光显微镜测定活性氧(reactive oxygen species,ROS)水平。流式细胞术测定分化标志物CD11b^(+)表达,检测细胞分化水平。结果(1)数据库分析结果显示,SMAD7在MDS患者中表达降低,并且与铁死亡有关。相较于Con组,MDS患者SMAD7表达下降(P<0.05),同时铁死亡负向调控基因谷胱甘肽过氧化酶4(glutathione peroxidase 4,GPX4)、铁蛋白重链(ferritin heavy chain,FTH1)表达下降(P<0.05),正向调控基因转铁蛋白受体(transferrin receptor protein1,TFRC)表达升高(P<0.05)。相较于人正常骨髓细胞HS-5,MDS细胞系SMAD7表达下降,GPX4、FTH1表达下降,而TFRC表达升高(P<0.05)。(2)经梯度浓度Fer-1处理后,MDS细胞系中GPX4与FTH1的表达呈浓度依赖性上调(P<0.05),TFRC显著下调(P<0.05),同时GSH含量与SOD活性升高,ROS水平与MDA含量下降(P<0.05),提示铁死亡被抑制。(3)过表达SMAD7后,结果显示GPX4、FTH1的表达升高,TFRC的表达降低(P<0.05),细胞的抗氧化能力提高,减少了铁死亡的发生。提高了CD11b^(+)表达(P<0.05),促进了细胞分化。(4)MDS细胞系经AC处理后GPX4与FTH1的表达呈浓度依赖性上调(P<0.05),但TFRC下调无统计学意义。AC处理还提高了细胞的抗氧化能力,同时提高了CD11b^(+)的含量(P<0.05),促进了细胞分化。结论AC激活MDS细胞系SMAD7,通过上调GPX4与FTH1并抑制TFRC,减轻氧化损伤和脂质过氧化,从而抑制MDS细胞铁死亡,同时激活SMAD7可以促进细胞分化。
Objective To investigate the relationship of aberrant SMAD family member 7(SMAD7)signaling pathway and ferroptosis in myelodysplastic syndromes(MDS)and evaluate the effect of asiaticoside(AC)-modulating SMAD7 up-regulation to suppress ferroptosis in MDS cell lines.Methods Publicly available MDS-related datasets from the Gene Expression Omnibus(GEO)database were analyzed to identify differentially expressed genes(DEGs)between MDS patients and healthy controls.These DEGs were crossreferenced with ferroptosis-associated genes from the Ferroptosis Database(FerrDb)to identify potential ferroptosis-related targets in MDS.Bone marrow mononuclear cells(BMMNCs)were isolated from 18 MDS patients freshly diagnosed in the First Medical Center of Chinese PLA General Hospitaland and from 16 healthy donors during October 2022 and November 2024.RT-qPCR was employed to detect the expression of SMAD7 and ferroptosis-related genes.Immunomagnetic bead sorting was applied to purify CD33^(+)cells,and then qPCR and Western blotting were utilized to measure the expression of SMAD7 and ferroptosis-related biomarkers at mRNA and protein levels.Human normal bone marrow cells(HS-5)and MDS cell lines(MUTZ-1,SKM-1)were treated with gradient concentrations of AC(SMAD7 activator)and ferrostatin-1(Fer-1,ferroptosis inhibitor),and SMAD7 overexpression plasmids were transfected into MDS cells.qPCR and Western blotting were utilized to measure the expression of SMAD7 and ferroptosis-related biomarkers at mRNA and protein levels,and the contents of glutathione(GSH),malondialdehyde(MDA),superoxide dismutase(SOD),and reactive oxygen species(ROS)were detected.Flow cytometry of CD11b^(+)was performed to measure cellular differentiation.Results①Bioinformatics analysis revealed significant down-regulation of SMAD7 in MDS patients,correlating with ferroptosis activation.Compared to the healthy controls,MDS patients exhibited decreased SMAD7 expression(P<0.05),reduced levels of negative regulators of ferroptosis,glutathione peroxidase 4(GPX4)and ferritin heavy chain(FTH1)(P<0.05),and elevated expression of its positive regulator transferrin receptor protein 1(TFRC)(P<0.05).Consistent with this,when compared with the normal human bone marrow stromal cell line HS-5,the MDS cell lines MUTZ-1 and SKM-1 exhibited declined expression of SMAD7,GPX4,and FTH1,alongside elevated expression of TFRC(P<0.05).②Treatment with gradient concentrations of the ferroptosis inhibitors ferrostatin-1(Fer-1),the expression levels of GPX4 and FTH1 in MDS cell lines were significantly upregulated in a concentration-dependent manner(P<0.05),while TFRC was markedly downregulated(P<0.05).Additionally,GSH content and SOD activity were enhanced,whereas ROS levels and MDA content were significantly reduced(P<0.05).These results suggest that Fer-1 effectively suppresses ferroptosis in MDS cells.③SMAD7 overexpression led to up-regulation of GPX4 and FTH1 ih MDS cell lines,while downregulation of TFRC,improved anti-oxidative ability and reduced ferroptosis,with enhanced CD11b^(+)expression and myeloid differentiation.④Following AC treatment,the expression levels of GPX4 and FTH1 in MDS cell lines were significantly upregulated in a concentration-dependent manner(P<0.05),whereas the downregulation of TFRC did not reach statistical significance.Additionally,AC treatment effectively enhanced the antioxidant capacity of the cells,increased the proportion of CD11b^(+)cells(P<0.05),and facilitated cellular differentiation.Conclusion AC activates SMAD7 in MDS cell lines,up-regulating GPX4 and FTH1 while suppressing TFRC expression.This mechanism alleviates oxidative damage and lipid peroxidation,thereby inhibiting ferroptosis in MDS cells.Concurrently,SMAD7 activation promotes cellular differentiation.
作者
王立烨
常青
董腾腾
刘亭
王驰
李绵洋
WANG Liye;CHANG Qing;DONG Tengteng;LIU Ting;WANG Chi;LI Mianyang(Department of Clinical Laboratory,First Medical Center of Chinese PLA General Hospital,Beijing,China)
出处
《陆军军医大学学报》
北大核心
2025年第12期1319-1331,共13页
Journal of Army Medical University
基金
北京市自然科学基金-昌平创新联合基金资助项目(L234048)。
