The Role Of Extracellular Peroxiredoxin 2 In Early Brain Injury After Experimental Subarachnoid Hemorrhage

Background: Subarachnoid hemorrhage(SAH) is one of three most life-threatening cerebrovascular diseases, with high mortality and disability rates. A great deal of erythrocytes and their lysate were left in the subarachnoid space after SAH. And these lysate could induce early brain injury(EBI) by a series of complicated pathological processes, including encephaledema, oxidative stress, inflammation and cytotoxicity etc.Peroxiredoxins(Prxs) are a ubiquitous family of antioxidant enzymes ineukaryotic cells, with functions that also control cytokine-induced peroxide levels andthereby mediate signal transduction involved in the occurrence and development ofmany diseases. Further studies showed Prxs lost their antioxidant capacity after releasefrom dying cells, and played some paradoxical roles. For example, Prxs were released extracellularly from dying cells after ischemia and then, paradoxically, became potent proinflammatory signals that initiated a destructive inflammatory response. The mechanism was that extracellular Prxs lost their protective function and acted as DAMPs by engaging TLR2 and TLR4 on infiltrating macrophages. And nuclear factor-κB(NF-κB)in these immune cells was activated and then led to production of proinflammatory cytokines, which, in turn, triggered a destructive inflammatory response. As a result, Prxs were further released extracellularly from dying cells and the levels of extracellular Prxs were elevated, which further enhanced the activation of the inflammatory cascade.Peroxiredoxin 2(Prx2), as one of the six members of Prxs, was the third most abundant protein in erythrocytes and was widely expressed in neurons. Because of the characteristics of the unique distribution in cells, Prx2 may play an unusal role in EBI after SAH, with the reasons as follows: subarachnoid erythrocytes and their lysate wereimportant causes to EBI after SAH and triggered activation of multiple mechanisms of brain injury, while unfortunately, neurons were the final target cells of these mechanisms;in other words, Prx2 was not only the protective antioxidant(when located intracellulary),but also the damage factor to neurons(when located extracellularly).Therefore, we put forward the following hypothesis. The levels of Prx2 in cerebrospinal fluid(CSF) and cortical tissue(extracellular Prx2) would be usually elevated after SAH, originating from the lysis of subarachnoid erythrocytes due to aging and swelling, and damaged neurons as a consequence of multiple mechanisms of injury. In addition to might lead directly to neuronal damage, these extracellular Prx2 might become potent proinflammatory signals, initiate and mediate a destructive inflammatory response involving DAMPs. Then, Prx2 would be further released extracellularly from dying neurons and finally cause lingering damage.Methods: Firstly, the expression of Prx2 in erythrocytes was assessed by Western blotting and the cell distribution of Prx2 in brain was assessed by immunofluorescent double staining. After the experimental SAH model in rabbits was estimated, the level of Prx2 in cortical tissue and the levels of Prx2, IL-6 and TNF-αin CSF were assessed by Western blotting and enzyme-linked immunosorbent assay(ELISA). The neural damage and cerebral vasospasm(CVS) were evaluated immunohistochemistry, nissl staining and H&E staining. Secondly, after the in vivo and in vitro SAH models were established, the expression of Prx2 in cortical tissue, neuron and basal was assessed by Western blotting and fluorescence quantitative PCR. Then, after BV2, neurons and neurons co-cultured with BV2 were induced by Prx2, we detected the expression of Toll-like receptor 4(TLR4),Myeloid differentiation factor 8(Myd88) and Nuclear factor- κ B(NF- κ B) in BV2, the expression of cleaved caspase 3 and bax in neurons co-cultured with BV2, the levels of IL-6, TNF-αin BV2 basal and neuronal damage by using Western blotting, ELISA and cell counting kit 8(CCK8). Finally, the level of Prx2 in CSF in patients after SAH was assessed by using Western blotting.Results: Our study verified again that Prx2 was exclusively expressed in neurons during neural cells, in addition to be abundant in erythrocytes. The mRNA expression of Prx2 was increased at 3h after SAH, accompanied by up-regulation of the protein expression of Prx2 at 12 h after SAH. Then Prx2 from necrotic neurons and lysis of erythrocytes constituted extracellular Prx2. Moreover, the level of Prx2 in CSF positively correlated with the levels of IL-6, TNF-α in CSF might involve in the mechanism of EBI after SAH.Further studies showed extracellular Prx2 could directly induce neuronal damage, and induce activation of the TLR4/NF- κB pathway and release of proinflammatory factors(IL-6, TNF-α) from activated microglia, which further aggravate neuronal damage. Finally,the level of Prx2 in CSF was the highest in Hunt-Hess II patients at 3d after SAH and the level of Prx2 in CSF at 3d after SAH was positively correlated to Hunt-Hess grades.Conclusion: Our research verified again that in addition to be abundant in erythrocytes,Prx2 was exclusively expressed in neurons during neural cells, and provided evidence, for the first time, that the level of extracellular Prx2 could be abnormally elevated, which originate from lysis of subarachnoid erythrocytes and necrotic neurons after SAH.Moreover, extracellular Prx2 could directly induce neuronal damage, and induceactivation of the TLR4/NF-κB pathway and release of proinflammatory factors(IL-6, TNF-α) from activated microglia, which further aggravate neuronal damage. In clinic, the level of Prx2 may be a credible biomarker for judging the severity of patients after SAH. These discoveries in this study help to improve the understanding of the complex mechanisms of EBI after SAH, and imply that the prognostic improvement of SAH patients would be acquired by intervening the proinflammatory pathway mediated by extracellular Prx2.