Xu Tao, Jing Wenjun, Chen Guilan
Instrumental Analysis Center of QAU, Qingdao 266109, China.
Department of Oncology, Qingdao Central Hospital, Qingdao University, Qingdao 266042, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2024 May;40(5):395-403.
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-1β and 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.
目的 本研究旨在探讨铁过载对脾脏损伤的影响以及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Fer-1治疗组和高铁饮食联合携带MPV17腺病毒注射组，每组5只。治疗8周后，摘取小鼠脾脏并固定；进行组织切片及HE染色以观察脾脏结构；用碘化丙啶（PI）染色检测CD3⁺ T细胞的细胞死亡情况；用C11 BODIPY581/591染色检测脂质过氧化水平；用qPCR检测溶质载体家族7成员11（SLC7A11）和前列腺素内过氧化物合酶2（PTGS2）的mRNA水平；用流式细胞术检测巨噬细胞表型转换（M1/M2）；用ELISA检测肿瘤坏死因子-α（TNF-α）、白细胞介素-1β（IL-1β）和白细胞介素-6（IL-6）水平。此外，增设高铁饮食联合细胞外信号调节激酶（ERK）抑制剂治疗组、ERK激动剂治疗组、β-半乳糖苷酶联合ERK激动剂治疗组以及MPV17过表达联合ERK激动剂治疗组。用蛋白质免疫印迹法检测MPV17、谷胱甘肽过氧化物酶4（GPX4）和磷酸化ERK（p-ERK）的蛋白水平；用JC-1染色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结构不规则，白髓几乎消失；细胞死亡、脂质过氧化物以及SLC7A11和PTGS2的表达水平升高；M1巨噬细胞与M2巨噬细胞的比例以及炎症因子水平均升高。在高铁饮食小鼠组中，Fer-1治疗或MPV17过表达部分恢复了脾脏的不规则结构，减少了CD3⁺ T细胞中的细胞死亡和脂质过氧化物，并降低了SLC7A11和PTGS2的表达水平；M1/M2巨噬细胞比例和炎症因子水平降低。高铁饮食降低了GPX4的蛋白水平，而p-ERK上调。抑制ERK可部分恢复GPX4的蛋白水平；ERK激动剂降低了GPX4的蛋白水平；MPV17抑制ERK信号传导，并部分恢复了GPX4的蛋白水平以及ERK激活诱导的CD3⁺ T细胞线粒体膜电位降低。结论 铁过载导致脾脏损伤及脾脏CD3⁺ T细胞铁死亡；MPV17通过阻断ERK信号通路预防铁过载小鼠脾脏损伤及脾脏CD3⁺ T细胞铁死亡。
