线粒体内膜蛋白17(MPV17)通过阻断ERK通路抑制铁过载小鼠脾脏CD3^(+)T细胞铁死亡

MPV17 inhibits iron overload-induced ferroptosis of splenic CD3^(+)T cells in mice by blocking ERK pathway

目的探索铁过载对小鼠脾脏损伤的影响及线粒体内膜蛋白17(MPV17)在铁过载小鼠脾脏CD3^(+)T细胞铁死亡中的作用。方法将小鼠随机分为正常饮食组、高铁饮食组、高铁饮食联合铁死亡抑制剂ferrostatin-1(Fer-1)处理组、高铁饮食联合MPV17腺病毒注射组,每组5只。喂食8周后,取脾脏组织并固定,通过组织切片和HE染色法观察脾脏结构,碘化丙啶(PI)染色检测脾脏CD3^(+)T细胞死亡,脂质过氧化荧光探针C11 BODIPY 581/591检测脂质氧化,实时定量PCR检测溶质载体家族7成员11(SLC7A11)及前列腺素内过氧化物合成酶2(PTGS2)mRNA水平,流式细胞术检测M1、M2巨噬细胞比例,ELISA实验检测肿瘤坏死因子α(TNF-α)、白细胞介素1β(IL-1β)及IL-6的含量。同时增加高铁饮食联合细胞外信号调节激酶(ERK)抑制剂处理组、ERK激活剂处理组、β半乳糖苷(β-gal)酶联合ERK激活剂处理组、MPV17联合ERK激活剂处理组,Western blot法检测MPV17、谷胱甘肽过氧化物酶4(GPX4)、磷酸化的ERK(p-ERK)水平,JC-1结合流式细胞术检测线粒体膜电位。结果与正常饮食组相比,高铁饮食组小鼠脾脏红髓形状不规则,白髓结构消失,脾脏CD3^(+)T细胞死亡增多,脂质过氧化物增多,SLC7A11及PTGS2表达升高,血液中M1/M2巨噬细胞比例升高,炎症因子含量升高;经Fer-1处理或过表达MPV17,部分恢复脾脏结构,CD3^(+)T细胞数量及脂质过氧化物减少,SLC7A11及PTGS2表达被抑制,M1/M2巨噬细胞比例及炎症因子的含量降低。高铁饮食导致GPX4表达降低,p-ERK表达升高,抑制ERK部分恢复GPX4表达,激活ERK降低GPX4表达;MPV17抑制ERK部分恢复GPX4表达,MPV17部分恢复因ERK激活导致的线粒体膜电势降低。结论铁过载可诱导小鼠脾脏CD3^(+)T细胞发生铁死亡,MPV17通过阻断ERK信号抑制高铁饮食诱导的脾脏CD3^(+)T细胞铁死亡。

Objective This work aimed to explore the effect of iron overload on splenic injury and the role of MPV17 in the ferroptosis of splenic CD3^(+)T cells from mice subjected to iron overload.Methods Mice were randomly divided into normal diet group,high-iron diet group,high-iron diet combined with Fer-1 treatment group,and high-iron diet combined with adenovirus harboring MPV17 injection group,with 5 mice in each group.After treatment for 8 weeks,mice spleens were harvested and fixed;Histological section and HE staining were performed to observe the structures of the spleens;Cell death of CD3^(+)T cells was detected by propidium iodide(PI)staining;The lipid peroxidation levels were detected by C11 BODIPY581/591 staining;The mRNA levels of Solute carrier family 7 member 11(SLC7A11)and prostaglandin-endoperoxide synthase 2(PTGS2)were detected by qPCR assays;The macrophage phenotype-switching(M1/M2)were detected by flow cytometry;The levels of TNF-α,IL-1βand IL-6 were measured by ELISA assays.Moreover,high-iron diet combined with extracellular signal-regulated kinase(ERK)inhibitor treatment group,ERK agonist treatment group,β-gal combined with ERK agonist treatment group,and MPV17 overexpression combined with ERK agonist treatment group were added.The protein levels of MPV17,glutathione peroxidase 4(GPX4)and phosphorylated ERK(p-ERK)were detected by Western blot;The mitochondrial membrane potential was detected by JC-1 staining and flow cytometry.Results Compared with the normal diet group,the red pulps of the mice spleens from the high-iron diet group showed irregular structures and the white pulps were almost missing;Cell death,lipid peroxides,and the expression levels of SLC7A11 and PTGS2 increased;Both the ratio of M1 macrophages to M2 macrophages and the levels of inflammatory factors increased.Fer-1 treatment or overexpression of MPV17 in the high-iron diet mice group partially recovered the irregular structures of the spleens,reduced cell death and lipid peroxides in CD3^(+)T cells,and decreased the expression levels of SLC7A11 and PTGS2;The ratio of M1/M2 macrophages and the levels of inflammatory factors were decreased.High-iron diet decreased the protein levels of GPX4 while p-ERK were up-regulated.Inhibition of ERK partially recovered the protein levels of GPX4;ERK agonist decreased the protein levels of GPX4;MPV17 inhibited the ERK signaling and partially recovered the protein levels of GPX4 and the decreased mitochondrial membrane potential of CD3^(+)T induced by ERK activation.Conclusion Iron overload resulted in splenic injury and ferroptosis in the splenic CD3^(+)T cells;MPV17 prevented splenic injury and ferroptosis of splenic CD3^(+)T cells of the iron overload mice through blocking ERK signaling pathway.