Inflammatory response mediated by oxidative stress is considered as an important pathogenesis of spinal cord injury (SCI). Advanced oxidation protein products (AOPPs) are novel markers of oxidative stress and their role in inflammatory response after SCI remained unclear. This study aimed to investigate the role of AOPPs in SCI pathogenesis and explore the possible underlying mechanisms. A C5 hemi-contusion injury was induced in Sprague-Dawley rats to confirm the involvement of AOPPs after SCI. For in vivo study, apocynin, the NADPH oxidase inhibitor was used to study the neuroprotective effects after SCI. For in vitro study, the BV2 microglia cell lines were pretreated with or without the inhibitor or transfected with or without small interference RNA (siRNA) and then stimulated with AOPPs. A combination of molecular and histological methods was used to clarify the mechanism and explore the signaling pathway both in vivo and in vitro. One-way analysis of variance (ANOVA) was conducted with Bonferroni post hoc tests to examine the differences between groups. The levels of AOPPs in plasma and cerebrospinal fluid as well as the contents in the spinal cord showed significant increase after SCI. Meanwhile, apocynin ameliorated tissue damage in the spinal cord after SCI, improving the functional recovery. Immunofluorescence staining and western blot analysis showed activation of microglia after SCI, which was in turn inhibited by apocynin. Pretreated BV2 cells with AOPPs triggered excessive generation of reactive oxygen species (ROS) by activating NADPH oxidase. Increased ROS induced p38 MAPK and JNK phosphorylation, subsequently triggering nuclear translocation of NF-κB p65 to express pro-inflammatory cytokines. Also, treatment of BV2 cells with AOPPs induced NLRP3 inflammasome activation and cleavage of Gasdermin-d (GSDMD), causing pyroptosis. This was confirmed by cleavage of caspase-1, production of downstream mature interleukin (IL)-1β and IL-18 as well as rupture of rapid cell membrane. Collectively, these data indicated AOPPs as biomarkers of oxidative stress, modulating inflammatory response in SCI by multiple signaling pathways, which also included the induction of NADPH oxidase dependent ROS, and NLRP3-mediated pyroptosis, and activation of MAPKs and NF-κB.

中文翻译：

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先进的氧化蛋白产物通过继发性脊髓损伤后的MAPKs-NF-κB信号传导途径诱导小胶质细胞介导的神经炎症和发烧

由氧化应激介导的炎症反应被认为是脊髓损伤（SCI）的重要发病机理。高级氧化蛋白产物（AOPPs）是氧化应激的新标记，尚不清楚SCI后它们在炎症反应中的作用。这项研究旨在调查AOPPs在SCI发病机制中的作用，并探讨可能的潜在机制。在Sprague-Dawley大鼠中诱发了C5半挫伤损伤，以确认SCI后AOPP的参与。在体内研究中，使用NADPH氧化酶抑制剂Apocynin来研究SCI后的神经保护作用。为了进行体外研究，将BV2小胶质细胞系用或不使用抑制剂预处理，或用或不使用小干扰RNA（siRNA）转染，然后用AOPP刺激。结合分子和组织学方法来阐明机理，并探索体内和体外的信号传导途径。使用Bonferroni事后检验进行单向方差分析（ANOVA），以检查组之间的差异。脊髓损伤后血浆和脑脊液中的AOPPs水平以及脊髓中的含量均显着增加。同时，Apocynin改善了脊髓损伤后脊髓的组织损伤，改善了功能恢复。免疫荧光染色和蛋白质印迹分析显示，SCI后小胶质细胞活化，而这又被阿朴西宁抑制。带有AOPP的BV2预处理细胞通过激活NADPH氧化酶触发了活性氧（ROS）的过量生成。ROS诱导的p38 MAPK和JNK磷酸化增加，随后触发NF-κBp65的核易位，以表达促炎性细胞因子。同样，用AOPP处理BV2细胞会诱导NLRP3炎性小体激活和Gasdermin-d（GSDMD）裂解，从而引起热凋亡。caspase-1的切割，下游成熟白介素（IL）-1β和IL-18的产生以及快速细胞膜的破裂证实了这一点。总的来说，这些数据表明AOPPs是氧化应激的生物标志物，通过多种信号途径调节SCI中的炎症反应，还包括诱导NADPH氧化酶依赖性ROS和NLRP3介导的凋亡，以及MAPKs和NF-κB的激活。引起细胞凋亡。caspase-1的切割，下游成熟白介素（IL）-1β和IL-18的产生以及快速细胞膜的破裂证实了这一点。总的来说，这些数据表明AOPPs是氧化应激的生物标志物，通过多种信号途径调节SCI中的炎症反应，还包括诱导NADPH氧化酶依赖性ROS和NLRP3介导的凋亡，以及MAPKs和NF-κB的激活。引起细胞凋亡。caspase-1的切割，下游成熟白介素（IL）-1β和IL-18的产生以及快速细胞膜的破裂证实了这一点。总的来说，这些数据表明AOPPs是氧化应激的生物标志物，通过多种信号途径调节SCI中的炎症反应，还包括诱导NADPH氧化酶依赖性ROS和NLRP3介导的凋亡，以及MAPKs和NF-κB的激活。