背景:免疫炎症反应在脊髓损伤(SCI)中起重要作用;然而,有益和有害的影响仍然存在争议。许多研究描述了中性粒细胞的作用,巨噬细胞,和T淋巴细胞在SCI后的免疫炎症反应中的作用,尽管人们对B淋巴细胞的作用知之甚少,SCI后易发生免疫抑制。
方法:建立SCI小鼠模型,HE染色和Nissl染色观察病理改变。检查脾脏的大小和形态,流式细胞术和ELISA检测SCI对脾功能和B细胞水平的影响。为探讨SCI后免疫抑制的具体机制,采用磁珠从SCI模型小鼠的脾脏中分离B细胞,并通过4D无标记定量蛋白质组学进行分析。检测炎性细胞因子和铁离子水平,通过蛋白质印迹法定量与Tom20途径相关的蛋白质的表达。为了阐明SCI后铁离子与B细胞焦亡之间的关系,我们使用了FeSO4和CCCP,诱导氧化应激刺激SCI,干扰B细胞过程。使用siRNA转染在B细胞和人B淋巴细胞瘤细胞中敲除Tom20(Tom20-KD)来验证Tom20的关键作用。为了进一步研究铁离子对SCI的影响,我们使用去铁胺(DFO)和葡聚糖铁(ID)干扰小鼠的SCI过程。分析脾B细胞中铁离子的水平和与Tom20-Bax-caspase-gasderminE(GSDME)途径相关的蛋白质的表达。
结果:SCI可损伤脾脏功能,导致B细胞水平降低;SCI上调B细胞线粒体中Tom20蛋白的表达;SCI可调节铁离子浓度,激活Tom20-Bax-caspase-GSDME通路,诱导B细胞的焦亡。铁离子通过激活Tom20-Bax-caspase-GSDME途径加重CCCP诱导的B细胞焦亡和人B淋巴细胞瘤的焦亡。DFO可以通过抑制Tom20-Bax-caspase-GSDME诱导的B细胞凋亡来减轻炎症反应并促进SCI后的修复。
结论:SCI后铁过载激活Tom20-Bax-caspase-GSDME通路,诱导B细胞凋亡,促进炎症,并加剧SCI引起的变化。这可能代表了SCI后诱导免疫炎症反应的新机制,并可能为SCI的治疗提供新的关键靶标。
BACKGROUND: Immune inflammatory responses play an important role in spinal cord injury (SCI); however, the beneficial and detrimental effects remain controversial. Many studies have described the role of neutrophils, macrophages, and T lymphocytes in immune inflammatory responses after SCI, although little is known about the role of B lymphocytes, and immunosuppression can easily occur after SCI.
METHODS: A mouse model of SCI was established, and HE staining and Nissl staining were performed to observe the pathological changes. The size and morphology of the spleen were examined, and the effects of SCI on spleen function and B cell levels were detected by flow cytometry and ELISA. To explore the specific mechanism of immunosuppression after SCI, B cells from the spleens of SCI model mice were isolated using magnetic beads and analyzed by 4D label-free quantitative proteomics. The level of inflammatory cytokines and iron ions were measured, and the expression of proteins related to the Tom20 pathway was quantified by western blotting. To clarify the relationship between iron ions and B cell pyroptosis after SCI, we used FeSO4 and CCCP, which induce oxidative stress to stimulate SCI, to interfere with B cell processes. siRNA transfection to knock down Tom20 (Tom20-KD) in B cells and human B lymphocytoma cell was used to verify the key role of Tom20. To further explore the effect of iron ions on SCI, we used deferoxamine (DFO) and iron dextran (ID) to interfere with SCI processes in mice. The level of iron ions in splenic B cells and the expression of proteins related to the Tom20-Bax-caspase-gasdermin E (GSDME) pathway were analyzed.
RESULTS: SCI could damage spleen function and lead to a decrease in B cell levels; SCI upregulated the expression of Tom20 protein in the mitochondria of B cells; SCI could regulate the concentration of iron ions and activate the Tom20-Bax-caspase-GSDME pathway to induce B cell pyroptosis. Iron ions aggravated CCCP-induced B cell pyroptosis and human B lymphocytoma pyroptosis by activating the Tom20-Bax-caspase-GSDME pathway. DFO could reduce inflammation and promote repair after SCI by inhibiting Tom20-Bax-caspase-GSDME-induced B cell pyroptosis.
CONCLUSIONS: Iron overload activates the Tom20-Bax-caspase-GSDME pathway after SCI, induces B cell pyroptosis, promotes inflammation, and aggravates the changes caused by SCI. This may represent a novel mechanism through which the immune inflammatory response is induced after SCI and may provide a new key target for the treatment of SCI.