Ferroptosis,一种程序性细胞死亡,以铁依赖性脂质ROS积累为特征,被认为是哺乳动物巨噬细胞抵抗细胞内细菌感染的重要细胞免疫。在这个过程中,脂质ROS氧化细菌生物膜以抑制细胞内细菌。然而,无脊椎动物铁死亡的功能仍然未知。在这项研究中,证实刺参腔体细胞存在铁细胞凋亡,并对其抗菌机理进行了研究。首先,我们的结果表明,用铁性凋亡诱导剂RSL3注射日本A后,谷胱甘肽过氧化物酶(AjGPX4)的表达显着抑制了0.21倍(p<0.01),而MDA的含量(3.70倍,p<0.01),亚铁(1.40倍,p<0.01),和脂质ROS(3.10倍,p<0.01)在此条件下均显着增加,同时伴有线粒体收缩和cr消失,表明日本血吸虫的腔体细胞中存在铁性凋亡。随后,亚铁含量(1.40倍,p<0.05),MDA(2.10倍,p<0.01),ROS(1.70倍,p<0.01),和脂质ROS(2.50倍,p<0.01)均显著增加,而在脾弧菌(AJ01)感染下,线粒体膜电位和GSH/GSSG显着降低了0.68倍(p<0.05)和0.69倍(p<0.01)。这个过程可以通过铁螯合剂去铁胺甲磺酸盐逆转,这表明AJ01可以诱导腔细胞铁凋亡。此外,结果表明,用RSL3和亚铁处理后,细胞内AJ01负荷明显降低至0.49倍(p<0.05)和0.06倍(p<0.01),这表明增强的铁死亡可以抑制细菌的生长。最后,亚细胞定位表明,亚铁外排蛋白铁转运蛋白(AjFPN)和细胞内AJ01共定位在腔体细胞中。AjFPN干扰后(0.58倍,p<0.01),细胞内AJ01中亚铁和脂质ROS水平的信号显着降低了0.38倍(p<0.01)和0.48倍(p<0.01),表明AjFPN是将铁凋亡引入细胞内细菌的重要因素。总的来说,我们的发现表明,铁性凋亡可以通过AjFPN抵抗细胞内AJ01感染。这些发现为水生动物抵抗细胞内细菌感染提供了一种新的防御机制。
Ferroptosis, a kind of programmed cell death, is characterized with iron-dependent lipid ROS buildup, which is considered as an important cellular immunity in resisting intracellular bacterial infection in mammalian macrophages. In this process, lipid ROS oxidizes the bacterial biofilm to inhibit intracellular bacteria. However, the function of ferroptosis in invertebrate remains unknown. In this study, the existence of ferroptosis in Apostichopus japonicus coelomocytes was confirmed, and its antibacterial mechanism was investigated. First, our results indicated that the expression of glutathione peroxidase (AjGPX4) was significantly inhibited by 0.21-fold (p < 0.01) after injecting A. japonicus with the ferroptosis inducer RSL3, and the contents of MDA (3.93-fold, p < 0.01), ferrous iron (1.40-fold, p < 0.01), and lipid ROS (3.10-fold, p < 0.01) were all significantly increased under this condition and simultaneously accompanied with mitochondrial contraction and disappearance of cristae, indicating the existence of ferroptosis in the coelomocytes of A. japonicus. Subsequently, the contents of ferrous iron (1.40-fold, p < 0.05), MDA (2.10-fold, p < 0.01), ROS (1.70-fold, p < 0.01), and lipid ROS (2.50-fold, p < 0.01) were all significantly increased, whereas the mitochondrial membrane potential and GSH/GSSG were markedly decreased by 0.68-fold (p < 0.05) and 0.69-fold (p < 0.01) under Vibrio splendidus (AJ01) infection. This process could be reversed by the iron-chelating agent deferoxamine mesylate, which indicated that AJ01 could induce coelomocytic ferroptosis. Moreover, the results demonstrated that the intracellular AJ01 load was clearly decreased to 0.49-fold (p < 0.05) and 0.06-fold (p < 0.01) after treating coelomocytes with RSL3 and ferrous iron, which indicated that enhanced ferroptosis could inhibit bacterial growth. Finally, subcellular localization demonstrated that ferrous iron efflux protein
ferroportin (AjFPN) and intracellular AJ01 were co-localized in coelomocytes. After AjFPN interference (0.58-fold, p < 0.01), the signals of ferrous iron and lipid ROS levels in intracellular AJ01 were significantly reduced by 0.38-fold (p < 0.01) and 0.48-fold (p < 0.01), indicating that AjFPN was an important factor in the introduction of ferroptosis into intracellular bacteria. Overall, our findings indicated that ferroptosis could resist intracellular AJ01 infection via AjFPN. These findings provide a novel defense mechanism for aquatic animals against intracellular bacterial infection.