The Role And Mechanism Of Advanced Oxidation Protein Products On Microglia Activation In Spinal Cord Injury Rats

BackgroundSpinal cord injury(SCI)is one of the serious injuries,accompanied by sudden loss of sensory,motor,and autonomic nerves.SCI exhibits great physical and mental burden on affected patients and regards as a challenging clinical problem.The pathological mechanism of SCI is complex.There is currently no effective treatment for SCI.Among all the mechanisms of secondary damage,the inflammatory response mediated by oxidative stress is the most important,and directly or indirectly controls the sequelae after SCI.As a hallmark of SCI pathology and a pivotal inflammatory cell in the central nervous system,microglia play a prominent role in neuroinflammation.Advanced oxidation protein products(AOPPs)are considered as novel markers of oxidative stress and regard as biomarkers of oxidative stress in different neuroinflammatory diseases.However,the mechanisms of AOPPs in the pathogenesis of SCI remains unclear.This study aimed to investigate the role of AOPPs in SCI pathogenesis and explore the possible underlying mechanisms.Materials and MethodsA C5 hemi-contusion injury was induced in Sprague-Dawley rats to confirm the involvement of AOPPs after SCI.For in vivo study,all animals were randomly divided into four experimental groups,with 24 rats per group:control group,Shamgroup,SCI group,and SCI+apocynin group.The spinal cord tissues,plasma,and cerebrospinal fluid were obtained on days 3,7,14,and 28 after SCI.Apocynin,the NADPH oxidase inhibitor was used to study the anti-oxidant and 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.ResultsThe 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 4.Increased ROS induced p38 MAPK and JNK phosphorylation,subsequently triggering nuclear translocation of NF-?B 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)-1? and IL-18 as well as rupture of rapid cell membrane.ConclusionsCollectively,the present study demonstrated that the content of AOPPs increased rapidly in rats after SCI.These data also 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 activation of MAPKs and NF-?B,and NLRP3-mediated pyroptosis.